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https://openalex.org/W2076012260	https://kclpure.kcl.ac.uk/portal/files/12225247/Anacker_Front_Behav_Neuro_2012.pdf	English	null	New models to investigate complex glucocorticoid receptor functions	Frontiers in behavioral neuroscience	2,012	cc-by	1,841	Citation for published version (APA): Anacker, C., & Pariante, C. M. (2012). New models to investigate complex glucocorticoid receptor functions. Frontiers in Behavioral Neuroscience, 6, Article 90. https://doi.org/10.3389/fnbeh.2012.00090 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. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Anacker, C., & Pariante, C. M. (2012). New models to investigate complex glucocorticoid receptor functions. Frontiers in Behavioral Neuroscience, 6, Article 90. https://doi.org/10.3389/fnbeh.2012.00090 A commentary on mechanism of action of antidepressants remains elusive and may possibly account for some of the behavioral changes upon antidepressant treatment in grs357 mutant ﬁsh, in which only GR transactivation is abolished. As the authors suggest, the role of serotonergic signaling on the neuroen- docrine system, behavior, and the regu- lation of GR function is likely to be of great importance for the effects of SSRI antidepressants (Laplante et al., 2002). At the same time, potential serotonin- independent molecular mechanisms of antidepressant action, such as liberation of G-proteins from membrane associated lipid rafts, have been suggested (Donati and Rasenick, 2005), and may represent possible alternative pathways involved in stress response regulation and GR activa- tion, which warrant further investigation. A zebraﬁsh model of glucocorticoid resistance shows serotonergic modulation of the stress response by Grifﬁths, B. B., Schoonheim, P. J., Ziv, L., Voelker, L., Baier, H., and Gahtan, E. (2012). Front. Behav. Neurosci. 6:68. doi: 10.3389/fnbeh.2012.00068 The signiﬁcance of the glucocorticoid receptor (GR) in stress response regula- tion and mood disorders has been demon- strated by many studies in animal and human models (Anacker et al., 2011a). The paper by Grifﬁths’ and colleagues adds to this evidence, by investigating GR function and its role in stress response regulation and antidepressant action in an exciting new zebraﬁsh model. The data suggest that some of the behavioral effects of ﬂuoxetine on revers- ing startle response abnormalities may be independent of GR transactivation in ﬁsh. However, it is important to note that many GR-mediated effects are indeed independent of direct GR binding to the DNA, particularly the glucocorticoid- mediated regulation of pomc expression (Tuckermann et al., 1999; Bilodeau et al., 2006). It thus remains to be elucidated whether GR protein-protein interactions, such as transrepression of the transcrip- tion factors NFkB, AP-1, or cyclic AMP response element binding protein (CREB), may indeed account for some of the SSRI effects on anxiety-like behavior in this study. Moreover, an increasing body of work suggests that a membrane-bound GR may mediate some of the rapid, non- genomic effects of glucocorticoids, partic- ularly glucocorticoid-induced changes in neuronal excitability and memory consol- idation (for comprehensive reviews, please see: Haller et al., 2008 and Groeneweg et al., 2012). Reviewed by: Phillip R. Zoladz, Ohio Northern University, USA Charlier D. Thierry, University of Liege, Belgium effectively counteracts anxiety-like behav- ior. These ﬁndings conﬁrm the crucial role of the GR in neuroendocrine reg- ulation and behavioral stress responses, and are in line with the authors’ previous work, in which they have identiﬁed stress axis abnormalities and increased freezing behavior upon exposure to a novel envi- ronment in the same mutant ﬁsh (Ziv et al., 2012). Their new ﬁndings thus con- solidate the use of the zebraﬁsh model to investigate GR-dependent regulation of the stress response. Christoph Anacker* and Carmine M. Pariante Department of Psychological Medicine, Section of Perinatal Psychiatry and Stress, Psychiatry and Immunology (SPI-Lab), Institute of Psychiatry, King’s College London, London, UK Correspondence: christoph.anacker@kcl.ac.uk Correspondence: christoph.anacker@kcl.ac.uk Edited by: Jozsef Haller, Institute of Experimental Medicine, Hungary Jozsef Haller, Institute of Experimental Medicine, Hungary General rights General rights Copyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associ ral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright condition of accessing publications that users recognize and abide by the legal requirements associated with these right •Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research. •You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal •Users may download and print one copy of any publication from the Research Portal for the purpose of private study •You may not further distribute the material or use it for any profit-making activity or commercial gain y y p g y •You may freely distribute the URL identifying the publication in the Research Portal Take down policy If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. p y If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we w the work immediately and investigate your claim. Download date: 24. Oct. 2024 A commentary on doi: 10.1016/j.yfrne.2007.10.004 Anacker, C., Zunszain, P. A., Carvalho, L. A., and Pariante, C. M. (2011a). The glucocorticoid recep- tor: pivot of depression and of antidepressant treatment? Psychoneuroendocrinology 36, 415–425. Kizil, C., Kaslin, J., Kroehne, V., and Brand, M. (2012). Adult neurogenesis and brain regeneration in zebraﬁsh. Dev. Neurobiol. 72, 429–461. Anacker, C., Zunszain, P. A., Cattaneo, A., Carvalho, L. A., Garabedian, M. J., Thuret, S., et al. (2011b). Antidepressants increase human hip- pocampal neurogenesis by activating the glucocor- ticoid receptor. Mol. Psychiatry 16, 738–750. Laplante, P., Diorio, J., and Meaney, M. J. (2002). Serotonin regulates hippocampal glucocorticoid receptor expression via a 5-HT7 receptor. Brain Res. Dev. Brain Res. 139, 199–203. Tuckermann, J. P., Reichardt, H. M., Arribas, R., Richter, K. H., Schutz, G., and Angel, P. (1999). The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin. J. Cell Biol. 147, 1365–1370. Bilodeau, S., Vallette-Kasic, S., Gauthier, Y., Figarella-Branger, D., Brue, T., Berthelet, F., et al. (2006). Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev. 20, 2871–2886. Ziv, L., Muto, A., Schoonheim, P. J., Meijsing, S. H., Strasser, D., Ingraham, H. A., et al. (2012). An affective disorder in zebraﬁsh with mutation of the glucocorticoid receptor. Mol. Psychiatry. doi: 10.1038/mp.2012.64. [Epub ahead of print]. In order to identify new treatment tar- gets and to elucidate the sophisticated heterogeneous mechanisms underlying depression pathogenesis, a cross-species investigation into the several layers of the complex nature of stress-induced biolog- ical abnormalities will be essential to our success. The paper by Grifﬁths’ and col- leagues, together with previous work on GR function in zebraﬁsh (Ziv et al., 2012), is a great example for such an approach, and offers a novel high-throughput plat- form in live ﬁsh to investigate drug responses and GR function in an in vivo system. Bury, N. R., and Sturm, A. (2007). Evolution of the corticosteroid receptor signalling pathway in ﬁsh. Gen. Comp. Endocrinol. 153, 47–56. Donati, R. J., and Rasenick, M. M. (2005). Chronic antidepressant treatment prevents accumulation of gsalpha in cholesterol-rich, cytoskeletal- associated, plasma membrane domains (lipid rafts). Neuropsychopharmacology 30, 1238–1245. Received: 29 November 2012; accepted: 12 December 2012; published online: 31 December 2012. Citation: Anacker C and Pariante CM (2012) New models to investigate complex glucocorticoid receptor functions. Front. Behav. Neurosci. 6:90. doi: 10.3389/ fnbeh.2012.00090 Groeneweg, F. A commentary on While previous studies have shown a crucial role for genomic GR effects in antidepressant action (Anacker et al., 2011b), the involvement of the membrane-bound form of the GR in the Of course, when modeling psychiatric disorders in ﬁsh, it needs to be carefully considered that the behavioral repertoire, the neural networks and the psycholog- ical responses to stress in teleosts are largely different from respective responses in the mammalian brain. Behavioral stud- ies in higher animals, such as rodents and primates, as well as cellular and molecular studies in human tissues, will therefore indeed remain indispensable for our understanding of stress-induced brain abnormalities, depression patho- physiology, and antidepressant treatment response. To this end, it will be interest- ing for future investigations to examine if other well established neurobiological correlates of depression, such as alter- ations in neuronal gene expression or adult neurogenesis, can also be mod- eled in comparable regions of the ﬁsh brain (Kizil et al., 2012). In addition, evolutionary differences in corticosteroid The main ﬁnding of this study is that mutant zebraﬁsh larvae with impaired GR transactivation, due to a single nucleotide substitution in a region essential for DNA binding (grs357 mutants), exhibit behav- ioral abnormalities and neuroendocrine dysfunction. Speciﬁcally, grs357 mutants show elevated startle responses, a sur- rogate measure of anxiety-like behav- ior in ﬁsh, as well as increased cortisol levels and impaired HPA axis feedback control, demonstrated by reduced sup- pression of pituitary pomc upon treat- ment with the synthetic glucocorticoid, betamethasone. The authors also test whether the selective serotonin reuptake inhibitor (SSRI) antidepressant, ﬂuoxe- tine, is able to reverse the neuroen- docrine and behavioral phenotype of grs357 mutants. Their ﬁndings suggest that ﬂuoxetine does not reverse neu- roendocrine abnormalities resulting from impaired GR transactivation, while it still December 2012 \| Volume 6 \| Article 90 \| 1 Frontiers in Behavioral Neuroscience Frontiers in Behavioral Neuroscience www.frontiersin.org www.frontiersin.org Anacker and Pariante Modelling GR functions receptor function have been demonstrated and need to be considered when investi- gating GR function across different phy- logenetic taxa (Bury and Sturm, 2007). Nevertheless, the zebraﬁsh is a useful model to investigate principle mecha- nisms underlying stress response regula- tion, and it offers a unique possibility for high-throughput drug screening in vivo, facilitating the discovery of new targets that alter neuroendocrine responses and proxy measures of stress-induced behav- ioral abnormalities. ﬁndings. Front. Neuroendocrinol. 29:273–291. doi: 10.1016/j.yfrne.2007.10.004 ﬁndings. Front. Neuroendocrinol. 29:273–291. December 2012 \| Volume 6 \| Article 90 \| 2 Frontiers in Behavioral Neuroscience A commentary on L., Karst, H., de Kloet, E. R., and Joels, M. (2012). Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signalling. Mol. Cell. Endocrinol. 350, 299–309. Copyright © 2012 Anacker and Pariante. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, pro- vided the original authors and source are credited and subject to any copyright notices concerning any third- party graphics etc. Haller, J., Mikics, E., and Makara, G. B. (2008). The effects of non-genomic glucocorticoid mechanisms on bodily functions and the cen- tral neural system. A critical evaluation of December 2012 \| Volume 6 \| Article 90 \| 2 Frontiers in Behavioral Neuroscience www.frontiersin.org www.frontiersin.org
https://openalex.org/W2992831949	https://europepmc.org/articles/pmc6944146?pdf=render	English	null	Prospective Early Clinical, Radiological, and Kinematic Pedobarographic Analysis Following Subtalar Arthroereises for Paediatric Pes Planovalgus	Curēus	2,019	cc-by	5,182	DOI: 10.7759/cureus.6309 Abstract Arthroereises implants mechanically block eversion and limit subtalar motion. They are used in children with pes planovalgus in order to correct the valgus deformity. In this study, we aimed to objectively assess children with flatfoot before and after the insertion of the Kalix II implant, clinically, radiologically and by kinematic pedobarographic analysis. Open Access Original Article Open Access Original Article Open Access Original Article Materials and methods Six children (12 feet) were treated by the insertion of the Kalix II implant (Integra LifeSciences, Plainsboro, NJ). Patients completed the Manchester Oxford Foot Questionnaire (MOXFQ) preoperatively and at six months post operatively. Radiological outcome was assessed by lateral (L) and anterior posterior (AP) foot weight-bearing radiographs taken pre operatively and post operatively. Pedobarographic data was obtained pre operatively and at six months post operatively using a 1 meter RS Scan Footscan (RSscan International, Olen, Belgium) pedobarograph. In addition, patients underwent gait analysis pre and post operatively. Prospective Early Clinical, Radiological, and Kinematic Pedobarographic Analysis Following Subtalar Arthroereises for Paediatric Pes Planovalgus Yvonne-Mary Papamerkouriou Dr , Rohan Rajan , Samena Chaudhry , Preetham Kodumuri , Helen Evans , Martin Kerr 1 2 2 2 3 3 1. Orthopaedics, Panagiotis and Aglaia Kyriakou Children's Hospital, Athens, GRC 2. Orthopaedics, Royal Derby Hospital, Derby, GBR 3. Physiotherapy, Royal Derby Hospital, Derby, GBR  Corresponding author: Yvonne-Mary Papamerkouriou Dr, ympapamerkouriou@yahoo.gr Results Mean age was 11.05 +/-3.24 years (range 6.2 to 15.5 years). In all cases, screw removal was carried out at between 15 to 18 months post insertion. The mean pre op MOXFQ score was 55.3 +/-9.68 which reduced to 34.3 +/-15.66 post operatively with a p value < 0.00001 which was statistically significant. Mean Meary's angle preop was -15.21+/-5.51 degrees which corrected to -7.57+/-4.62 post op with a p value=0.00001. The mean calcaneal pitch before surgery was 11.96+/-3.8 which increased to 14.98+/-3.85 with a p value =0.00067. The first MTH: fifth MTH peak pressure ratio pre operatively was 4.53+/-2.78 which was found to reduce significantly post operatively to 1.35+/-0.97 (p=0.04), indicating a lateral shift of the foot pressures. Received 11/13/2019 Review began 11/28/2019 Review ended 12/05/2019 Published 12/06/2019 Received 11/13/2019 Review began 11/28/2019 Review ended 12/05/2019 Published 12/06/2019 © Copyright 2019 Papamerkouriou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Flatfoot is a common paediatric problem. In the vast majority of cases, these are physiological in nature and improve spontaneously as the child grows. A differentiation between pes planus - flatfoot and pes planovalgus - flat foot with valgus of the calcaneus on weight bearing needs to be made. Clinical evaluation is made to differentiate between a rigid and a flexible flat foot. In stance, the heel is found to be in valgus, which corrects into neutral or varus on forefoot weight bearing with restoration of the medial longitudinal arch, in flexible pes planovalgus. Children with this deformity usually present with medial midfoot weight bearing, mild genu valgum and parental concern. Tarsal coalition is the usual cause for a rigid pes plano valgus, where this correction does not occur. These patients usually present with a stiff painful flat foot. Consideration should also be given towards Achilles and gastrocnemius contractures (differentiated by the Silfverskiold test) associated with this condition. As the calcaneus is in valgus, the Achilles tendon is found to be contracted [1]. The subtalar joint comprises the talus, calcaneus and navicular and the intervening interosseous talocalcaneal and calcaneonavicular ligaments (spring ligament) and multiple joint capsules. These function as a unit. Flatfoot should be thought of as a three-dimensional deformity where the hindfoot is pronated, the subtalar joint is dorsiflexed and externally rotated, the midfoot abducted and forefoot supinated [2]. Initially, conservative treatment is advocated such as physiotherapy, reassurance, medial arch supports or UCBL orthotics. However, there is no evidence supporting the use of orthotics to correct this deformity [3]. It has been suggested that orthotics could, in fact, lead to dependency and long term negative psychological effects [3-5]. When these have failed, in patients with continued pain and dysfunction, surgical intervention is offered. Arthroereisis procedures were introduced between 1946 and 1977 to restrict excessive subtalar joint eversion by placing a bony block in the sinus tarsi [6-9]. Bone grafts were found to resorb over time leading to recurrence. Arthroereisis with synthetic implants was introduced in the late 1970s as these do not resorb, allowing for a greater time for bone remodeling [2]. Subotnick first described using a block of silicone elastomer in the sinus tarsi [10]. Although considered extra articular, if inserted appropriately, it mechanically blocks eversion and blocks subtalar joint motion with an intra articular effect [11-12]. Conclusion Papamerkouriou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. There were statistically significant improvements in the patient-reported MOXFQ, radiological improvements, and pedobarographic changes, indicating a lateral shift of the foot pressures. There were no complications. Categories: Orthopedics, Pediatric Surgery Categories: Orthopedics, Pediatric Surgery Keywords: pes planovalgus, arthroereisis, pedobarograph 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 Materials And Methods Six children (12 feet) with symptomatic painful correctible flexible pes planovalgus were included in our prospective study where the Kalix II subtalar arthroereisis implant consisting of a tapared tintanium alloy body and ultra-high molecular weight polyethelene shell was employed. All patients had a failed trial of conservative treatment including orthoses and physiotherapy. None of our patients had tarsal coalition. All our patients were assessed especially not to require any adjunctive soft tissue procedures in addition to the insertion of the subtalar arthroereisis implant. This was important in order to allow us to assess specifically the changes afforded by the implant alone, post operatively. All patients were given a general anaesthetic and were positioned supine. Image intensifier was used in theatres. A 1-cm incision was performed just anterior and plantar to the tip of the lateral malleolus, allowing for blunt dissection down to the sinus tarsi. The Kalix II Viladot lever was inserted from the anterior to posterior direction into the sinus tarsi to elevate the talus whilst the forefoot was pronated to restore the medial longitudinal arch. Trial implants of increasing size were inserted after the removal of the Viladot lever until a stable trial was found. The appropriate implant was then inserted with the lateral border of the implant being flush with the lateral border of the talus as confirmed by image intensifier. Surgical incision was closed in layers with subcuticular closure to the skin. A below-knee weight-bearing plaster cast was applied for the first four weeks post operatively. This was mainly to rest the soft tissues and avoid early extrusion, as well as to assist in pain relief. All patients were asked to complete the validated patient-reported Manchester Oxford Foot Questionnaire (MOXFQ) preoperatively and at six months post operatively. MOXFQ is a 16- item patient-reported outcome (PRO) measure developed and validated for use in studies assessing outcome following foot and/or ankle corrective surgery. Scores for each domain are calculated by summing the responses to each item within a given domain. Raw scores can be converted to a 0-100 metric where 100=most severe. Radiological outcome was assessed by lateral (L) and anterior posterior (AP) foot weight bearing radiographs taken pre operatively and post operatively. Introduction During pronation, the sinus tarsi closes as the lateral process of the talus glides anteriorly towards the prethalamic surface of the calcaneus. During foot supination, the sinus tarsi opens. An arthroereisis implant limits this motion, limiting hindfoot pronation. This prevents the lateral process of the talus from advancing towards the floor of the sinus tarsi, causing the subtalar joint to be inverted. It is postulated that extra articular arthroereises implants temporarily correct the valgus deformity in children allowing for bone remodeling to lead to a permanent correction following its removal [13]. Sixty percent of the talar surface is covered by articular cartilage which moves simultaneously with the peritalar structures [13]. The subtalar joint is a single axis joint acting as a hinge connecting the talus and calcaneus. When the subtalar joint axis is inclined 45 degrees from the transverse plane, rotation of the vertical part of the joint is coupled to equal rotation of the horizontal part of the joint [14]. A more horizontally aligned axis such as in planovalgus causes a greater rotation of the horizontal part for a given rotation of the vertical part. This 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 2 of 18 allows greater supination/pronation at the longitudinal axis of the horizontal segment for a given external/internal rotation of the vertical segment. allows greater supination/pronation at the longitudinal axis of the horizontal segment for a given external/internal rotation of the vertical segment. Vogler classified the biomechanical mode of action of subtalar arthroereisis implants into three distinct modes: “axis altering”, “impact blocking” and “self locking” [15]. This last mechanism of action is that which is employed by the implant in our study as it is inserted into the main axis of the sinus tarsi, supporting the talar neck, thus avoiding contact between the lateral process of the talus and the sinus tarsi floor, limiting talar adduction and plantar flexion. In this study, we aimed to objectively assess children with flatfoot before and after treatment by insertion of the Kalix II (Integra LifeSciences, Plainsboro, NJ) arthroereisis implant, clinically, radiologically and by kinematic pedobarographic analysis. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 MOXFQ scores The mean pre-op MOXFQ score was 55.3 +/-9.68 which reduced to 34.3 +/-15.66 post op with a p value < 0.00001 which was found to be a statistically significant result. In the MOXFQ the maximum score is 100 which corresponds to the most severe result. Results All six children in our prospective study underwent bilateral procedures. Mean age 11.05 +/- 3.24 years (range 6.2 to 15.5 years). In all cases, screw removal was carried out between 15 to 18 months post insertion. There were no complications in our cohort of patients. Materials And Methods In each case, the Meary's angle (talus - first metatarsal angle) and calcaneal pitch (angle formed by line passing from plantar most surface of calcaneus to inferior border of calcaneocuboid joint and line from plantar surface of calcaneus to plantar surface of fifth metatarsal head) of the lateral weight-bearing radiographs were calculated. Children underwent gait analysis pre and post operatively. Pedobarographic data was obtained pre operatively and at 6 months post implant insertion at our gait laboratory using a 1 meter RS Scan Footscan (RSscan International, Olen, Belgium) pedobarograph, capturing data at 200 Hz. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 3 of 18 Radiographic correction Mean Meary's angle preop was -15.21°+/-5.51° degrees which corrected to -7.57°+/-4.62° post- op with a p value=0.00001. In a normal foot, Meary’s angle is 0°. The negative value indicates the convex of the angle is downward, as is expected in flatfoot. In our study, whereas Meary’s angle did not correct to normal, it reduced, indicating improvement. The mean calcaneal pitch before surgery was 11.96°+/-3.8° which increased to 14.98°+/-3.85° post op with a p value =0.00067. Angles between 10° and 20° are indicative of pes planus. In our study, the angle increased, indicating improvement even though, it remained below the normal range. A minimum of four passes were made to capture at least six clear footprints on each side (right and left). Parametric data was expressed as mean+/- standard deviation (SD). The SPSS 17.0 software (SPSS, Chicago, IL, USA) was employed for statistical analysis. Paired Student’s t-test was used for comparisons for pre operative and post-operative results. A p value of <0.05 was considered statistically significant. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 Pedobarographic data We analysed the peak pressures under the first and fifth metatarsal heads (MTH) pre operatively and post operatively in kilopascals (kPa). Before surgery, the mean first MTH peak pressure was 218.42+/-109.56 which reduced postoperatively to 113.82+/-79.71 (p=0.0016). The mean preoperative fifth MTH peak pressure was 65.08+/-32.83 increasing to 88.58+/- 59.17 (p=0.281). The first MTH: fifth MTH peak pressure ratio pre operatively was 4.53+/-2.78 which was found to reduce significantly post operatively to 1.35+/-0.97 (p=0.04), indicating a lateral shift of the foot pressures (Table 1). 4 of 18 Pre op (+/-SD) Post op (+/-SD) P value MOXFQ 55.3 (9.68) 34.3 (15.66) 0.00001 Meary angle -15.21 (5.51) -7.57 (4.62) 0.00001 Calcaneal pitch 11.96 (3.8) 14.98 (3.85) 0.00067 1st MTH peak pressure (kPa) 218.42 (109.56) 113.82 (79.71) 0.0016 5th MTH peak pressure (kPa) 65.08 (32.83) 88.58 (59.17) 0.281 1st:5th MTH PP ratio 4.53 (2.78) 1.35 (0.97) 0.04 TABLE 1: Results Pre- and post-operative MOXFQ, radiological, and kinematic data. MOXFQ: Manchester Oxford Foot Questionnaire; MTH: metatarsal heads. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 TABLE 1: Results Pre- and post-operative MOXFQ, radiological, and kinematic data. Pre- and post-operative MOXFQ, radiological, and kinematic data. MOXFQ: Manchester Oxford Foot Questionnaire; MTH: metatarsal heads. MOXFQ: Manchester Oxford Foot Questionnaire; MTH: metatarsal heads. We allowed for post operative swelling to settle before performing the above measurement at around the six-month post operative period. We postulated that before corrective surgery, there is an increase in pedobarographic pressures on the medial side of the foot as it is pronated in pes planovalgus. These should be reduced as the foot is corrected into some supination, allowing for a reduction in medial pressures and an increase in lateral pressures of the foot. This is apparent in the pedobarographic images of one of the patients in our cohort, before and after the procedure (Figures 1-2). Our study has demonstrated a statistically significant reduction in the peak first metatarsal head pressure post operatively while the increase in the peak fifth metatarsal head pressure post operatively was not statistically significant. However, when we analysed the ratio of the first to fifth metatarsal peak pressures, which we feel is a more meaningful measure of the redistribution of the foot pressures than the raw individual metatarsal head pressures, we found a statistically significant redistribution of foot pressures towards the lateral side of the foot and away from the medial side of the foot. FIGURE 1: Pre-operative pedobarograph pressure images Pre-operative pedobarograph pressure images of one of the patients in our cohort. There is an increase in pedobarographic pressures under the first metatarsal head (MTT) and a decrease under the fifth MTT. FIGURE 1: Pre-operative pedobarograph pressure images Pre-operative pedobarograph pressure images of one of the patients in our cohort. There is an increase in pedobarographic pressures under the first metatarsal head (MTT) and a decrease under the fifth MTT. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 5 of 18 FIGURE 2: Post-operative pedobarograph pressure images Post-operative pedobarograph pressure images of the same patient. There is a decrease in pressures under the first metatarsal head (MTT) and an increase under the fifth MTT head indicating a lateral shift of pressures. FIGURE 2: Post-operative pedobarograph pressure images Post-operative pedobarograph pressure images of the same patient. There is a decrease in pressures under the first metatarsal head (MTT) and an increase under the fifth MTT head indicating a lateral shift of pressures. In addition, we observed the limiting of hindfoot pronation, as demonstrated by the pre- and post-surgery gait analysis graphs of the aforementioned patient (Figures 3-4). Gait analysis photographs indicate a reduction in heel valgus (as seen in Figures 5-6) as well as an improvement in the medial arch post operatively (Figures 7-10) in the same patient. 6 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 3: Pre-operative gait analysis graphs Pre-operative gait analysis graphs of the aforementioned patient indicating increased hindfoot pronation. FIGURE 3: Pre-operative gait analysis graphs Pre-operative gait analysis graphs of the aforementioned patient indicating increased hindfoot pronation. 7 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 4: Post-operative gait analysis graphs Post-operative gait analysis graphs of the aforementioned patient indicating a decrease in hindfoot pronation. FIGURE 4: Post-operative gait analysis graphs FIGURE 4: Post-operative gait analysis graphs Post-operative gait analysis graphs of the aforementioned patient indicating a decrease in hindfoot pronation. Post-operative gait analysis graphs of the aforementioned patient indicating a decrease in hindfoot pronation. 8 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 5: Pre-operative gait analysis photo of heel valgus Pre-operative gait analysis photo of the same patient indicating prominent heel valgus. FIGURE 5: Pre-operative gait analysis photo of heel valgus Pre-operative gait analysis photo of the same patient indicating prominent heel valgus. 9 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 6: Post-operative gait analysis photo of heel valgus Post-operative gait analysis photo of the same patient indicating a decrease in heel valgus. FIGURE 6: Post-operative gait analysis photo of heel valgus Post-operative gait analysis photo of the same patient indicating a decrease in heel valgus. 10 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 7: Right foot pre-operative gait analysis photo of the medial arch Right foot gait analysis photo of the medial arch of the same patient where there is evident flatening of the arch. FIGURE 7: Right foot pre-operative gait analysis photo of the medial arch Right foot gait analysis photo of the medial arch of the same patient where there is evident flatening of the arch. 11 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 FIGURE 8: Right foot post-operative gait analysis photo of the medial arch Right foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. FIGURE 8: Right foot post-operative gait analysis photo of the medial arch Right foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. FIGURE 8: Right foot post-operative gait analysis photo of the medial arch Right foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. 12 of 18 FIGURE 9: Left foot pre-operative gait analysis photo of the medial arch Left foot pre-operative gait analysis photo of the same patient indicating flattening of the medial arch. FIGURE 9: Left foot pre-operative gait analysis photo of the medial arch Left foot pre-operative gait analysis photo of the same patient indicating flattening of the medial arch. 13 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 decrease in Meary's angle indicating improvement; however, there is a slight worsening in the calcaneal pitch, as indicated by its decrease. FIGURE 12: X-rays of the left foot Meary's angle and calcaneal pitch A) Left foot pre-operative radiological angles, namely Meary's angle and calcaneal pitch, in a lateral standing X-rays of the foot. B) Left foot post-operative radiological angles. There is an improvement in both angles, indicated by the decrease in Meary's angle and increase of calcaneal pitch. FIGURE 12: X-rays of the left foot Meary's angle and calcaneal pitch A) Left foot pre-operative radiological angles, namely Meary's angle and calcaneal pitch, in a lateral standing X-rays of the foot. B) Left foot post-operative radiological angles. There is an improvement in both angles, indicated by the decrease in Meary's angle and increase of calcaneal pitch. FIGURE 12 X f th l ft f t M ' l d l l FIGURE 12: X-rays of the left foot Meary's angle and calcaneal pitch A) Left foot pre-operative radiological angles, namely Meary's angle and calcaneal pitch, in a lateral standing X-rays of the foot. B) Left foot post-operative radiological angles. There is an improvement in both angles, indicated by the decrease in Meary's angle and increase of calcaneal pitch. FIGURE 10: Left foot post-operative gait analysis photo of the medial arch Left foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. FIGURE 10: Left foot post-operative gait analysis photo of the medial arch Left foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. FIGURE 10: Left foot post-operative gait analysis photo of the medial arch Left foot post-operative gait analysis photo of the medial arch indicating elevation of the arch. Furthermore, our prospective study has demonstrated an improvement in the radiological calcaneal pitch and Meary’s angle measured at six months post surgery. With respect to radiological angles, although in specific cases the post operative angles revealed some under correction, the improvement post operatively was statistically significant overall. We have included X-rays of the already stated patient where there was an improvement in Meary’s angle post operatively in both feet. Calcaneal pitch improved slightly in the left foot but worsened in the right (Figures 11-12). FIGURE 11: X-rays of the right foot Meary's angle and calcaneal pitch A) Right foot pre-operative radiological angles, specifically Meary's angle and calcaneal pitch in a lateral standing X-ray of the foot. B) Right foot post-operative radiological angles. There is a FIGURE 11: X-rays of the right foot Meary's angle and calcaneal pitch A) Right foot pre-operative radiological angles, specifically Meary's angle and calcaneal pitch in a lateral standing X-ray of the foot. B) Right foot post-operative radiological angles. There is a 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 14 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 removal of the implants or even not removing them [28]. Older studies recommended leaving the implant in situ for up to two years before removal allowing for bone and soft tissue remodelling [10,8]. More recent literature recommends removal between 6-18 months [29-30]. A recent literature review reported concerns about a high complication rate in 4%-18% of cases [24]. Reported complications include malposition of the implant, improper correction of the deformity, extrusion of the implant from the sinus tarsi, foreign body reaction to the implant, peroneal spasm and persistent foot pain. These complications are usually treated by implant removal. We removed the implants at around the 18 month period, following literature recommendations, as we were concerned about any polyethelene debris. To date, we found no other study in the existing literature evaluating the outcomes of arthroereisis by pedobarographic evaluation. Our study was primarily concerned with the objective changes or improvement in the pedobarographic pressures following the insertion of the subtalar arthroereisis implant. Limitations of this study include the small numbers treated, although they provided adequate statistical analysis, as well as the lack of a control group of normal-arched children. We continue to follow up with these patients until skeletal maturity and post removal of the implant. We hope to be able to report on their pedobarographic changes at one and two years post removal of the implant with a larger series of patients. However, we felt it important to highlight the improvements in pedobarographic, radiological and MOXFQ outcome in the early stages pre removal of the implant. Discussion Arthroereisis for the treatment of severe pes planovalgus has been used for over 50 years. The use of implant materials and shapes vary but no study has demonstrated an advantage of one over another [16]. Vedantam et al. reported satisfactory results in 96% of feet in a study of 78 children with neuromuscular flexible flatfeet where 140 arthroereisis procedures were performed utilizing STA-peg implants. Assessment was based on radiological angle improvement as well as reduction of hindfoot valgus and pain [17]. Giannini et al. reported 4- year results of subtalar arthroereisis for 21 children with bilateral flexible flatfeet using a bioresorbable implant, finding improvement in clinical results, radiological angles and footprint grades [18]. Fernandez de Retana et al. found that the Viladot foot prints and radiographic angles improved post operatively, in a study of 97 feet where the Kalix implant was employed [19]. The Viladot 4 category footprint classification used is a simple visual model which does not allow quantifying of pre and post operative differences. Many other publications for subtalar arthroereisis include Achilles tendon lengthening [12,20-23]. Overall, these studies have demonstrated an increase in dorsiflexion, decreased foot pain, improvement of radiographic angles and improvement in foot print following this procedure [18,21,24-25]. De Pellegrin et al. found that following removal of the implant there was the maintenance of the correction in an evaluation of 76 patients (121 feet) who were followed up after screw removal, which occurred on average 2.9 years after SESA (subtalar extra-articular screw arthroereisis). The evaluation was based on radiological angles [26]. Cao et al. compared radiographic results as well as pain and function pre and post application of the Kalix II arthroereisis system in 27 feet. The study concluded that the application of the Kalix II system combined with dissection of accessory navicular and reconstruction of the tibialis posterior tendon is an effective therapy for juvenile flexible flatfoot [27]. Bernasconi et al., in their review of the role of arthroereisis of the subtalar joint for flatfoot, have concluded that only level IV and V evidence is available. There is only one level II comparative non-randomised study, which does not provide strong recommendation. It was suggested that a validated patient outcome measure needed to be used as many of the studies still used non-validated scores. No firm recommendations were made about the timing for the 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 15 of 18 Disclosures Human subjects: Consent was obtained by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Conclusions In our small prospective series, there were no complications. We have been impressed particularly by the objective statistically significant improvements in the patient-reported MOXFQ, radiological improvements, and pedobarographic changes. We feel that the arthroereisis procedure is safe and simple if performed correctly, with attention to the correct placement of the implant in carefully selected, appropriate patients. We intend to follow up on this study over a longer period. 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 1. Vulcano E, Maccario C, Myerson MS: How to approach the pediatric flatfoot. World J Orthop. 2016, 7:1-7. doi: 10.5312/wjo.v7.i1.1 2. Mosca VS: Flexible flatfoot in children and adolescents . J Child Orthop. 2010, 4:107-121. 10.1007/s11832-010-0239-9 3. Helfet AJ: A new way of treating flat feet in children . Lancet. 1956, 270:262-264. https://doi.org/10.1016/s0140-6736(56)91187-4 4. Rao UB, Joseph B: The influence of footwear on the prevalence of flat foot. A survey of 2300 children. J Bone Joint Surg Br. 1992, 74:525-527. https://doi.org/10.1302/0301- 620X.74B4.1624509 5. Driano AN, Staheli L, Staheli LT: Psychosocial development and corrective shoewear use in childhood. J Pediatr Orthop. 1998, 18:346-349. http://dx.doi.org/10.1097/01241398- 199805000-00014 6. Haraldsson S: Pes plano-valgus staticus juvenilis and its operative treatment . Acta Orthop Scand. 1965, 35:234-256. 10.3109/17453676508989356 7. Chambers EF: An operation for the correction of flexible flat feet of adolescents . West J Surg Obstet Gynecol. 1946, 54:77-86. 8. LeLievre J: Current concepts and correction in the valgus foot. Clin Orthop Relat Res. 1970, 70:43-55. 9. Miller GR: The operative treatment of hypermobile flatfeet in the young child . 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Needleman RL: Current topic review: subtalar arthroereisis for the correction of flexible flatfoot. Foot Ankle Int. 2005, 26:336-46. 10.1177/107110070502600411 17. Vedantam R, Capelli AM, Schoenecker PL: Subtalar arthroereisis for the correction of planovalgus foot in children with neuromuscular disorders. J Pediatr Orthop. 1998, 18:294- 298. 10.1097/01241398-199805000-00004 18. Giannini S, Ceccarelli F, Benedetti MG, Catani F, Faldini C: Surgical treatment of flexible flatfoot in children:a four-year follow-up study. J Bone Joint Surg Am. 2001, 83:73-9. 10.2106/00004623-200100022-00003 19. Fernandez de Retana P, Alvarez F, Viladot R: Subtalar arthroereisis in pediatric flatfoot reconstruction. Foot Ankle Clin N Am. 2010, 15:323-335. 10.1016/j.fcl.2010.01.001 21. Gutierrez PR, Lara MH: Giannini: prosthesis for flatfoot. Foot Ankle Int. 2005, 26:918-26. https://doi.org/10.1177/107110070502601104 22. Cicchinelli LD, Huerta JP, Garcıa-Carmona FJ, Morato DF: Analysis of gastrocnemius recession and medial column procedures as adjuncts in arthroereisis for the correction of pediatric pes planovalgus: a radiographic retrospective study. J Foot Ankle Surg. 2008, 47:385-91. 10.1053/j.jfas.2008.06.002 23. References 3. Helfet AJ: A new way of treating flat feet in children . Lancet. 1956, 270:262-264. https://doi org/10 1016/s0140 6736(56)91187 4 3. Helfet AJ: A new way of treating flat feet in childre https://doi.org/10.1016/s0140-6736(56)91187-4 https://doi.org/10.1016/s0140-6736(56)91187-4 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 16 of 18 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 Nelson SC, Haycock DM, Little ER: Flexible flatfoot treatment with arthroereisis: radiographic improvement and child health survey analysis. 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Bernasconi A, Lintz F, Sadile F: The role of arthroreisis of the subtalar joint for flatfoot in 28. Bernasconi A, Lintz F, Sadile F: The role of arthroreisis of the subtalar joint for flatfoot in 2019 Papamerkouriou et al. Cureus 11(12): e6309. DOI 10.7759/cureus.6309 17 of 18 29. Zhu Y, Xu XY: Treatment of stage II adult acquired flatfoot deformity with subtalar arthroreisis. Foot Ankle Spec. 2015, 8:194-202. 10.1177/1938640014548320 30. Schon LC: Subtalar arthroreisis: a new exploration of an old concept . Foot Ankle Clin. 2007, 12:329-339. 10.1016/j.fcl.2007.03.011 18 of 18
https://openalex.org/W2785283050	https://europepmc.org/articles/pmc5853880?pdf=render	English	null	An Instrument to Measure Maturity of Integrated Care: A First Validation Study	International journal of integrated care	2,018	cc-by	14,639	* Department of Family Medicine and Chronic Care, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, B-1090 Brussels, BE † Department Patient and Care, Maastricht University Medical Center, Maastricht, NL ‡ Panaxea B.V., Amsterdam, NL Corresponding author: Liset Grooten, MSc (fgrooten@vub.ac.be) Grooten, L, et al. An Instrument to Measure Maturity of Integrated Care: A First Validation Study. International Journal of Integrated Care, 2018; 18(1): 10, 1–20. DOI: https://doi.org/10.5334/ijic.3063 RESEARCH AND THEORY Keywords: integrated care; measurement; maturity; instrument; validity Integrated care initiatives are being developed around the world [3–5]. Countries in Europe have endeavoured to improve the performance of their health systems. Many have implemented different types of integrated care programmes representing a diversity in their nature and scope of approaches [6]. This is not surprising since the transition to integrated care is a complex undertaking, while sufficient support of a systematic understanding of integration in health systems is scarce [7–9]. The complexity of integration is reflected in the definition of integrated care provided by Kodner [10, p. 12]: “[a] multi- level, multi-modal, demand driven and patient-centred strategy designed to address complex and costly health needs by achieving better coordination of services across the entire care continuum. Not an end in itself, integrated care is a means of optimizing system performance and attaining quality patient outcomes.” As a response to the call for the establishment of a common language and framework of integrated care to better understand integrated care and guide empirical research [10, 11], several studies have attempted to clarify the concepts underpinning integrated care [8, 12]. Liset Grooten, Liesbeth Borgermans and Hubertus JM Vrijhoef,†,‡ Liset Grooten, Liesbeth Borgermans* and Hubertus JM Vrijhoef,†,‡ Introduction: Lessons captured from interviews with 12 European regions are represented in a new instrument, the B3-Maturity Model (B3-MM). B3-MM aims to assess maturity along 12 dimensions reflecting the various aspects that need to be managed in order to deliver integrated care. The objective of the study was to test the content validity of B3-MM as part of SCIROCCO (Scaling Integrated Care into Context), a European Union funded project. ) p p j Methods: A literature review was conducted to compare B3-MM’s 12 dimensions and their measurement scales with existing measures and instruments that focus on assessing the development of integrated care. Subsequently, a three-round survey conducted through a Delphi study with international experts in the field of integrated care was performed to test the relevance of: 1) the dimensions, 2) the maturity indicators and 3) the assessment scale used in B3-MM.i ) Results: The 11 articles included in the literature review confirmed all the dimensions described in the original version of B3-MM. The Delphi study rounds resulted in various phrasing amendments of indicators and assessment scale. Full agreement among the experts on the relevance of the 12 B3-MM dimensions, their indicators, and assessment scale was reached after the third Delphi round. Conclusion and discussion: The B3-MM dimensions, maturity indicators and assessment scale showed satisfactory content validity. While the B3-MM is a unique instrument based on existing knowledge and experiences of regions in integrated care, further testing is needed to explore other measurement properties of B3-MM. Keywords: integrated care; measurement; maturity; instrument; validity Grooten, L, et al. An Instrument to Measure Maturity of Integrated Care: A First Validation Study. International Journal of Integrated Care, 2018; 18(1): 10, 1–20. DOI: https://doi.org/10.5334/ijic.3063 Introduction Health systems around the world are under great pressure to drive forward transformation in order to meet the evolving needs of their populations. The traditional disease orientated approaches currently provided no longer suffice to meet the people’s needs [1]. In many countries care is too often fragmented and has clear deficiencies in quality, inducing low responsiveness of the health system and low satisfaction with health services [2]. To address these challenges, the transformation towards integrated care has the potential to repair deficiencies in order to obtain accessible, quality, effective and sustainable health care. In particular, the conceptual framework of Valentijn et al. can be used to aid an understanding of the concept Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 2 of 20 of integrated care [8] and shows the complexity of what transformation to integrated care delivery entails. The framework identifies key elements for achieving integrated service delivery which are organised into six dimensions of integration. The features have complementary roles on the micro (clinical integration), meso (professional and organisational integration), and macro (system integration) levels to deliver comprehensive services that address the needs of people and populations. Functional and normative integration establish connectivity across the micro, meso and macro level. Transitioning to integrated care involves various related activities taking place at different levels of the health system and including diverse actors and organisations with various perspectives on integrated care. in the context of Dutch disease management programs and it is not clear how the approach extends to complex co-morbidities and long-term conditions. Furthermore, to transfer knowledge about successful integrated care interventions to other settings and thus support the development and scaling up of these interventions, it is important to consider the specific local conditions that influence the implementation and sustainability of a particular integrated care intervention [6]. A second model, has been developed by the B3 Action Group on Integrated Care of the European Innovation Partnership on Active and Healthy Aging (EIP on AHA) [24]. Unique to the B3-Maturity Model (B3-MM), is that it is derived from a pragmatic bottom-up approach with decision-makers involved in integrated care delivery from 12 European countries. These experts were interviewed about how healthcare systems are attempting to deliver more integrated care services to citizens. Introduction The rich collection of lessons learned are structured into 12 dimensions and reflect the various activities that need to be managed in order to deliver integrated care [25]. The B3-MM explicitly focuses on the need to understand the context and environment (i.e. the regional delivery system and political and organisational environment) of integrated care interventions. The goal of the B3-MM is to provide a self-assessment tool for European regions to assess their maturity in the provision of integrated care, thereby revealing strengths and areas for improvement. To demonstrate B3-MM’s full potential as a tool for measuring the maturity of integrated care, testing and validation of the tool is, however, needed. To achieve a successful transformation to more integrated care systems, insights are needed into what factors contribute to the progress and success of integrated care interventions. However, there is a lack of substantiation of the working mechanisms in integrated care, partly stemming from poor or non-existent evaluation and measurement of integrated care interventions [13]. and measurement of integrated care interventions [13]. Capturing the complexity of measuring integrated care is a challenging task. A first difficulty is that measurement is complicated by the conceptual haziness adhering to integrated care which thus limits the theoretical foundations of existing instruments [14]. A second difficulty is the demonstration of association between the changes in services as part of the integration efforts’ and their outcomes [15–17]. The linkage between changes in services and service outcomes is problematic because most patient or service user outcomes do not emerge from linear cause and effect chains [18]. Current measures of quality in health care, such as the structure-process- outcome model, do not clarify the underlying mechanisms governing the components of integrated care [19–22]. A sound analytical method for evaluating the outcomes of integrated care programmes, which would provide insight into why and where they are effective, is lacking [22]. Moreover, a recent review by Bautista et al., which has compared 200+ instruments of integrated care by looking at their measurement properties, found that most measurement properties of existing instruments need to be improved [23]. Hence the need for measurement, preferably based on sound evidence, of integrated care interventions which captures the complexity of integrated care as reflected in its multiple components and dynamic nature. This measurement will enable identification of potential problems in progress. indicators and assessment scale of B3-MM according to international experts in the field of integrated care? healthcare system, and allocating a measure of ‘maturity’ (on a 0–5 scale), it should become possible to develop a simple graphical representation (i.e. spider diagram) of maturity level of the region/healthcare system, including its strengths and weaknesses in the path towards integrated care delivery. Using these insights, and comparing the findings with other regions that have conducted the same self-assessment process, should inform the complementary regions or healthcare systems about the possibility to progress with further knowledge transfer activities in order to improve their maturity in integrated care. The process of information sharing on lessons learned could help other regions is expected to speed up the adoption of integrated care. Theory and methods Theoretical background Strategy for expansion of integrated care Since 2013, the B3 Action Group on Integrated Care of the EIP on AHA has been collecting good practices in integrated care in Europe [25]. The extensive collection of good practices has provided a better understanding of the existing solutions, resources and expertise in integrated care delivery. However, the collected good practices are often limited to a particular pilot, project or region and the ambitions of the EIP on AHA and the B3 Action Group in particular is to promote the scaling up of these local initiatives throughout Europe [25]. In order to meet this ambition, the challenge remains on how to best leverage the existing evidence and support scaling up of good practices in Europe. Introduction The insight obtained into the relevant success factors will support the further development of integrated care. Validity is an important feature in selecting or applying an instrument and is defined as ‘the degree to which an instrument truly measures the construct(s) it purports to measure’ [26]. The construct is a well-defined and precisely demarcated subject of measurement. Three forms of validity can be determined: content validity, criterion validity and construct validity [27]. The purpose of a content validation study is to determine whether the instrument adequately represents the construct under study [28]. Assessing content validity of an instrument is useful as it provides information on the representativeness and clarity of each item of the instrument. Furthermore, substantial suggestions are obtained to improve the measure, saving numerous revisions of the untested measure through several pilot evaluation studies [29]. The improved instrument can then be used in a pilot study to assess other psychometric properties. This study has two objectives. As part of the European project SCIROCCO [30], it is to test the appropriateness of B3-MM’s dimensions, maturity indicators and assessment scale. Moreover, it also aims to test the content validity of B3-MM. In doing so, this paper reports on the following research questions fundamental to the study: A number of measurement models may be helpful in this activity. One model which has taken the complex dynamic and multiformity nature of integrated care into account and provides insight in the development process of integrated care, by describing four developmental phases, has been designed by Minkman et al. [12]. This model intends to be used as a quality management model for integrated care supporting the further development of integrated care practices. The model is, however, developed and used 1. How is maturity of integrated care measured by instruments identified in the scientific and non- scientific literature? 2. How relevant are the dimensions, maturity Art. 10, page 3 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care indicators and assessment scale of B3-MM according to international experts in the field of integrated care? Maturity Models h f Maturity Models The art of measuring maturity displayed in the Capability Maturity Model (CMM) was introduced in the mid 80’s by the Software Engineering Institute (SEI) – Carnegie Mellon University [33]. Since then, several disciplines, especially those in the field of information systems, have successfully used maturity models as a way to assess the value and improve the competence of organisations [34]. A maturity model is regarded as a conceptual model which is characterised by the display of several maturity levels representing the developmental capabilities of organisations [35]. A limited number of studies have adapted these maturity models to the healthcare domain or proposed healthcare specific maturity models [36]. Blondiau et al. [37, p. 2] state that these healthcare specific maturity models show “a staged representation of an actual state in relation to a potentially achievable goal state and a description of steps required to achieve this objective.” Several models already exist, including the HIMSS Analytics Continuity of Care Model [38], TEMPEST (an Integrative model for health technology assessment) [39], and Maturity Matrix to Support Health and Social Care Integrated Care Partnerships [40]. However, the three above mentioned models tend to be either too simplistic [38, 40], focusing for example only on the functionality of IT systems, or rather complex, given that they offer a volume of different factors to consider [39]. There is an increase in literature describing frameworks for scaling health interventions, the majority of which has an explicit focus on scaling up health actions in low- and middle-income country contexts [31]. However, literature is sparse on scaling up long-term care innovations in developed healthcare systems [32]. A 2016 study by Nolte et al. [4], examining three pilots in integrated care delivery, focussing on their development, implementation and sustainability including how they impacted the wider system context. It showed that the wider dissemination of the projects studied occurred in an incremental and somewhat random way. To guide the necessary transformation a formal strategy for expansion is needed [4], preferably based on sound evidence. Figure 1: Dimensions of the B3-MM (retrieved from: http://www.scirocco-project.eu/maturitymodel/). STRUCTURE & GOVERNANCE REMOVAL OF INHIBITORS POPULATION APPROACH READINESS TO CHANGE CAPACITY BUILDING INNOVATION MANAGEMENT EVALUATION METHODS FINANCE & FUNDING CITIZEN EMPOWERMENT INFORMATION & eHEALTH SERVICES BREADTH OF AMBITION STANDARDISATION & SIMPLIFICATION Figure 1: Dimensions of the B3-MM (retrieved from: http://www.scirocco-project.eu/maturitymodel/). The B3 Maturity Model The B3 Maturity Model Recognizing the need for a structured approach which could stimulate path-breaking changes towards more sustainable health and care systems, partners of the B3 Action Group on Integrated Care of the EIP on AHA developed the B3-MM (to obtain a more standardised approach for scaling-up integrated care throughout Europe). The B3-MM is derived from an observational study, based on interviews with decision-makers in 12 European countries, or regions within a country, responsible for health care (namely Attica, the Basque Country, Catalonia, Galicia, Northern Ireland Saxony, Medical Delta, Olomouc region, Puglia region, Scotland, Skane, and South Denmark) over 18 months in 2014–2015. The interviews involved asking three sets of questions, to uncover i) the extent of integration already achieved, ii) the journey taken to get to this point, and iii) a view of future plans and investments. The outcomes of the study served as the baseline for the development of the B3-MM [25]. The maturity model intends to serve as a self- assessment tool for regions or health care systems that aim to assess progress along 12 dimensions. These dimensions reflect the various aspects to be managed in order to deliver integrated care (Figure 1) [30]. By considering each dimension, assessing the current situation within a By regarding the development, implementation and scaling up of health innovations as a multi-stage process [41], the rationale of the B3-MM being a maturity model should be found in the evolution of integrated care services. The B3-MM displays the development of a regional system on several dimensions to achieve integrated care delivery. The B3-MM is considered to be a practical model, and thus easy to use, to comprehensively assess the maturity of the progress of a regional system thereby uncovering gaps and areas for improvement in the development of integrated care delivery. The insights gained by employing the B3-MM are intended to be used as a starting point from which regions with complementary strengths and weakness could be matched and start to share their lessons learned on specific areas. In doing so, the B3-MM is intended to be used to guide the process on how developments in one jurisdiction can inform developments in other regions. This process Art. 10, page 4 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Figure 1: Dimensions of the B3-MM (retrieved from: http://www.scirocco-project.eu/maturitymodel/). The B3 Maturity Model STRUCTURE & GOVERNANCE REMOVAL OF INHIBITORS POPULATION APPROACH READINESS TO CHANGE CAPACITY BUILDING INNOVATION MANAGEMENT EVALUATION METHODS FINANCE & FUNDING CITIZEN EMPOWERMENT INFORMATION & eHEALTH SERVICES BREADTH OF AMBITION STANDARDISATION & SIMPLIFICATION Remarks interested in describing and comparing the dimensions, indicators, measurement scales, and the psychometric property content validity of the selected measures and instruments. Data extraction and analysis of the literature review Data were extracted by looking for descriptions on dimensions, indicators, and measurement scales in the selected articles which matched with the 12 dimensions, maturity indicators and assessment scale of the B3-MM. We marked all matching items and listed them in a table developed in MS EXCEL. Descriptions on dimensions, indicators or measurement scales in the selected articles which did not match, but which could nevertheless provide an addition to B3-MM, were also identified. Furthermore, we evaluated the overall quality of the measurement property content validity (definition in Box 1) of the instruments identified in the narrative review based on the criteria used by Bautista et al. [23]. Data extraction and analysis of the literature review Data were extracted by looking for descriptions on dimensions, indicators, and measurement scales in the selected articles which matched with the 12 dimensions, maturity indicators and assessment scale of the B3-MM. We marked all matching items and listed them in a table developed in MS EXCEL. Descriptions on dimensions, indicators or measurement scales in the selected articles which did not match, but which could nevertheless provide an addition to B3-MM, were also identified. Furthermore, we evaluated the overall quality of the measurement property content validity (definition in Box 1) of the instruments identified in the narrative review based on the criteria used by Bautista et al. [23]. The literature search consisted of two parts. For the first part, we built on the work of Bautista et al. [23] who recently conducted a systematic review in MEDLINE/PubMed on measurement properties of instruments measuring integrated care. The authors selected articles from the systematic literature review which focused on measures and instruments of the development of integrated care with indications of “maturity”, “phase”, “level”, or “degree” of integrated care. To broaden the search for articles, a narrative review was undertaken. In narrative reviews, the authors have the objective to identify, evaluate and synthesize what is already known about a topic [46]. The preliminary search started in the electronic databases Cochrane, Google, Google Scholar, GreyLit, IDEA and OpenGrey using a combination of search terms, as shown in Table 1. The final search was restricted to the databases which retrieved adequate hits; Google (Filter: English only), Google Scholar and IDEA. Remarks The search terms used included terms referring to the construct, integrated care, and terms referring to an instrument. We used the terms from the study of Bautista et al. [23] who derived the terms from the work of Uijen et al. [47] and Terwee et al. [48]. We added search terms indicating a measurement feature of an instrument. The final key terms used in the ultimate search strategy are presented in Table 5. Box 1: Definition of measurement property content validity (adapted from Uijen et al.) [47] Content validity: the degree to which the content of an instrument is an adequate reflection of the construct to be measured. In quality assessment, there is an important distinction between the quality of a study on measurement properties and the quality of an instrument [49]. In the article by Bautista et al. [23], the quality assessment of the studies and the instruments is guided by the COnsensus-based Standards for the selection of health status Measurement INstruments (COSMIN) [26, 28, 50, 51]. In this study, the overall quality of the content validity for the instruments was assessed by the researchers (LG and HV) using the criteria for the levels of evidence and overall assessment of measurement properties of instrument (Table 2) by determining four factors [23, 27, 47, 52]. The first factor includes the number of validation studies per instrument. Snowball sampling and hand searching in Google and Google Scholar were performed to identify validation studies on the retrieved instruments from the narrative search. The second factor concerns the assessment of methodological quality of the studies relating to content validity. This assessment was based on the criteria of the COSMIN checklist [28] using the four-point scale in the COSMIN checklist. A study was rated as poor, fair, good, or excellent according to its measurement property content validity. The third factor is about the assessment of the direction of results of the measurement property content validity (whether positive or negative). This was rated using the modified criteria as presented in Table 3 [47]. To be included in the review, we used the two eligible article criteria: 1. availability of full-text English document; (Due to the large number of hits, we limited the search to that of English language only when possible); g g g y p ) 2. description of items/constructs/measurement scales of measures and/or instruments on the maturity of integrated care. Methods [48] Terms reflecting “maturity” Component Terms Construct Integrated care, coordination of care, continuity of care, patient centered care Instrument Questionnaire, measure, survey, instrument Feature Degree, maturity model, level, phase Methods allows a tailored approach for regions in their journeys towards (more) integrated care as the regions can decide for themselves which areas attention should be paid to in order to speed up the adoption of or the development towards integrated care. Depending on the local needs of the regions, these tailored approaches can operate at the several dimensions and levels of integrated care [8]. Sensitivity to the differences between countries in terms of local needs and context to obtain a tailored approach for achieving progress in integrated care is important, as external contextual factors have been found to support the successful implementation of an integrated care model [42]. allows a tailored approach for regions in their journeys towards (more) integrated care as the regions can decide for themselves which areas attention should be paid to in order to speed up the adoption of or the development towards integrated care. Depending on the local needs of the regions, these tailored approaches can operate at the several dimensions and levels of integrated care [8]. Sensitivity to the differences between countries in terms of local needs and context to obtain a tailored approach for achieving progress in integrated care is important, as external contextual factors have been found to support the successful implementation of an integrated care model [42]. In this study, we conducted a literature review and a Delphi study to test the content validity of B3-MM as instrument to measure the level of maturity of integrated care. Content validity can be determined using both quantitative or qualitative methods [43]. A qualitative approach consists of an accurate analysis of the representativeness and clarity of items in the literature and by consultation of experts [44]. Evidence of content validity is usually obtained by having knowledgeable people look at the test items and make judgments about the appropriateness of each item and overall coverage of the domain [45]. Literature review A review was conducted to identify articles, papers and/or reports focusing on measures and instruments of the maturity of integrated care. Moreover, we were Art. 10, page 5 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Table 1: Search terms used in narrative literature review. Remarks Based on the work of Uijen et al. [47] modified by Bautista et al. [23] User-defined based on Terwee et al. Remarks First, one researcher (LG) screened the titles and abstract of the articles from the main search in the three databases to identify articles for full text read. Two researchers (LG and HV) independently screened the full texts to select articles to be included in the final review. Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 6 of 20 Table 2: Criteria for the level of evidence and overall assessment of measurement properties. Table 2: Criteria for the level of evidence and overall assessment of measurement properties. Criteriaa Overall assessment Level of evidence Consistent findings in multiple studies of good methodological quality OR in one study of excellent methodological quality +++ or – – – Strong Consistent findings in multiple studies of fair methodological quality OR in one study of good methodological quality ++ or – – Moderate One study of fair methodological quality + or – Limited Conflicting findings from multiple studies +/– Conflicting Only studies of poor methodological quality OR only indeterminate results from multiple studies regardless of methodological quality ? Unknown Measurement property not assessed 0 Not assessed a Adapted from Uijen et al. [47]. Table 3: Criteria for rating the adequacy of the reported measurement properties. Measurement property Reported Result Quality criteria [47] Content validity + The target population considers all items in the questionnaire to be relevant AND considers the questionnaire to be complete ? No target population involvement – The target population considers items in the questionnaire to be irrelevant OR considers the questionnaire to be incomplete 0 Did not assess content validity Measurement property Reported Result Quality criteria [47] The fourth factor entails the assessment of the consistency of several studies on the same instrument. The fourth factor entails the assessment of the consistency of several studies on the same instrument. First Delphi round In the first Delphi round, experts were asked to rank the relevance of the dimensions, indicators and assessment scale of B3-MM to assess maturity of integrate care on a 9-point Likert scale (1 = Extremely irrelevant to 9 = Extremely relevant). The Likert scale corresponds to the conventional format used for comparative assessment and prioritisation of different health options (such as technologies) [54]. The survey started with general questions (including age, country of employment, disciplinary field, and years of experience) and continued with statements on the relevance of components of the B3-MM. Remarks These statements were presented in three different parts. The first part (A) considered statements on the relevance of the 12 dimensions (12 statements); the second part (B) reflected statements on the relevance of each indicator on the maturity scales on every dimension used in B3-MM (72 statements); the third part (C) included statements on the relevance of the assessment scale (12 statements). The survey concluded with a set of open-ended questions. One question included a possible addition to the assessment scale which was retrieved from the literature review on existing tools and measures by Ahgren & Axelsson [55]. Experts were asked to assess if a part of the measurement scale used in the tool of Ahgren & Axelsson [55], referring to the assessment of both the actual rank and the optimum rank of integration, could provide a meaningful addition to the assessment scale as Delphi study To test the appropriateness of the items of the B3-MM to measure maturity of integrated care, an international Delphi study was performed. The Delphi technique is a widely used research method in healthcare research, which consists of “a series of data collection ‘rounds’ to capture and structure the knowledge and opinions of a ‘panel’ of participants on a topic with which they are perceived to have expertise” [42, p. 208]. Types of experts two active members of the SCIROCCO consortium (who had participated in the first round) were excluded from further participation. Again, the experts were given one and a half weeks to complete the second Delphi round. used in B3-MM. Finally, experts were asked if they had any additional comments/suggestions on B3-MM or the survey. The survey was anonymised and a single reminder email message was sent to the experts. To diminish potential misunderstandings concerning the interpretation of the survey, the first survey round was pre-tested by two researchers (YM, LB). The survey was adjusted to reflect their feedback, including a clearer introduction to part B and C of the survey about statements on the assessment of the relevance of each indicator and scale. Experts were invited to the first survey in three different streams due to the arrival of late responses to the call for experts. The respondents were given one and a half weeks to complete the first survey. Third Delphi round Third Delphi round The third Delphi round was conducted to explore the level of agreement among experts on the items with insufficient agreement in the second Delphi round. These items were rephrased by partners in the SCIROCCO consortium. Using the same 9-point Likert scale, experts were asked to reassess the relevance of the refined features of the B3-MM. The 13 experts who participated in the second round were re-invited to participate in the third Delphi round. The invitation included a report on the outcomes of the previous round, including (1) a median agreement rating (IQR) on every statement which was included in the second round, (2) the level of agreement among the experts, (3) the level of disagreement among the experts, and (4) whether consensus had been achieved. Experts were given the opportunity to provide feedback on the survey. Due to the project’s deadlines and the small number of statements in the third round, experts were given one week to complete the last round. Selection of experts The experts were selected on basis of relevant experience in scientific research or having a practical background (medicine, nursing, managerial, policy making) with relevant experience in the development, implementation and/or monitoring of integrated care interventions. An overview of the type of experts who were invited to the first round of the Delphi survey is presented in Table 4. A total number of 55 experts received the email invitation that included information about the purpose and process of the study and a link to an online version of the questionnaire in SurveyMonkey. We asked the experts to commit their participation in two planned Delphi rounds. Art. 10, page 7 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Table 4: List of experts in the first Delphi round. Types of experts Number of experts selected Experts retrieved from Corresponding/first author of scientific articles (researchers with experience in the measurement or development of integrated care) 10 Articles included in the literature review used in the study Experts with practical experience in the development, implementation and/or monitoring of integrated care interventions 10 SCIROCCO consortium partners Experts from the B3 Action Group on Integrated care 11 SCIROCCO consortium partners* Experts with experience in the field of Information and eHealth services in the field of integrated care 10 SCIROCCO consortium partners* Members of the SCIROCCO advisory board 5 SCIROCCO consortium partners* Researchers with expertise in measurement of development of integrated care 9 A convenience sample provided by one of the researchers * Basque Country (ESP), Norrbotten Lans Landsting (SE), Puglia region (IT), Olomouc region (CZ) and Scotland (UK). Number of experts selected Experts retrieved from Second Delphi round p The items for which insufficient agreement was found were rephrased by partners of the SCIROCCO consortium and presented to experts in the second Delphi round. A total number of 44 experts were invited to the second round. They were asked to reassess the relevance of the refined maturity indicators of the B3-MM items on the same 9-point Likert scale. Furthermore, they were asked to what extent they considered the addition to the assessment scale relevant by assessing both the actual rank and the optimum rank of integration using the B3-MM. Again, the experts were asked if they had any comments on the rephrased items or feedback on the survey. The second invitation included a report on the outcomes of round one of the Delphi exercise, including (1) a median agreement rating (interquartile range (IQR)) on every statement, (2) the level of agreement among the experts, (3) the level of disagreement among experts, and (4) whether consensus had been achieved. After discussion among the researchers and members of the SCIROCCO consortium, it was decided to exclude certain participants from the exercise due to a perceived conflict of interest: five members from the SCIROCCO advisory board (who had not participated in the first round of the exercise) and Data analysis of the Delphi study Before conducting the Delphi survey, we defined the conditions of agreement among experts to be applied during the three Delphi rounds. In order to determine consensus within a Delphi study, many studies use a predefined level of agreement among the experts [56]. However, no standard threshold for consensus is offered by the literature [53], with thresholds for consensus ranging from 55%–100% [57]. In our study we decided on using a 75% cut off point, which is suggested and used by several studies to clearly differentiate the consensus and non-consensus results [53, 58, 59]. Art. 10, page 8 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Figure 2: Flowchart calculation of consensus. Second Delphi round Calculaon of median Placement in category Median 1-3 Median 4-6 Median 1-3 Round 2: Equivocal Median in category 4-6 Less than 75% of scores in one of the categories Round 2: Equivocal More than 15% of scores in opposite category Round 2: Equivocal Median 1-3 Median 7-9 Median in category 1-3 or 7-9 More than 75% of scores in one of the categories Less than 15% of scores in opposite category Median 1-3: Irrelevant Median 7-9: Relevant CONSENSUS Median 7-9 Median 7-9 Calculaon of median Calculaon of median Placement in category Median in category 4-6 Round 2: Equivocal Less than 75% of scores in one of the categories More than 75% of scores in one of the categories More than 15% of scores in opposite category Round 2: Equivocal Less than 15% of scores in opposite category Figure 2: Flowchart calculation of consensus. Analysis were performed in MS Excel. Under Belgium law no ethical approval is required to interview experts as part of a Delphi panel. The 9-point scale was classified in three options; 1–3 as irrelevant, 4–6 as equivocal and 7–9 as relevant. The experts’ overall consensus on every statement on the items in the B3-MM was analysed using the median of the group’s scores and the “level of agreement” reached. Agreement among the experts on every statement on the items in the maturity model was reached when more than 75% of the experts’ ratings were within the same three-point range (that is, 1–3, 4–6, or 7–9) as well as the observed median. Several studies use a cut-off point of more than 75% of participants scoring 7 to 9, and include the condition (without disagreement) that less than 15% of the participants should have a scoring of between 1 to 3 [60, 61]. In this study, we used the 75% threshold for reaching consensus, including the condition that less than 15% of the participants should have a scoring in the opposite range of that scale (Figure 2). Furthermore, the qualitative comments derived from the answers to the question on the optimum and actual rank, and the comments/suggestions on B3-MM and feedback on the survey were analysed using a qualitative approach. Literature review Out of the 300 articles included in the study of Bautista et al. [23] a total of seven articles were selected for our review [55, 62–67]. From the narrative search, an additional number of four articles were retrieved. One duplicate full-text article from Bainbridge et al. [68] selected from Google and Google Scholar described a framework to guide evaluation and a more recent study was available describing the instrument which was based on this framework [69]. We included this article in the review instead of the initial full-text article retrieved. Details on the review process are presented in Figure 3. The combination of final search terms used for each database, date searched and the hits retrieved are shown in Table 5. The characteristics of the selected articles are shown in Appendix A. Art. 10, page 9 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Figure 3: Flowchart narrative review process. Figure 3: Flowchart narrative review process. review concerning validation of the DMIC, three more validation studies were found. The results on the quality of the studies, the direction of results and the overall quality of the measurement property content validity of the instruments are shown in Table 7. Overall, there is considerable similarity between the content of the original B3-MM model, and the instruments described in the articles selected from the literature review. All 12 dimensions and the related indicators described by the B3-MM corresponded with the content of the 11 retrieved articles (Table 6). Two dimensions of the B3-MM (“Information and eHealth services” and “Breadth of Ambition”) were described by all 11 articles. The content of over half of the articles matched with descriptions of ten of the dimensions. Less than half of the selected articles described items which matched with the two dimensions, “Population Approach” and “Innovation Management”. Apart from looking for matching descriptions, we searched for the use of possible dimensions, indicators or measurement scales which are not part of B3-MM (as it existed at the start of the project), and could complement or refine the B3-MM. One measurement scale was found which could provide a complement to the B3-MM: it was retrieved from the study of Ahgren & Axelsson [55]. They use a measurement model that can be used to evaluate the degree of integration, focusing on the functional aspects of clinical integration in arrangements of integrated care. Literature review In their model, the actual and the optimum rank of integration between units of the health authority are rated. This measurement feature could provide an extension to the B3-MM. It would enable the B3-MM to assess both the actual rank and the optimum rank of integration. Thus, it would provide a contextual explanation for the current situation in integrated care delivery while measuring the maturity of integrated care. This issue was further explored in the first two rounds of the Delphi study. First round A total of 31 experts responded to the first survey round (response rate 56%). Three experts did not complete the survey. Furthermore, two experts were excluded due to a conflict of interest. The final analysis included 26 experts (84% completion rate). Reasons for non-participation included one delivery failure, one retirement, and two time constraints. The rest of the respondents did not provide reasons for not participating. The outcomes on every statement of the first Delphi round are shown in Appendix B. Sufficient agreement was found among the experts on all 12 dimensions of B3-MM. Insufficient level of agreement was found for the first few indicators per dimension. Additionally, sufficient agreement was found on the assessment scale of the dimensions, except for the scale of “Innovation Management”. Comments and suggestions with regard to the dimensions, indicators or assessment scale of B3-MM were provided by 17 out of 26 experts (65.4%). Although three experts provided positive comments with regard to the B3-MM, three other experts commented that some dimensions were unclear or that indicators in some of the dimensions were already covered by other dimensions. A total of five experts commented that some indicators/scales were ambiguous or contradictory and did not follow a logical structure. From the experts who provided feedback to the survey, two experts stated that the survey was difficult to understand and four experts did not fully understand the scale assessment in part C. Regarding the assessment of the measurement property content validity of the instruments, we retrieved the data on the assessment of the overall quality rating score from the review of Bautista et al. [23] for the seven instruments selected from their study. Out of the 4 articles retrieved from the narrative review, three instruments were identified. No other validation studies on those three instruments were found by the hand searches and snowball sampling. In the dissertation included in the did not fully understand the scale assessment in part C. Regarding answers to the question about assessing the actual and optimum rank of integration, 22 out of 26 experts (84.6%) agreed that the actual and the optimum ranks of integration should be taken into account when Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 10 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Table 5: Oversight narrative review search terms and hits. First round Information and e-Health Services 11 [12, 55, 62–67, 69–71] 1.1 No connected health services, just isolated medical record systems 1.2 No integrated services used, only pilots/local services 1.3 eHealth deployed in some areas, but limited to specific organisations or patients 1.4 Voluntary use of regional/national eHealth services across the healthcare system 1.5 Mandated or funded use of regional/national eHealth infrastructure across the healthcare system 1.6 Universal, at-scale regional/national eHealth services used by all integrated care stakeholders 4. Standardisation & Simplification 7 [12, 64, 65, 67, 69–71] 1.1 No systematic attempt to standardise the use of citizen health & care data, or to simplify systems in use 1.2 Debate on information standards (e.g., coding, formatting); exploration of options for consolidating ICT 1.3 A recommended set of agreed information standards at local level; a few local attempts at ICT consolidation 1.4 A recommended set of agreed information standards at regional/national level; some shared procurements of new systems at regional/national level; some large-scale consolidations of ICT underway 1.5 A unified set of agreed standards to be used for system implementations specified in procurement documents; many shared procurements of new systems; consolidated data centres and shared services widely deployed 1.6 A unified and mandated set of agreed standards to be used for system implementations fully incorporated into procurement processes; clear strategy for regional/national procurement of new systems; consolidated datacentres and shared services (including the cloud) is normal practice. 5. Finance & Funding 8 [12, 55, 63, 64, 67, 69–71] 1.1 No special funding allocated or available 1.2 Fragmented innovation funding, mostly for pilots 1.3 Consolidated innovation funding available through competitions/grants for individual care providers 1.4 Regional/national (or European) funding or PPP for testing and for scaling-up 1.5 Regional/national funding for scaling-up and on-going operations 1.6 Secure multi-year budget, accessible to all stakeholders, to enable further service development (Contd.) Table 6: Overview of articles matching descriptions with B3-MM. Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 1. Readiness to change to enable more integrated care 8 [12, 55, 64, 65, 67, 69–71] 1.1 No acknowledgement of crisis 1.2 Crisis recognized, but no clear vision or strategic plan 1.3 Dialogue and consensus-building underway; plan being developed 1.4 Vision or plan embedded in policy; leaders and champions emerging 1.5 Leadership, vision and plan clear to the general public; pressure for change 1.6 Political consensus; public support; visible stakeholder engagement 2. First round Database Final used search term combination/string Date search Hits Filter Selected articles based on title/abstract Selected articles after full text selection Grey literature Peer-reviewed literature Total included in review IDEA “integrated care” 26-7-2016 126 None 2 1 0 1 1 GOOGLE integrated care or coordination of care or continuity of care or patient centered care and measure or instrument or survey or questionnaire and degree or maturity model or level or phase 1-8-2016 164 English only 6 (1 duplicate with Google Scholar) 2 0 2 (1 dissertation) 2 Google Scholar (“integrated care” or “coordination of care” or “continuity of care” or “patient centered care”) and (measure or instrument) 29-7-2016 141 None 3 (1 duplicate with Google) 1 (Retrieved from the article of Bainbridge et al. [68]) 0 1 1 Table 5: Oversight narrative review search terms and hits. Table 5: Oversight narrative review search terms and hits. Database Final used search term combination/string Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 11 of 20 Table 6: Overview of articles matching descriptions with B3-MM. Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 1. Readiness to change to enable more integrated care 8 [12, 55, 64, 65, 67, 69–71] 1.1 No acknowledgement of crisis 1.2 Crisis recognized, but no clear vision or strategic plan 1.3 Dialogue and consensus-building underway; plan being developed 1.4 Vision or plan embedded in policy; leaders and champions emerging 1.5 Leadership, vision and plan clear to the general public; pressure for change 1.6 Political consensus; public support; visible stakeholder engagement 2. Structure and Governance 6 [12, 55, 64, 67, 69, 71] 1.1 No overall attempt to manage the move to integrated care 1.2 Change underway, but with fragmented organisations & plans 1.3 Formation of task forces, alliances and other informal ways of collaborating 1.4 Governance established at a regional or national level 1.5 Roadmap for a change programme defined and broadly accepted 1.6 Full, integrated programme established, with funding and a clear mandate 3. First round 10, page 12 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 6. Removal of inhibitor 7 [12, 55, 64, 67, 69–71] 1.1 All projects delayed or cancelled due to inhibitors 1.2 Some projects delayed or cancelled due to inhibitors 1.3 Process for identifying inhibitors in place 1.4 Strategy for removing inhibitors agreed at a high level 1.5 Solutions for removal of inhibitors developed and commonly used 1.6 High completion rate of projects & programmes; inhibitors no longer an issue for service development 7. Population Approach 5 [12, 66, 69–71] 1.1 No consideration of population health in service provision 1.2 A population focus of risk stratification but no risk stratification tools 1.3 Individual risk stratification for the most frequent service users 1.4 Group risk stratification for those who are at risk of becoming frequent service users 1.5 Population-wide risk stratification started but not fully acted on 1.6 Whole population stratification deployed and fully implemented. 8. Citizen empowerment 7 [12, 62, 65–67, 69, 71] 1.1 No systematic plan for empowerment 1.2 Citizens are not involved in decision-making processes and do not participate in the co-design of their services 1.3 Policies to support citizens’ empowerment and protect their rights, but may not reflect their real needs 1.4 Incentives and tools to motivate and support citizens to co-create health and participate in decision- making processes 1.5 Citizens are supported and involved in decision-making processes, and have access to information and health data 1.6 Citizens are involved in decision-making processes, and their needs are frequently monitored and reflected in service delivery and policy-making. 9. Evaluation methods 6 [12, 64, 67, 69–71] 1.1 No routine evaluation 1.2 Evaluation exists, but not as a part of a systematic approach 1.3 Evaluation established as part of a systematic approach 1.4 Some initiatives and services are evaluated as part of a systematic approach 1.5 Most initiatives are subject to a systematic approach to evaluation; published results 1.6 A systematic approach to evaluation, responsiveness to the evaluation outcomes, and evaluation of the desired impact on service redesign (i.e. a closed loop process) 10. First round Structure and Governance 6 [12, 55, 64, 67, 69, 71] 1.1 No overall attempt to manage the move to integrated care 1.2 Change underway, but with fragmented organisations & plans 1.3 Formation of task forces, alliances and other informal ways of collaborating 1.4 Governance established at a regional or national level 1.5 Roadmap for a change programme defined and broadly accepted 1.6 Full, integrated programme established, with funding and a clear mandate 3. Information and e-Health Services 11 [12, 55, 62–67, 69–71] 1.1 No connected health services, just isolated medical record systems 1.2 No integrated services used, only pilots/local services 1.3 eHealth deployed in some areas, but limited to specific organisations or patients 1.4 Voluntary use of regional/national eHealth services across the healthcare system 1.5 Mandated or funded use of regional/national eHealth infrastructure across the healthcare system 1.6 Universal, at-scale regional/national eHealth services used by all integrated care stakeholders 4. Standardisation & Simplification 7 [12, 64, 65, 67, 69–71] 1.1 No systematic attempt to standardise the use of citizen health & care data, or to simplify systems in use 1.2 Debate on information standards (e.g., coding, formatting); exploration of options for consolidating ICT 1.3 A recommended set of agreed information standards at local level; a few local attempts at ICT consolidation 1.4 A recommended set of agreed information standards at regional/national level; some shared procurements of new systems at regional/national level; some large-scale consolidations of ICT underway 1.5 A unified set of agreed standards to be used for system implementations specified in procurement documents; many shared procurements of new systems; consolidated data centres and shared services widely deployed 1.6 A unified and mandated set of agreed standards to be used for system implementations fully incorporated into procurement processes; clear strategy for regional/national procurement of new systems; consolidated datacentres and shared services (including the cloud) is normal practice. 5. Finance & Funding 8 [12, 55, 63, 64, 67, 69–71] 1.1 No special funding allocated or available 1.2 Fragmented innovation funding, mostly for pilots 1.3 Consolidated innovation funding available through competitions/grants for individual care providers 1.4 Regional/national (or European) funding or PPP for testing and for scaling-up 1.5 Regional/national funding for scaling-up and on-going operations 1.6 Secure multi-year budget, accessible to all stakeholders, to enable further service development (Contd.) 6 [12, 55, 64, 67, 69, 71] 11 [12, 55, 62–67, 69–71] (Contd.) Art. First round Breadth of ambition 11 [12, 55, 62–67, 69–71] 1.1 No level of integration 1.2 Services in silos; the citizen or their family as the integrator of services 1.3 Integration within the same level of care (e.g., primary care) 1.4 Integration between care levels (e.g., between primary and secondary care) 1.5 Integration includes both social care service and health care service needs 1.6 Fully integrated health & social care services (Contd.) Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 6. Removal of inhibitor 7 [12, 55, 64, 67, 69–71] 1.1 All projects delayed or cancelled due to inhibitors 1.2 Some projects delayed or cancelled due to inhibitors 1.3 Process for identifying inhibitors in place 1.4 Strategy for removing inhibitors agreed at a high level 1.5 Solutions for removal of inhibitors developed and commonly used 1.6 High completion rate of projects & programmes; inhibitors no longer an issue for service development 7. Population Approach 5 [12, 66, 69–71] 1.1 No consideration of population health in service provision 1.2 A population focus of risk stratification but no risk stratification tools 1.3 Individual risk stratification for the most frequent service users 1.4 Group risk stratification for those who are at risk of becoming frequent service users 1.5 Population-wide risk stratification started but not fully acted on 1.6 Whole population stratification deployed and fully implemented. 8. Citizen empowerment 7 [12, 62, 65–67, 69, 71] 1.1 No systematic plan for empowerment 1.2 Citizens are not involved in decision-making processes and do not participate in the co-design of their services 1.3 Policies to support citizens’ empowerment and protect their rights, but may not reflect their real needs 1.4 Incentives and tools to motivate and support citizens to co-create health and participate in decision- making processes 1.5 Citizens are supported and involved in decision-making processes, and have access to information and health data 1.6 Citizens are involved in decision-making processes, and their needs are frequently monitored and reflected in service delivery and policy-making. 9. First round [23]) Author (name of first author only used) [reference] Number of validation studies Methodological quality of studies on content validity (COSMIN checklist [51]) Direction of results (Table 3) of measurement property content validity Overall quality measurement property content validity score (Table 2) Scale of Functional integration Ahgren[55] 1 Fair a ? DELTA service user assessment Ahgren [62] 1 Fair a + Human Service Integration Measure Browne [63] 1 Excellent a ? Unnamed1 Lukas [64] 1 Fair a + Dual Diagnosis Capability in Health Care Settings (DDCHCS) McGovern [65] 1 Not assessed a 0 Patient Perceptions of Integrated Care Survey (PPICS) Singer [66] 1 Fair a + Unnamed2 Uyei [67] 1 Good a ? Instruments (derived from the narrative review) HCP integration survey Bainbridge [69] 1 Fair ? ? Unnamed3 Calciolari [70] 1 Fair ? ? Development Model of Integrated Care (DMIC) 5 +++ Minkman [12] Excellent + Minkman [12] Excellent + Minkman [12] Excellent + Minkman [12] Excellent + Longpré [71] Fair ? Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 11. Innovation management 4 [12, 64, 69, 71] 1.1 No plan for innovation management 1.2 Isolated innovations across the region/country, but limited visibility 1.3 Innovations are captured and published as good practice 1.4 Innovation is governed and encouraged at a region/country level 1.5 Formalised innovation management process in place 1.6 Extensive open innovation combined with supporting procurement & the diffusion of good practice. 12. Capacity building 8 [12, 62–65, 67, 69, 71] 1.1 No plan for capacity-building 1.2 Single organisational initiatives engaged in process improvement 1.3 Some mechanisms for sharing knowledge among organisations 1.4 Systematic learning about IT; integrated care and change management 1.5 Knowledge shared, skills retained and lower turnover of experienced staff 1.6 A ‘learning healthcare system’ involving reflection and continuous improvement Number of article(s) [Reference] 4 [12, 64, 69, 71] 4 [12, 64, 69, 71] 8 [12, 62–65, 67, 69, 71] Table 7: Number of validation studies, the methodological quality of the studies, the direction (positive or negative) of results of the measurement properties and overall quality measurement property content validity score. Instrument (data derived from Bautista et al. First round Evaluation methods 6 [12, 64, 67, 69–71] 1.1 No routine evaluation 1.2 Evaluation exists, but not as a part of a systematic approach 1.3 Evaluation established as part of a systematic approach 1.4 Some initiatives and services are evaluated as part of a systematic approach 1.5 Most initiatives are subject to a systematic approach to evaluation; published results 1.6 A systematic approach to evaluation, responsiveness to the evaluation outcomes, and evaluation of the desired impact on service redesign (i.e. a closed loop process) 10. Breadth of ambition 11 [12, 55, 62–67, 69–71] 1.1 No level of integration 1.2 Services in silos; the citizen or their family as the integrator of services 1.3 Integration within the same level of care (e.g., primary care) 1.4 Integration between care levels (e.g., between primary and secondary care) 1.5 Integration includes both social care service and health care service needs 1.6 Fully integrated health & social care services (Contd.) Dimensions and related indicators as described in B3-MM [30] 5 [12, 66, 69–71] (Contd.) Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 13 of 20 Art. 10, page 13 of 20 Dimensions and related indicators as described in B3-MM [30] Number of article(s) [Reference] 11. Innovation management 4 [12, 64, 69, 71] 1.1 No plan for innovation management 1.2 Isolated innovations across the region/country, but limited visibility 1.3 Innovations are captured and published as good practice 1.4 Innovation is governed and encouraged at a region/country level 1.5 Formalised innovation management process in place 1.6 Extensive open innovation combined with supporting procurement & the diffusion of good practice. 12. Capacity building 8 [12, 62–65, 67, 69, 71] 1.1 No plan for capacity-building 1.2 Single organisational initiatives engaged in process improvement 1.3 Some mechanisms for sharing knowledge among organisations 1.4 Systematic learning about IT; integrated care and change management 1.5 Knowledge shared, skills retained and lower turnover of experienced staff 1.6 A ‘learning healthcare system’ involving reflection and continuous improvement Table 7: Number of validation studies, the methodological quality of the studies, the direction (positive or negative) of results of the measurement properties and overall quality measurement property content validity score. Instrument (data derived from Bautista et al. First round [23]) Author (name of first author only used) [reference] Number of validation studies Methodological quality of studies on content validity (COSMIN checklist [51]) Direction of results (Table 3) of measurement property content validity Overall quality measurement property content validity score (Table 2) Scale of Functional integration Ahgren[55] 1 Fair a ? DELTA service user assessment Ahgren [62] 1 Fair a + Human Service Integration Measure Browne [63] 1 Excellent a ? Unnamed1 Lukas [64] 1 Fair a + Dual Diagnosis Capability in Health Care Settings (DDCHCS) McGovern [65] 1 Not assessed a 0 Patient Perceptions of Integrated Care Survey (PPICS) Singer [66] 1 Fair a + Unnamed2 Uyei [67] 1 Good a ? Instruments (derived from the narrative review) HCP integration survey Bainbridge [69] 1 Fair ? ? Unnamed3 Calciolari [70] 1 Fair ? ? Development Model of Integrated Care (DMIC) 5 +++ Minkman [12] Excellent + Minkman [12] Excellent + Minkman [12] Excellent + Minkman [12] Excellent + Longpré [71] Fair ? a Data on direction of results per instrument was summarised in the review of Bautista et al. [23]. No individual data per instrument was provided. a Data on direction of results per instrument was summarised in the review of Bautista et al. [23]. No individual data per instrument was provided. a Data on direction of results per instrument was summarised in the review of Bautista et al. [23]. No individual data per instrument was provided. Grooten et al: An Instrument to Measure Maturity of Integrated Care Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 14 of 20 measuring maturity of integrated care in a region or country. due to time constraints. The rest of the potential respondents did not provide reasons for not participating. The outcomes for every statement of the second Delphi round are shown in Appendix C. Sufficient agreement was found among experts on the rephrased indicators, except for the two rephrased indicators, 8.2 and 9.1. Furthermore, 92.3% of the experts scored between 7–9 (median 7) in response to the question on the relevance of assessing both the actual rank and the optimum rank Discussion This study reports on the content validity of the B3-MM instrument, developed to measure the level of maturity of integrated care. The literature review and Delphi study allowed the assessment of the content validity of B3-MM and enabled the instrument to be enhanced. Following on from the review, the dimensions and indicators of the maturity model correspond to the items of instruments measuring maturity of integrated care in the academic literature. The results of the Delphi study showed that all the dimensions of the B3-MM are considered relevant by experts in the field of integrated care. Initially in the first Delphi round, there was insufficient agreement on the first few maturity indicators on every dimension whereas, after rephrasing the indicators during the second and third Delphi rounds, experts agreed that all the indicators were relevant for the assessment of the maturity of integrated care. As a result, B3-MM is considered to be a comprehensive instrument consisting of a wide range of dimensions applicable to the development of integrated care. A few limitations need to be considered with regard to this study. The review was based on search terms derived from a systematic literature review which enabled a broad search in several databases. However, a first limitation of the narrative review is the focus on English language studies, which may have led to a language bias. A second limitation is that literature represent a large diversity of concepts (methods and measurements) concerning the measurement of integrated care [73]. Since “the definition and application of the concept of integrated care is influenced by the background and health care systems of the various authors” [12, p. 8], the data extraction from the literature conducted by the researchers is inevitably subjective. This is a disputable characteristic of any review that addresses complex interventions focusing on the items described for instruments in different contexts. A third limitation is that the review is susceptible to publication bias, although the search has been broadened to include literature found through various search engines. Concerning the overall assessment of the quality of the measurement property content validity we used data obtained from the review of Bautista et al. [23] on the score for the instruments and applied their criteria to the assessment of the instruments retrieved from our narrative review. Second round A total of 14 experts responded to the second survey round (response rate 34%). One expert did not complete the survey. The final analysis included 13 experts (92.9% completion rate). One expert was not able to participate Table 8: Characteristics of experts in Delphi rounds 1, 2 and 3 (in % unless stated otherwise). Characteristic Category Expert group first round (n = 26) Expert group second round (n = 13) Expert group third round (n = 10) Age (year) Min–Max 36–71 36–71 36–71 Average (sd) 49.23 (11.73) 52.69 (13.22) 52.60 (13.43) <40 23.1 23.1 20 40–50 30.8 23.1 30 >50 46.2 53.8 50 Gender Male 30.8 46.2 50.0 Female 69.2 53.8 50.0 Country Belgium 3.8 7.7 10 Canada 7.7 7.7 10 Czech Republic 3.8 7.7 10 Finland 3.8 0 0 Germany 3.8 0 0 Italy 15.4 15.4 0 Luxembourg 3.8 0 0 Netherlands 7.7 0 0 Netherlands and USA 3.8 7.7 10 Portugal 7.7 7.7 10 Spain 7.7 15.4 20 Sweden 7.7 0 0 UK 15.4 23.1 20 USA 7.7 7.7 10 Professional Affiliation Medicine 15.4 15.4 20 Nursing 7.7 7.7 10 Policy 7.7 15.4 0 Managerial 15.4 23.1 20 Research 46.2 30.8 40 Other 7.7 7.7 10 Years of experience <1 0 0 0 1–5 38.5 23.1 30 5–10 26.9 23.1 20 >10 34.6 53.8 50 Table 8: Characteristics of experts in Delphi rounds 1, 2 and 3 (in % unless stated otherwise). Art. 10, page 15 of 20 Art. 10, page 15 of 20 Grooten et al: An Instrument to Measure Maturity of Integrated Care not mean that the quality of the instruments is low, but rather that there is a need for high quality studies that can adequately assess the measurement properties and eventually the instrument quality. Moreover, out of the 300 articles retrieved in the literature review undertaken by Bautista et al. [23], only seven articles were included in this review. The need for high quality studies on measurement properties and the small number of selected articles indicates that the measurement of maturity in integrated care is not yet strongly developed in the academic literature. The complexity of the development, implementation and scale-up of the multi-stage process of integrated care makes the measurement of the maturity of integrated care a difficult exercise. Third round A total of 10 experts participated in the third Delphi round (response rate 76.9%). The rest of the potential respondents did not provide reasons for not participating. Sufficient agreement was found on both of the two rephrased indicators 8.2 and 9.1 (Appendix D). The main characteristics of the expert group who participated in the first, second and Delphi round are presented in Table 8. Second round However, if integrated care initiatives are to make a significant contribution to the transformation of health systems, solid measurement of the maturity of integrated care should become an essential element of their development. Measurement of the maturity of integrated care provides insight into both the problems experienced and the success factors that work when making progress on the development of integrated care services. It provides the knowledge needed to guide further development of integrated care initiatives in appropriate directions. of integration, by applying B3-MM to provide a contextual explanation for the current situation while measuring maturity of integrated care. A total of six experts provided comments on the rephrased indicators. Three experts indicated that the rephrasing of the indicators was performed well. Furthermore, two experts emphasised that some of the rephrased indicators could still be made more explicit to distinguish these indicators clearly from the other indicators in their scale. Discussion In the first step, five participating health care regions in Europe are asked to use the tool for self-assessment of the of maturity level of the region or healthcare system in the path towards integrated care delivery. Based on the outcomes of the tool, the regions with complementary levels of maturity are matched. SCIROCCO will then organize twinning and coaching to facilitate shared learning among the regions. The SCIROCCO project will explore how matching the complementary strengths and weaknesses of regions can deliver two major benefits: a strong basis for successful twinning and coaching that facilitates shared learning and a practical support for the scaling up of good practices that promote active and healthy ageing and participation in the community [30]. As the B3-MM will be used as a starting point from which regions will be matched and shared learning will be facilitated, insight in the measurement properties of the tool is a prerequisite to ensure a valid and reliable assessment of the maturity level of the regional healthcare system. This will enable the more tailored process of achieving progress in the path towards integrated care for health care regions. number of rounds to be organised regarding the Delphi method; “rather the Delphi method appears to be related to common sense and practical possibilities” [46, p. 208]. Furthermore, the sample of the expert panels in the Delphi method are not being judged in terms of being representative samples for statistical purposes, but rather assessed on the qualities of the expert [75]. Although, we tried to reduce possible artefacts, a few limitations need to be considered for the Delphi study. To reach a reliable consensus in Delphi studies, it is important to establish a balance among the participants who represent a particular topic. The balance between the expert types who were recruited for the Delphi study and who participated in the first Delphi round was as follows: about half of the respondents who participated included researchers with experience in the measurement or development of integrated care. The other half consisted of a pool of experts who were recruited via the SCIROCCO consortium partners (i.e. a mix of experts with a practical experience in the development, implementation and/or monitoring of integrated care interventions, with experience in the field of Information and eHealth services or experts from the B3 Action Group on Integrated care). Discussion The assessment is therefore subject to possible inconsistency although we tried to diminish this by discussing the assessment of the instruments among the researchers (LG and HV). The items included in another instrument, called the DMIC, described in two articles, matched all the dimensions of the B3-MM [12, 71]. While the DMIC is regarded as a validated generic quality management model for integrated care, the model was developed and widely used in the Netherlands [72]. In comparison, the B3-MM is of a wider scope, developed on basis of lessons learned in achieving integrated care by 12 different European regions. In line with other studies [23, 47], a variety in the constructs and elements measured by the selected instruments was observed in this study. Furthermore, the level of evidence on the overall quality of the measurement property content validity for only one out of ten instruments assessed in this study, was found to be strong. In their systematic review of measurement properties of care continuity instruments, Uijen et al. [47] indicated that these findings on the levels of evidence do The Delphi technique has long been regarded as an appropriate research technique to reach consensus amongst groups of experts and has been widely applied in health and social studies [74]. However, there are currently no universally agreed criteria for the selection of experts; no directives on the minimum or maximum number of experts on a panel; and no firm guidelines on the correct Grooten et al: An Instrument to Measure Maturity of Integrated Care Art. 10, page 16 of 20 the B3-MM is a unique instrument based on existing knowledge and lessons learned in implementing integrated care, further research on its measurement properties is needed to enhance the quality of the B3-MM as instrument. The determination of the validity of an instrument measuring a construct is important. This further research on its measurement properties should preferably be guided by the COSMIN manual [51]. Moreover, in the SCIROCCO project, the use of the B3-MM instrument will be further explored as a tool to facilitate the exchange of good practices and scaling-up of integrated care processes in Europe. SCIROCCO will do so by testing a step-based strategy. Additional Files The additional files for this article can be found as follows: Discussion The agreement found among the experts on the items of the B3-MM represents the majority opinion of the experts, yet, it does not mean the ‘right’ answers have been found [53]. The results may be biased due to the recruitment strategy that involved partners of the consortium; however, it may be expected that the experts provided their nuanced opinions garnered from their expertise. Furthermore, we provided room for the experts’ comments and suggestions as well as ensured that the Delphi rounds were completed anonymously without the influence of other panel members, to obtain a reliable and diverse collection of opinions. Additionally, a gradual decline in the number of experts participating in each Delphi round was observed. Although we provided experts with more than a week for responding and sent reminders, by asking for their participation in several rounds the Delphi technique asks much more dedication from respondents than does a simple survey, and the potential for low responses increases considerably [53]. A final limitation to the study is that a few expert respondents found the survey difficult to understand, which indicates that it is not evident that the instrument is easy to understand. The different backgrounds of the experts, concerning their fields of experience and origins (including variations in the types of health care systems, social values, and on-going health reform) may have also an influence on the way in which the instrument is interpreted. To obtain an adequate understanding of the instrument among its users, a clear manual explaining the meaning and application of the instrument would be desirable. • Appendix A: Characteristics of articles identified. DOI: https://doi.org/10.5334/ijic.3063.s1 • Appendix B: Outcomes Delphi round 1. DOI: https://doi.org/10.5334/ijic.3063.s2 Acknowledgements We acknowledge the contribution of the following researchers participating in SCIROCCO: • NHS 24 – Donna Henderson, Andrea Pavlickova • UEDIN –Cristina-Adriana Alexandru, Stuart Anderson • UVEG – Elisa Vaila Cotanda, Tamara Alhambra • EHTEL – Marc Lange, Diane Whitehouse • Kronikgune – Esteban de Manuel Keenoy, Jon Txarramendieta Suarez, Ane Fullaondo Zabala • Osakidetza – Igor Zabala, Ainhoa Martin • Ares Puglia – Francesca Avolio, Anna Elisabetta Graps • FNOL – Zdenek Gutter, Michal Stybnar • NLL – Lisa Lundgren, Ann-Charlotte Kassberg • Vrije Universiteit Brussel – Yannick Marchal • NHS 24 – Donna Henderson, Andrea Pavlickova • UEDIN –Cristina-Adriana Alexandru, Stuart Anderson • UVEG – Elisa Vaila Cotanda, Tamara Alhambra • EHTEL – Marc Lange, Diane Whitehouse • Kronikgune – Esteban de Manuel Keenoy, Jon Txarramendieta Suarez, Ane Fullaondo Zabala • Osakidetza – Igor Zabala, Ainhoa Martin • Ares Puglia – Francesca Avolio, Anna Elisabetta Graps • FNOL – Zdenek Gutter, Michal Stybnar • NLL – Lisa Lundgren, Ann-Charlotte Kassberg • Vrije Universiteit Brussel – Yannick Marchal • Appendix A: Characteristics of articles identified. 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DOI: https://doi.org/10.5334/ijic.3063 How to cite this article: Grooten, L, Borgermans, L and Vrijhoef, HJM. An Instrument to Measure Maturity of Integrated Care: A First Validation Study. International Journal of Integrated Care, 2018; 18(1): 10, 1–20. How to cite this article: Grooten, L, Borgermans, L and Vrijhoef, HJM. An Instrument to Measure Maturity of Integrated Care: A First Validation Study. International Journal of Integrated Care, 2018; 18(1): 10, 1–20. DOI: https://doi.org/10.5334/ijic.3063 References DOI: https://doi.org/10.5334/ijic.3063 Submitted: 23 February 2017 Accepted: 28 November 2017 Published: 25 January 2018 Submitted: 23 February 2017 Accepted: 28 November 2017 Published: 25 January 2018 Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. International Journal of Integrated Care is a peer-reviewed open access journal published by Ubiquity Press. OPEN ACCESS
https://openalex.org/W4396229337	https://www.frontiersin.org/journals/public-health/articles/10.3389/fpubh.2024.1216164/pdf	English	null	Secular trends in physical growth, biological maturation, and intelligence in children and adolescents born between 1978 and 1993	Frontiers in public health	2,024	cc-by	9,199	TYPE Original Research PUBLISHED 29 April 2024 DOI 10.3389/fpubh.2024.1216164 TYPE Original Research PUBLISHED 29 April 2024 DOI 10.3389/fpubh.2024.1216164 Flynn effect, cognitive functioning, IQ, height, weight, head circumference, skeletal maturation, birth weight KEYWORDS Flynn effect, cognitive functioning, IQ, height, weight, head circumference, skeletal maturation, birth weight OPEN ACCESS OPEN ACCESS EDITED BY Jakob Pietschnig, University of Vienna, Austria REVIEWED BY Dieter Wolke, University of Warwick, United Kingdom Joe Rodgers, Vanderbilt University, United States CORRESPONDENCE Oskar G. Jenni oskar.jenni@kispi.uzh.ch †These authors share last authorship RECEIVED 03 May 2023 ACCEPTED 12 April 2024 PUBLISHED 29 April 2024 Dominique A. Eichelberger 1, Aziz Chaouch 2, Valentin Rousson 2, Tanja H. Kakebeeke 1,3, Jon Caflisch 1,3, Flavia M. Wehrle 1,3,4† and Oskar G. Jenni 1,3,5† 1 Child Development Center, University Children's Hospital Zurich, Zurich, Switzerland, 2 Department of Epidemiology and Health Systems, Quantitative Research, Center for Primary Care and Public Health (Unisanté), University of Lausanne, Lausanne, Switzerland, 3 Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland, 4 Department of Neonatology and Intensive Care, University Children’s Hospital Zurich, Zurich, Switzerland, 5 Faculty of Medicine, University of Zurich, Zurich, Switzerland CITATION Eichelberger DA, Chaouch A, Rousson V, Kakebeeke TH, Caflisch J, Wehrle FM and Jenni OG (2024) Secular trends in physical growth, biological maturation, and intelligence in children and adolescents born between 1978 and 1993. Introduction: Human physical growth, biological maturation, and intelligence have been documented as increasing for over 100 years. Comparing the timing of secular trends in these characteristics could provide insight into what underlies them. However, they have not been examined in parallel in the same cohort during different developmental phases. Thus, the aim of this study was to examine secular trends in body height, weight, and head circumference, biological maturation, and intelligence by assessing these traits concurrently at four points during development: the ages of 4, 9, 14, and 18 years. Front. Public Health 12:1216164. doi: 10.3389/fpubh.2024.1216164 Front. Public Health 12:1216164. doi: 10.3389/fpubh.2024.1216164 KEYWORDS Secular trends in physical growth, biological maturation, and intelligence in children and adolescents born between 1978 and 1993 OPEN ACCESS EDITED BY Jakob Pietschnig, University of Vienna, Austria REVIEWED BY Dieter Wolke, University of Warwick, United Kingdom Joe Rodgers, Vanderbilt University, United States *CORRESPONDENCE Oskar G. Jenni oskar.jenni@kispi.uzh.ch †These authors share last authorship RECEIVED 03 May 2023 ACCEPTED 12 April 2024 PUBLISHED 29 April 2024 CITATION Eichelberger DA, Chaouch A, Rousson V, Kakebeeke TH, Caflisch J, Wehrle FM and Jenni OG (2024) Secular trends in physical growth, biological maturation, and intelligence in children and adolescents born between 1978 and 1993. 1 Introduction conscripts in Northern European countries. In fact, Sundet et al. (14) showed that the increase in intelligence and height followed an identical pattern among Norwegian military draftees between 1950 and 1990. However, the increase in height was driven primarily by the upper half of the height distribution, while the lower part of the IQ distribution was responsible for the IQ gain. Rönnlund et al. (15) also found parallel secular trends in the two domains between 1970 and 1979, but the rise in intelligence test scores continued during 1980– 1993, when the secular trend in height was no longer observed. Therefore, both studies concluded that improved nutrition, better medical care, and perhaps other biological factors might not have been the primary cause for the Flynn effect, because secular trends in growth were not observed in the same periods and to the same extent. For over 100 years, substantial upward trends have been observed in a range of human traits, skills, and performances. Typical examples of these secular trends include the substantial gain in life expectancy since the beginning of the 19th century (1), the large generational increases in human growth characteristics such as body height and weight as well as the acceleration in biological maturation (2), the improved physiological strength as measured in sport achievements over many decades (1), and the rise in populations’ average intelligence since 1900 (3, 4). This secular trend was named the Flynn effect after James Flynn (5). Environmental factors have frequently been proposed as the main cause of the positive secular trends in human characteristics; this includes the improved quality of nutrition, sanitation, hygiene and health care, the decreased rate of infections and other diseases, the higher value and better structure of educational systems, the reduced size of families and higher standard of child rearing, the increased overall complexity of environments, and ultimately, the economic advance of societies (6). In contrast to these environmental explanatory factors, increases in genetic diversity and consequently genetic advantages resulting from demographic trends towards more random mating in recent societies were also proposed (7). However, a single explanation does, likely, not suffice to fully account for the positive secular trends in populations’ traits but a combination of multiple aspects that may vary across domains may be relevant (3). 1 Introduction Previous findings on secular trends in traits assessed concurrently in the same cohort are limited to adults, mainly conscripts aged 18–20 years old (14–18). However, secular trends have been suggested to vary with age. In the Netherlands, for example, Woodley and Meisenberg (19) found that intelligence test scores between 1950 and 1990 increased more among 30-year-olds than 5- to 6-year-olds, and they even decreased among 14- to 16-year-olds, indicating a negative trend. However, findings about age-specific secular trends in intelligence remain inconsistent, in particular in recently published meta-analyses: Whereas Pietschnig and Voracek (3) reported larger secular trends in intelligence in adults than in children and adolescents, Trahan et al. (4) could not confirm these findings. One reason for the discrepancies between studies may be the frequent inclusion of cross-sectional studies, which are prone to sample composition effects. In the most recent decades, secular trends in a number of traits have been observed to slow, stop, and even reverse. For example, in the Northern European countries, the rise in adult height has reached a plateau (8), and intelligence test scores have even declined over time (9). The slowing and flattening of secular trends since the end of the 20th century may be explained by the fact that in industrialized populations with the highest living standards, an increasing proportion of individuals have reached the upper limit of their genetic and biological potential, and consequently, the benefits of environmental factors are now fading (1, 9). Notably, a reverse in the direction of secular trends (i.e., negative secular trends) is expected to stem from a different set of processes because the factors that previously contributed to an increase are unlikely to explain a subsequent decline (10). Larger secular trends for height have been reported during childhood than during adulthood, with a maximum at puberty (2, 20, 21). These age-related differences suggest that beyond environmental factors, maturational tempo may contribute to secular trends in growth parameters during development (2, 21–26). In fact, several studies have confirmed a secular trend in skeletal maturity, a biological indicator of the tempo of growth and maturation (27, 28). COPYRIGHT © 2024 Eichelberger, Chaouch, Rousson, Kakebeeke, Caflisch, Wehrle and Jenni. 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. © 2024 Eichelberger, Chaouch, Rousson, Kakebeeke, Caflisch, Wehrle and Jenni. This is an open-access article distributed under the terms of the Creative Commons Attribution Methods: Data derived from growth measures, bone age as an indicator of biological maturation, and full-scale intelligence tests were drawn from 236 participants of the Zurich Longitudinal Studies born between 1978 and 1993. In addition, birth weight was analyzed as an indicator of prenatal conditions. Results: Secular trends for height and weight at 4 years were positive (0.35 SD increase per decade for height and an insignificant 0.27 SD increase per decade for weight) and remained similar at 9 and 14 years (height: 0.46 SD and 0.38 SD increase per decade; weight: 0.51 SD and 0.51 SD increase per decade, respectively) as well as for weight at age 18 years (0.36 SD increase per decade). In contrast, the secular trend in height was no longer evident at age 18 years (0.09 SD increase per decade). Secular trends for biological maturation at 14 years were similar to those of height and weight (0.54 SD increase per decade). At 18 years, the trend was non-significant (0.38 SD increase per decade). For intelligence, a positive secular trend was found at 4 years (0.54 SD increase per decade). In contrast, negative secular trends were observed at 9 years (0.54 SD decrease per decade) and 14 years (0.60 SD decrease per decade). No secular trend was observed at any of the four ages for head circumference (0.01, 0.24, 0.17, and − 0.04 SD increase per decade, respectively) and birth weight (0.01 SD decrease per decade). Discussion: The different patterns of changes in physical growth, biological maturation, and intelligence between 1978 and 1993 indicate that distinct mechanisms underlie these secular trends. Flynn effect, cognitive functioning, IQ, height, weight, head circumference, skeletal maturation, birth weight Frontiers in Public Health 01 frontiersin.org Eichelberger et al. 10.3389/fpubh.2024.1216164 Eichelberger et al. Frontiers in Public Health 1 Introduction The present study aims to advance the understanding of similarities and dissimilarities in the timing of secular trends in growth parameters including height, weight, and head circumference, as well as in biological maturation (measured by bone age), and intelligence by assessing these traits longitudinally during four developmental phases, at ages of 4, 9, 14, and 18 years, in a single cohort born between 1978 and 1993. Mingroni was among the first who did not only focus on single secular trends, but also explored secular changes in multiple variables such as height, head circumference, intelligence, myopia, asthma, autism and others at the same time (7, 11). In fact, the concurrent investigation of secular changes in multiple traits may contribute to identifying the explanatory processes underlying them. More specifically, if a similar timing is apparent in multiple traits, likely, a common mechanism underlies the respective secular trends. Conversely, if the timing is different for different traits, this may suggest the effect of different factors. Particularly, the comparison of secular trends in growth with maturational and intelligence parameters is interesting because they have all been attributed to improved nutrition and health care [see, e.g., (12)], while other authors have postulated that improved education and increased overall complexity of environments have led to the changes of the intellectual capacity across generations [see, e.g., (13)]. However, to date very few studies have compared secular trends in multiple traits concurrently in the same cohort. Notable exceptions are the studies of Eichelberger et al. 2.2 Assessments Data on child development were assessed numerous times between birth and 18 years of age. All developmental assessments were conducted by a psychologist or a developmental pediatrician at the University Children’s Hospital Zurich, Switzerland. For the current analyses, examinations at the ages of 4, 9, 14, and 18 years were used because they included the assessment of height, weight, head circumference, biological maturation, as well as intellectual abilities [see (29) for details on the full study protocol]. We use yij to denote the outcome observed on child i at age j = 1 (4 years), j = 2 (9 years) or j = 3 (14 years); ti the year of birth of child i; sexi the sex (female = 0, male = 1) of the child; and sesi their SES. We consider this regression model, ( ) ( )( ) i ij 0j 1j 2j i 3j i 4j i i ij t 1985 y sex ses 5 10 ses 5 ses 5 - æ ö = b + b + b + b - ç ÷ è ø + b - > + e Height was measured in the standing position to the nearest millimeter with a stadiometer. For the weight measurement, the children stood on a beam scale with an accuracy of 0.1 kilograms. Head size was measured at the widest possible circumference of the head: encompassing the broadest part of the forehead above the eyebrow, above the ears, and the most prominent part of the back of the head. The head measurement was taken three times, and the largest measurement to the nearest millimeter was selected. A more detailed description of the measurement of height, weight, and head circumference can be found in Prader et al. (31). where ( ) T j 0j 1j 2j 3j 4j , , , , = b b b b b b is a vector of regression coefficients and ij e is a residual error term with mean zero and variance 2 j . s Note that in this model, 0j b is a global intercept term and b1j quantifies the contrast between two cohorts of age j with same sex and SES born 10 years apart, expressed in original outcome units. 1 This number differs from that published in Wehrle et al. (29) (N = 327) because subsequent data checks revealed that 31 participants had incorrectly been listed as enrolled at birth. No data had been assessed in these individuals. One participant declined the continued use of the childhood data for future research projects and is thus no longer considered part of ZLS-3. 2 Methods 2.1 Sample The data originates from the Zurich Longitudinal Studies (ZLS), a set of three cohort studies that examined physical, motor, and mental development and social environment from birth to young adulthood (29). The third ZLS cohort (ZLS-3) covers the largest range of birth years, from 1973 to 2002, and consequently was considered for the current analyses. Individuals included in the ZLS-3 cohort are the offspring of participants of the first ZLS cohort (ZLS-1). ZLS-1 participants were enrolled into the ZLS between 1954 and 1961 as a representative sample of the population of Zurich with regard to parental occupation (30) and if they were born into a Swiss family 02 frontiersin.org Eichelberger et al. 10.3389/fpubh.2024.1216164 The full-scale IQ is estimated from the verbal and the performance IQ. The WISC-R is normed for children aged 6 to 16 years. residing in Zurich. Consequently, all participants in the ZLS-3 cohort have at least one Swiss parent. In total, 295 infants from 161 families were enrolled at birth.1 The current analyses only included those who did not have a developmental disability or genetic syndrome with known effects on intellectual ability or growth and who were fluent in German, because intelligence testing was conducted in German. Additionally, birth weight was collected from the birth report and used as an indicator of prenatal conditions. Childhood socioeconomic status (SES) was estimated from parental occupation and maternal education at ages 1 or 3 months on a scale ranging from 2 to 10 with higher scores indicating lower SES, following Largo et al. (38). The vast majority of the ZLS-3 cohort, 92.2%, was born between 1978 and 1993, with less than five children being born each year outside of this range. We therefore restricted the analyses to the 236 children and adolescents from 132 families born during this period (Supplementary Figure S1). We report the results of a sensitivity analysis that incorporated participants born before 1978 and after 1993 in Supplementary material S1. 2.3 Statistical analysis We defined the secular trend as the effect of the year of birth on height, logarithm of weight, head circumference, biological maturation, and full-scale IQ among participants of the same age after conditioning on sex and SES. The year of birth was considered as a continuous variable with decimals so that, for instance, a child born on 26.04.1985 had a year of birth equal to 1985.315. Because the IQ tests were not the same at each age, we analyzed each age group separately. For the sake of comparability, we also analyzed data from the other outcomes separately in each age group. frontiersin.org Frontiers in Public Health 2.2 Assessments Additionally, 2j b and 3j b refer to the sex and linear SES effect on the outcome, respectively, and 4j b allows SES to have a different linear effect on the outcome below and above the median value of 5. This last coefficient was included in the model only when statistically significant (p < 0.050). A model including an interaction term between the year of birth and sex, thus allowing for different secular trends in males and females, was also tested, but the interaction term was never found to be statistically significant. Therefore, we report results obtained for the simpler model without such interaction. Because study participants were nested within 132 families, error terms from individuals belonging to the same family were allowed to be correlated using an exchangeable covariance structure. We used Generalized Estimating Equations (39) fitted separately at each age to obtain estimates of the vector of regression coefficients j b and the residual variance 2 j . s The same procedure was applied for birth weight. Biological maturation was quantified as skeletal maturity (32) and measured by bone age at ages 14 and 18 years. Bone age was analyzed from hand x-rays of the left hand using the software BoneXpert (33, 34). Intellectual abilities were assessed using age-appropriate and standardized full-scale IQ instruments: at 4 years the Snijders–Oomen Non-Verbal Intelligence Test [SON; (35)] and at 9, and 14 years, the Wechsler-Intelligence Scale for Children-Revised [WISC-R; German version; (36, 37)] was used. The SON is a non-verbal intelligence test based on five sub-tests (sorting, mosaic, combination, memory, and copying) and is normed for children aged 2.5 to 7 years. The WISC-R comprises six subtests to estimate verbal IQ (information, comprehension, arithmetic, similarities, vocabulary, and digit span) and five subtests to estimate performance IQ (coding, picture completion, picture arrangement, block design, and object assembly). j Comparing secular trend estimates for outcomes measured on different scales requires such estimates to be reported on a relative (i.e., unitless) scale. Two types of standardization can be used for this purpose: an external out-of-sample standardization or an internal in-sample one. In this study, we used an internal standardization with the relative secular trend at age j defined as the ratio 1j j / s b . 2.2 Assessments This ratio quantifies the average change in the outcome between two cohorts born 10 years apart as a function of the in-sample Frontiers in Public Health 03 frontiersin.org Eichelberger et al. 10.3389/fpubh.2024.1216164 10.3389/fpubh.2024.1216164 Table 2 and Figure 1 present the relative secular trend estimates of height, weight, head circumference, biological maturation, and intellectual abilities at ages 4, 9, 14, and 18 years with 95% confidence intervals (CI). within-cohort inter-individual variability after controlling for sex and SES differences. Inference was performed by approximating the variance of the ratio 1j j / s b using the Delta method (40). The relative secular trend estimate can be interpreted as a regular standardized difference in means [Cohen’s d; (41)], with values of 0.2, 0.5, and 0.8 referring to small, moderate, and large effects, respectively. We direct interested readers to the Supplementary material S2 for a comparison of results obtained with internal and external standardizations. The secular trends in height and weight at age 4 years were positive (0.35 SD increase per decade for height and a statistically insignificant 0.27 SD increase per decade for weight, respectively) and remained about the same at ages 9 and 14 years (for height: 0.46 SD and 0.38 SD increase per decade; for weight: 0.51 SD and 0.51 SD increase per decade, respectively). Weight at age 18 years also remained similar (0.36 SD increase per decade). In contrast, the secular trend in height was no longer evident at age 18 (0.09 SD increase per decade). For head circumference, no statistically significant secular trends were found for the four developmental periods (0.01, 0.24, 0.17, and − 0.04 SD increase per decade for 4, 9, 14, and 18 years, respectively). For birth weight, no secular trend was found (0.01 SD decrease per decade). Secular trends for biological maturation at 14 years were similar to those of height, and weight (0.54 SD increase per decade), and remained positive at age 18 years (statistically insignificant 0.38 SD increase per decade).i All statistical analyses were carried out in R version 4.2.1 (42) using a level of statistical significance of 5%. 2.4 Ethics The study was reviewed and approved by the Ethical Committee of the Canton of Zurich, Switzerland (Basec-Nr. 2018-00686); further details on the informed consent procedure since the initiation of the ZLS in 1954 are provided in Wehrle et al. (29). Frontiers in Public Health erage change in the outcome value between two cohorts born 10 years apart divided by the within-cohort standard deviations, after controlling for sex and SES. 3 Results Statistically significant secular trends for intellectual abilities were found for all three available age periods: At 4 years, the secular trend was positive, with a 0.54 SD. However, negative trends were observed at 9 years (0.54 SD decrease per decade) and at 14 years (0.60 SD decrease per decade). The estimates for verbal and performance IQ assessed with the WISC-R at 9 and at 14 years indicated a negative Table 1 provides a descriptive summary at the four assessment time-points of the participant characteristics: sex distribution, SES, growth parameters, bone age, and IQ score. Between the ages of 4 and 18 years, 35 participants (14.8%) dropped out of the study. scores, and growth parameter at the four assessment time-points, for participants born between 1978 and 1993. TABLE 1 Demographic information, IQ scores, and growth parameter at the four assessment time-points, for participants born between 1978 and 1993. TABLE 1 Demographic information, IQ scores, and growth parameter at the four assessment time points, for participants born between 1978 and 1993. 4 Year 9 Year 14 Year 18 Year Number of participants (N) 229 218 205 200 Males [n (%)] 120 (52%) 114 (52%) 108 (53%) 102 (51%) Family SES (Mdn [IQR]) 5 [4, 6] 5 [4, 6] 5 [4, 6] 5 [4, 6] Birth weight [g]; M (SD) 3289 (434) 3282 (431) 3273 (426) 3264 (412) Head circumference [cm]; M (SD) 50.7 (1.3) 52.8 (1.3) 54.9 (1.5) 56.2 (1.7) Height males [cm]; M (SD) 104.2 (4.0) 134.2 (5.4) 163.6 (8.4) 177.4 (6.7) Height females [cm]; M (SD) 102.9 (3.5) 133.7 (4.7) 161.8 (5.3) 165.5 (4.8) Weight males [kg]; M (SD) 16.8 (1.9) 28.9 (4.6) 52.2 (10.9) 69.7 (11.7) Weight females [kg]; M (SD) 16.1 (1.7) 29.2 (5.2) 51.5 (8.9) 58.7 (8.4) Bone age [years]; M (SD) Not available Not available 13.9 (1.1) 17.5 (0.9) Full scale IQ; M (SD) 116.1 (11.4) 104.1 (11.3) 111.3 (10.8) Not available Mdn, median; IQR, interquartile range; M, mean; SD, Standard Deviation; SES, socioeconomic status based on a composite score of parents’ education and occupation, with scores ranging Mdn, median; IQR, interquartile range; M, mean; SD, Standard Deviation; SES, socioeconomic status based on a composite score of parents’ education and occupation, with scores ranging from 2 to 10 (higher scores indicating a lower SES). The estimates refer to the average change in the outcome value between two cohorts born 10 years apart divided by the within-cohort standard deviations, after cont 3 Results TABLE 2 Relative secular trend estimates (with 95% confidence interval) for height, weight, head circumference, bone age, and full scale IQ at four different ages (4, 9, 14, and 18 years). secular trend estimates (with 95% confidence interval) for height, weight, head circumference, bone age, and full scale IQ at four 9, 14, and 18 years). different ages (4, 9, 14, and 18 years). Age 4 years Age 9 years Age 14 years Age 18 years Height 0.35 (0.00; 0.69) 0.46 (0.11; 0.82) 0.38 (0.03; 0.72) 0.09 (−0.28; 0.45) Weight 0.27 (−0.07; 0.61) 0.51 (0.18; 0.85) 0.51 (0.17; 0.84) 0.36 (0.00; 0.73) Head circumference 0.01 (−0.35; 0.37) 0.24 (−0.09; 0.58) 0.17 (−0.18; 0.53) −0.04 (−0.41; 0.32) Bone age Not available Not available 0.54 (0.15; 0.93) 0.38 (−0.01; 0.77) Full scale IQ 0.54 (0.17; 0.92) −0.54 (−0.90; −0.17) −0.60 (−1.00; −0.21) Not available The estimates refer to the average change in the outcome value between two cohorts born 10 years apart divided by the within-cohort standard deviations, after controlling for sex and SES. 04 Frontiers in Public Health frontiersin.org Eichelberger et al. 10.3389/fpubh.2024.1216164 FIGURE 1 Relative secular trend estimates for height, weight, head circumference, bone age and full scale IQ at four different ages (4, 9, 14, and 18 years). The average relative change over 10 years can be interpreted as a standardized difference in means [Cohen’s d; (41)], with values of 0.2, 0.5, and 0.8 referring to small, moderate, and large effects, respectively. FIGURE 1 Relative secular trend estimates for height, weight, head circumference, bone age and full scale IQ at four different ages (4, 9, 14, and 18 years). The average relative change over 10 years can be interpreted as a standardized difference in means [Cohen’s d; (41)], with values of 0.2, 0.5, and 0.8 referring to small, moderate, and large effects, respectively. secular trend for performance IQ at 9 years (0.61 SD decrease per decade) and for verbal IQ at 14 years (0.85 SD decrease per decade) whereas the negative secular trend estimates for verbal IQ at 9 years and performance IQ at 14 years were statistically insignificant (see Supplementary Table S1 for details). of 178 cm for men and 166 cm for women after 1970. 4 Discussion The aim of these analyses was to examine secular trends of growth measures, biological maturation, and intelligence concurrently during four developmental phases: at 4, 9, 14, and 18 years. The analyses examined data from participants of the third cohort of the ZLS, who were born between 1978 and 1993 and assessed between 1982 and 2011 [see (29) for details on the ZLS cohorts]. Notably, this is the first study to investigate secular trends in several traits during four phases of child development in a single cohort. Positive secular trends were observed in height and weight at 4, 9, and 14 years. In contrast, at age 18 years, the secular trend of weight remained similar while the secular trend of height became negligible. No secular trends were observed in head circumference at any of the ages or in birth weight. A secular trend in bone age was apparent at the age of 14 years while it was only marginally significant at 18 years. For intelligence, a positive secular trend was apparent at age 4 years. In contrast, a negative secular trend was found at ages 9 and 14 years. The positive secular trends that we found for weight in the current study are in line with those found in the adult population in Switzerland (45). This likely reflects the growing obesity epidemic worldwide (46), including Switzerland (45). Only since about 2012, after the cohort studied here reached adulthood, has the secular trend in children’s weight leveled off (8, 47, 48). In line with recent studies, we found no evidence for a secular trend in birth weight, an important parameter of child growth in utero. Birth weight was monitored in Norway in registry studies between 1860 and 1984. It declined from 1860 to 1900 and increased again by about 150 g between 1900 and 1940, but it has remained largely constant since then (49). Birth weight has also changed little over recent decades in other countries (49–51). In contrast to a number of previous studies that have shown an increase in head circumference increased in recent decades [see (52) for a review], we did not observe a secular trend in head circumference. A positive secular trend in height was found in this cohort of Swiss children and adolescents. In contrast, no secular height trend was observed at 18 years, which is in agreement with Vinci et al. (43). 3 Results A possible reason for the ongoing secular trend in children and adolescents is that such changes are still present in the pubertal growth spurt in adolescence and in the developmental tempo during childhood (21, 44). In fact, we also found a secular trend in skeletal maturity as measured by bone age, which is considered a reliable indicator of the maturational tempo of growth (32). Positive secular trends in bone age measures between the 1960s and 2000 have been reported in two previous studies from Taiwan and South Africa (27, 28). The pattern in the ZLS-3 of a positive secular height trend in childhood and adolescence in the absence of such a trend in young adulthood may be due to the fact that children and adolescents born in more recent years (1990s) were biologically more mature (i.e., showed earlier maturation) compared to individuals born longer ago (1970s).h Secular trend estimations for height and weight at 14 years became non-significant when adjusted for biological maturation (quantified as bone age at 14 years) while the positive secular trend remained similar for IQ (Supplementary Table S2). Frontiers in Public Health frontiersin.org 4 Discussion Although two large meta- analyses have reported average Flynn effects of about three IQ points per decade since the 1930s (3, 4), the secular trend in intelligence has not always been linear, and seems to have slowed so much over the last 30 years that it has even become negative. A systematic review in 2016 described this reverse Flynn effect in seven European countries among individuals tested between 1975 and 2012 (9). A more recent review found secular IQ declines in even more countries in Africa, Europe, and North and South America (65).ht The Netherlands are often described as the country that experienced an IQ decline as early as 1975 [e.g., (19)]. In fact, analysis of secular trends in this country between 1968 and 2005 showed a pattern very similar to the findings of our study; although IQ was quite stable among preschoolers, a negative secular trend emerged among the 14-year-olds (19). While improvements in education may have contributed to raises in IQ in the past (56–58), the IQ decline observed in our cohort (and in other’s) is unlikely to be attributable to changes in the educational structures in Switzerland as they have improved over the course of the 20th century rather than worsened. For example, the average class size declined steadily from 55 to 20 students per class between 1870 and 1980 (66, 67). Also, the sharp decline within a relatively short period of time cannot be attributed to a poorer educational system. Similarly, other factors related to the living standard that may have previously contributed to the positive secular trends in IQ, such as improved nutrition, hygiene status or the medical care system (1), are unlikely to have declined in Switzerland since the mid-1970s, when the participants of the current study were born. Thus, they cannot explain the negative secular trend in IQ seen in the current study at 9 and 14 years. In fact, Rodgers argued that the same factors contributing to positive secular trends may be implicated in the slowing and flattening of these trends, however, they certainly cannot be responsible for their turn-around. Rather, an entirely different set of explanations may require consideration (10). Exploring the reasons for the secular trend pattern in IQ that we found in the current study is challenging: Differential secular trends became apparent at 4 years (positive trend) and at 9 and 14 years (negative trends). 4 Discussion These authors described a steady increase in the height of young Swiss adults after 1937 which slowed and nearly stopped at an average height Together, the findings of positive secular trends at most developmental phases in height, weight and biological maturation, of no secular trends in head circumference as well as in birthweight and the differential secular trends at different developmental phases in 05 frontiersin.org 10.3389/fpubh.2024.1216164 Eichelberger et al. intelligence make it unlikely that any single environmental factor is the primary driver of these changes. improved abilities and gain access to progressively superior resources, thereby amplifying their own children’s intellectual growth. Several studies have, indeed, provided evidence for a parental contribution to secular trends in IQ (61–64). Swiss parents have likely also become increasingly involved in supporting their children’s development and have created more stimulating and nurturing environments for their young children, leading to the positive secular trend in early childhood that was found in the current study. The hypotheses that have been put forward to explain the positive secular trends in various traits across the 20th century include, among others, improved nutritional quality and quantity [i.e., “nutrition hypothesis” advocated by Lynn (12, 53, 54)], better health and health care (55), and advances in education and the educational system (56– 58). It has been argued that high-income countries, including Switzerland, are currently approaching an asymptote: with environmental improvements having unfolded over past decades, large parts of the population have now reached their genetic and biological potential, and any further improvements in those environmental conditions may no longer result in an upwards trend in those traits (1, 9). This “environmental saturation effect” was formalized by Irving Gottesman’s genome-environment interaction model (59), postulating a maximum developmental potential that can be realized to a large extent if living conditions are favorable. In fact, such an effect may be reasonably assumed to underlie the secular trends in some of the growth parameters assessed in the current study, most notably in height (i.e., no secular trend in adult height measured at 18 years) and head circumference (i.e., no secular trend at any developmental phase). The negative secular trend that we found in 9- and 14-year-olds is consistent with recent studies, especially in the European Nordic countries [see, e.g., (9) for a general review]. 4 Discussion To thoroughly discuss this finding, first, a methodological issue must be considered, namely, the use of different instruments. At 4 years, the SON was used, a nonverbal test, providing a single IQ score, without any further distinction into different IQ domains (13). In contrast, at 9 and 14 years, the WISC-R was used, and its full-scale IQ combines estimates of verbal and performance IQ (36, 37). Previously, larger IQ gains have been described for fluid or performance IQ than for crystallized or verbal IQ (3). This raises the question whether the different aspects of IQ captured by the SON and the WISC-R could explain the differences in the secular trend patterns. When separately investigating the secular trends in verbal and performance IQ at 9 and 14 years in the current study, a negative trend was observed in both IQ domains, although only reaching statistical significance for performance IQ at 9 years and for verbal IQ at 14 years. Performance IQ may be considered conceptually overlapping with fluid IQ, including some very similar subtests (e.g., mosaics and block design in the SON and the WISC-R, respectively). This suggests the differential directions of the secular trends at 4 years (positive trend assessed with SON) versus at 9 and at 14 years (negative trend assessed with WISC-R) are, likely, not explained solely by the different tests used at different developmental periods. Alternative lines of explanation are discussed in the following.h f p y q One new explanation that has been brought forward to explain the observed negative secular trends in IQ in recent decades is that of dysgenic effects: disadvantageous genetic effects of higher reproduction rates of low-ability as opposed to high-ability individuals within populations (68–70). However, this cannot be responsible for the negative secular trends observed in 9- and 14-year-olds because of the positive trend at 4 years in the identical cohort. In any case, selection-driven genetic changes in the population of the ZLS-3 are implausible considering the relatively short time span and the large magnitude of the effect. Moreover, because the sample is relatively homogenous and born in the greater Zurich area, changes in the sample composition through immigration or methodological artifacts due to, for instance, recruitment biases between cohorts can be excluded. The positive secular trend in intelligence at 4 years may have resulted from changes in child rearing and parenting from the 1970s onwards. Frontiers in Public Health Ethics statement The studies involving humans were approved by Ethical Committee of the Canton of Zurich, Switzerland (Basec-Nr. 2018- 00686). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. 4 Discussion This idea was summarized by Rodgers and O’Keefe (60) in their integrative theory termed ‘Parental Executive Model’. Their theory posits that parents are in general highly motivated to facilitate their children’s intellectual development, and consequently that they use resources available to them, namely, those that have previously been proposed by other theories to explain the Flynn effect, such as improved nutrition, education, technology, healthcare, and so forth. Additionally, a cross-generational feedback loop exists, whereby children who are “better nurtured for intelligence” (10, p. 5) have 06 frontiersin.org Eichelberger et al. 10.3389/fpubh.2024.1216164 Eichelberger et al. It has further been suggested that fluid and crystallized IQ undergo differential secular trends (e.g., illustrated in 3, Figure 1) and that the respective divergence may contribute to varying trend patterns (10). While our data showed no differential effect for verbal and performance IQ, it cannot contribute to this exact discussion as the assessment instruments are not designed to distinguish between fluid and crystallized IQ. Further studies are needed to systematically investigate this, alongside identifying other potential explanatory processes underlying the differential patterns of secular trends across different traits, including IQ. use same-aged control groups from similar birth years. Such an approach is critical to ensure that interpretations of cohort differences (e.g., due to improved medical care or after interventions) are not confounded by secular trends [e.g., (73, 74)]. Overall, the findings of the current analyses highlight the need for further research to monitor secular trends of human characteristics. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. As already outlined above, the ultimate cause of secular trends suggested by Flynn (13)—the industrial revolution, economic progress, educational standards, and the complexity of society—may have reached a maximum since the 1980s, at least in developed countries, and the upper limit of children’s developmental and intellectual capacities may have been reached in recent decades. Consequently, any further changes in the environment may now result in a weakening (i.e., slowing/flattening) or in random increases and declines of secular trends. Frontiers in Public Health 5 Strengths and limitations A particular strength of this study is the use of longitudinal data with a consistent sample composition over time. Furthermore, the use of in-sample standardization when defining secular trend estimates enhanced the comparability of estimates across traits because the within-cohort variability s j at age j is not affected by secular trends and is estimated in the same sample for all traits. Author contributions DE contributed to the formulation and development of the research goals, conceived and designed the analysis of the data samples, contributed to data collection of some samples, drafted and edited the manuscript. AC performed the statistical analysis of the entire data set, drafted and edited the manuscript. VR supervised the statistical analysis of the data, reviewed and edited the manuscript. TK contributed to the formulation and development of the research goals, and reviewed and edited the manuscript. JC contributed to the formulation and development of the research goals, performed data collection of the children and adolescents, reviewed, and edited the manuscript. OJ is the principal investigator of the Zurich Longitudinal Studies (ZLS), contributed to the formulation and development of the research goals, and drafted and edited the manuscript. FW is the current project leader of the ZLS, contributed to the formulation and development of the research goals, and drafted and edited the manuscript. All authors contributed to the article and approved the submitted version. The current study is limited by a relatively small sample size and a limited range of birth years between 1978 and 1993. However, previous studies were also able to identify secular trends in various traits across comparable ranges [e.g., (17, 71, 72)]. The cohort included in the current analyses (ZLS-3) consists of the offspring of the ZLS-1 cohort. This unique study setting makes it challenging to replicate the findings in an independent sample. Further, the assessment of IQ with different tests at different developmental phases complicates the comparison of the full-scale IQs across ages. 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Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh.2024.1216164/ full#supplementary-material The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 6 Conclusion The ZLS have been or are funded by the Swiss National Science Foundation (32473B_129956 and 32003B-112324), the International Children’s Center in Paris, the Department of Education of the Canton of Zurich, Switzerland, the University of Zurich, Switzerland, the Hermann Klaus-Stiftung, the Hartmann Müller-Stiftung, the Remo Largo Stiftung für Entwicklungspädiatrie, the Foundation for Research in Science and the Humanities at the University of Zurich, the Maiores Stiftung, the Baugarten Stiftung, the Children’s Research Center of the University Children’s Hospital Zurich, the Velux Stiftung, and the Stiftung Für das Kind Giedion Risch. 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https://openalex.org/W4230983267	https://www.qeios.com/read/IMBFY1/pdf	English	null	Concomitant Medication Use Duration	Definitions	2,020	cc-by	58	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Concomitant Medication Use Duration National Cancer Institute Qeios ID: IMBFY1 · https://doi.org/10.32388/IMBFY1 Source National Cancer Institute. Concomitant Medication Use Duration. NCI Thesaurus. Code C83224. The period of time from start to finish of concomitant medication usage. Qeios ID: IMBFY1 · https://doi.org/10.32388/IMBFY1 1/1
https://openalex.org/W2978805953	https://ejournal.undip.ac.id/index.php/ksa/article/download/24796/15853	Indonesian	null	Kinetics of Transesterification Reaction of Soybean Oil into Biodiesel with CaO Catalyst	Jurnal Kimia Sains dan Aplikasi/Jurnal kimia sains dan aplikasi	2,019	cc-by-sa	4,745	Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 ISSN: 1410-8917 e-ISSN: 2597-9914 ISSN: 1410-8917 e-ISSN: 2597-9914 Kata Kunci: katalis CaO; fluidized bed; CSTR; kecepatan reaksi; minyak kedelai; transesterifikasi Abstract Title: Kinetics of Transesterification Reaction of Soybean Oil into Biodiesel with CaO Catalyst The use of biodiesel as an alternative fuel is expected to reduce dependence on fossil fuels. This study aims to examine the kinetics of the transesterification reaction of soybean oil with methanol using a heterogeneous CaO solid base catalyst to determine the corresponding reaction rate law equation. Testing the kinetics of the reaction to determine the corresponding reaction rate law equation was fitted in the transesterification reaction of soybean oil and methanol with a solid CaO catalyst. and reaction time (for taking product concentration data) of 180 minutes. The results of dependency analysis of the reaction rate to the reactants showed that methanol was adsorbed on the surface of the catalyst and triglycerides were not adsorbed on the surface of the catalyst, so that the possible reaction mechanism of the catalytic reaction follows the Eley-Rideal mechanism. Final equation of the reaction rate law (- rA) model after synchronizing the equation to experimental kinetics reaction data is: - rA=(4,7901(0,7240CACB-(CCCD/248,2845)))/(0,7240CB+(CC/2,4342)+1). The final reaction rate law model can be used in reactor design (the relationship between the weight of the catalyst needed to achieve a certain triglyceride conversion and predicting the reactor volume required). Keywords: CaO catalyst; fluidized bed; CSTR; reaction rate law; soybean oil; transesterification Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 213 Kinetika Reaksi Transesterifikasi Minyak Kedelai Menjadi Biodiesel dengan Katalis CaO Setiarto Pratigto a,1, I. Istadi a,2 Setiarto Pratigto a,1, I. Istadi a,2 a Departemen Teknik Kimia, Fakultas Teknik Universitas Diponegoro, Jl. Prof. Soedarto, SH. Kampus UNDIP Tembalang, Semarang. Indonesia 50275 * Corresponding author: (1) setiartopratigto@students.undip.ac.id (2) istadi@che.undip.ac.id 1. Pendahuluan Biodiesel adalah senyawa ester asam lemak yang dihasilkan dari proses transesterifikasi minyak (trigliserida) maupun esterifikasi asam lemak yang berasal dari minyak nabati atau hewani dengan alkohol rantai pendek. Kandungan asam lemak bebas atau free fatty acid (FFA) bahan baku merupakan salah satu faktor penentu jenis proses pembuatan biodiesel. Minyak murni (refined vegetable oil) pada umumnya, memiliki kadar FFA rendah (< 2%) sehingga dapat langsung diproses dengan metode transesterifikasi. Jika kadar FFA minyak tersebut masih tinggi (>2%), sebelumnya perlu dilakukan proses esterifikasi. Berdasarkan kondisi tersebut, proses produksi biodiesel perlu dilakukan analisis kadar FFA terlebih dahulu. Bahan baku dalam pembuatan biodiesel misalnya minyak goreng bekas [1], minyak jarak [2], minyak kacang tanah [3], minyak sawit [4], minyak kelapa, minyak kedelai [5, 6, 7], minyak biji kapuk, minyak biji karet, minyak kemiri sunan, minyak rapeseed [8], dan minyak bunga matahari [9]. Pada penelitian ini digunakan minyak kedelai murni (refined soybean oil) karena kajian ini hanya berfokus pada kinetika reaksi transesterifikasi, namun bukan pada pengembangan prosesnya. Penelitian ini mengkaji kinetika reaksi transesterifikasi (penentuan persamaan hukum kecepatan reaksi) minyak kedelai dengan metanol menggunakan katalis kalsium oksida dengan memperhatikan parameter-parameter suhu reaksi, rasio mol reaktan, waktu reaksi, dan konsentrasi katalis terhadap konversi trigliserida. Hasil analisis ketergantungan kecenderungan kecepatan reaksi terhadap masing-masing perbandingan reaktan akan menentukan mekanisme reaksi katalisis (adsorpsi, desorpsi, reaksi permukaan) yang sesuai sehingga dapat menentukan model bentuk persamaan kecepatan reaksi transesterifikasi. Oleh karena itu, penelitian ini bertujuan untuk mengkaji kinetika reaksi transesterifikasi minyak kedelai menjadi biodiesel menggunakan katalis padat CaO untuk mendapatkan model persamaan kecepatan reaksi heterogen. Kajian kinetika penelitian ini meliputi: prediksi mekanisme yang sesuai dengan data eksperimen melalui analisis ketergantungan perubahan laju reaksi terhadap perubahan masing-masing reaktan dan perumusan persamaan akhir laju reaksi. Persamaan laju reaksi yang dihasilkan dapat dipakai pada desain perancangan reaktor. Penggunaan katalis homogen memiliki kekurangan sebab katalis terlarut sempurna dalam gliserol dan larut sebagian dalam biodiesel sehingga sulit dipisahkan pada proses pemurnian produk. Katalis heterogen lebih mudah dalam pemisahan dan pemurnian produk biodiesel yang dapat mengurangi biaya produksi biodiesel, dapat digunakan kembali, eco-friendly, dan ramah lingkungan [10]. Kalsium oksida (CaO) banyak digunakan untuk reaksi transesterifikasi, karena memiliki kekuatan basa yang relatif tinggi, ramah lingkungan, kelarutan yang rendah dalam metanol, dan dapat disintesis dari sumber yang murah seperti batu kapur, kalsium hidroksida, batu gamping, dan yang lainnya yang mengandung kalsium karbonat (CaCO3; mineral kalsit) [11, 12]. 2.2. Pembuatan dan Karakterisasi Katalis CaO Proses pembuatan katalis CaO mengikuti metode yang dilakukan oleh Kouzu dkk. [5] dengan cara kalsinasi padatan CaCO3 dalam furnace pada suhu 800°C selama 2 jam. Setelah dikalsinasi selama 2 jam, padatan CaO dibiarkan mencapai suhu kamar 27°C dan dimasukkan ke dalam desikator untuk mencegah terjadinya kontak antara permukaan katalis dengan uap air di dalam ruangan, yang mengakibatkan menurunnya kekuatan basa katalis. Luas permukaan katalis, volume pori total, dan ukuran pori dianalisis dengan metode Barrett, Joyner, Halenda (BJH) menggunakan peralatan Quantachrome NOVA tipe 1200e, struktur kristal dan kristalinitas menggunakan metode X-ray Difraction (XRD) (Shimadzu), dan morfologi permukaan katalis dianalisis menggunakan Scanning Electron Microscopy (SEM) (JEOL). Abstrak Kata Kunci: katalis CaO; fluidized bed; CSTR; kecepatan reaksi; minyak kedelai; transesterifikasi Penggunaan biodiesel sebagai bahan bakar alternatif diharapkan dapat mengurangi ketergantungan pada bahan bakar fosil. Penelitian ini bertujuan mengkaji kinetika reaksi transesterifikasi minyak kedelai dengan metanol menggunakan katalis basa padat heterogen CaO untuk menentukan persamaan kecepatan reaksi yang bersesuaian. Pengujian kinetika reaksi untuk menentukan persamaan kecepatan reaksi yang bersesuaian diuji pada reaksi transesterifikasi minyak kedelai dan metanol dengan katalis padat CaO meliputi analisis ketergantungan kecepatan reaksi terhadap perbandingan mol reaktan pada parameter proses suhu reaksi 60°C, berat katalis 3% (%b/v), dan waktu reaksi (untuk pengambilan data konsentrasi produk) 180 menit. Hasil analisis ketergantungan reaktan-reaktan menunjukkan bahwa metanol teradsorpsi di permukaan katalis dan trigliserida tidak teradsorpsi di permukaan katalis, sehingga mekanisme reaksi katalitik mungkin terjadi mengikuti mekanisme Eley-Rideal. Bentuk persamaan kecepatan akhir laju reaksi (-rA) setelah sinkronisasi model persamaan dengan data-data eksperimen kinetika reaksi adalah: - rA=(4,7901(0,7240CACB-(CCCD/248,2845)))/(0,7240CB+(CC/2,4342)+1). Persamaan Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 214 akhir kecepatan reaksi tersebut dapat digunakan dalam perancangan reaktor (hubungan antara berat katalis yang diperlukan untuk mencapai konversi trigliserida tertentu dan memprediksi volume reaktor). akhir kecepatan reaksi tersebut dapat digunakan dalam perancangan reaktor (hubungan antara berat katalis yang diperlukan untuk mencapai konversi trigliserida tertentu dan memprediksi volume reaktor). Penelitian-penelitian tersebut tidak mengkaji prediksi mekanisme reaksi yang terjadi. 1. Pendahuluan Katalis CaO dapat digunakan pada reaksi transesterifikasi karena sifat basa yang kuat [13, 14]. Katalis CaO dapat dihasilkan dari dekomposisi CaCO3 pada suhu yang tinggi [14]. Peneliti- peneliti sebelumnya tentang penggunaan CaO sebagai katalis pada proses produksi biodiesel belum memfokuskan kajiannya pada kinetika untuk memperoleh persamaan kecepatan reaksi heterogen yang memperhatikan fenomena-fenomena adsorpsi-desorpsi reaktan dan/atau produk, namun kebanyakan masih bersifat global atau terlalu sederhana. Pendekatan kinetika penelitian ini berbeda dengan peneliti sebelumnya yang kebanyakan merumuskan kinetika kecepatan reaksi berdasarkan hasil pengamatan eksperimen secara empiris menggunakan asumsi model kinetika reaksi katalisis yang terlalu sederhana (misalnya: simple dan Power Law) [15, 16, 17, 18, 19]. 2.1. Bahan Bahan-bahan penelitian yang digunakan dalam penelitian ini adalah minyak kedelai 100%, CaCO3 (Merck, 99%), dan Metanol (99,9%, Merck). 3.1. Karakterisasi Katalis Gambar 1. Rangkaian alat reaksi transesterifikasi: (1) labu leher tiga, (2) heating mantle, (3) termometer, (4) motor penggerak, (5) motor controller, (6) condenser reflux, (7) elbow, (8) pengaduk, (9) statif dan klem, (10) adapter, dan (11) rangka portable. Katalis CaO yang digunakan pada transesterifikasi minyak kedelai menjadi biodiesel bersifat basa kuat dan mempunyai kristalinitas yang baik jika dikalsinasi pada suhu 800°C dengan struktur dominan adalah CaO disamping masih ada struktur lainnya (CaCO3) dan Ca(OH)2 (berdasarkan hasil karakterisasi XRD pada Gambar 2) [20, 21]. Katalis CaO ini juga mempunyai mempunyai morfologi permukaan yang seragam berdasarkan pada hasil pengujian SEM (Gambar 3). Katalis CaO ini mempunyai luas permukaan 37,983 m3/g, volume pori 0,141 cm3/g, dan radius pori 19,084 Å berdasarkan pengujian menggunakan metode BJH. Logam oksida dengan luas permukaan 37,983 m3/g dapat menjamin bahwa reaksi transesterifkasi dapat berjalan dengan baik di permukaan katalis dan di dalam pori katalis. Gambar 1. Rangkaian alat reaksi transesterifikasi: (1) labu leher tiga, (2) heating mantle, (3) termometer, (4) motor penggerak, (5) motor controller, (6) condenser reflux, (7) elbow, (8) pengaduk, (9) statif dan klem, (10) adapter, dan (11) rangka portable. Gambar 2. Struktur kristal katalis CaO dengan X-ray Diffraction Gambar 3. Morfologi permukaan katalis CaO dengan SEM 3 2 Analisis Ketergantungan Kecepatan Reaksi 2θ (o) Intensity (a.u.) Gambar 2. Struktur kristal katalis CaO dengan X-ray Diffraction 2θ (o) Intensity (a.u.) Gambar 2. Struktur kristal katalis CaO dengan X-ray Diffraction 2θ (o) Intensity (a.u.) Minyak kedelai, metanol, dan katalis CaO dimasukkan ke dalam labu leher tiga. Perbandingan mol umpan trigliserida (A) terhadap metanol (B) bervariasi sesuai variabel penelitian pada analisis ketergantungan terhadap reaktan. Larutan dipanaskan sampai suhu stabil pada 60°C (di bawah titik didih metanol 64,7°C) dan diaduk selama 3 jam. Produk didinginkan dan dipisahkan pada corong pemisah, didiamkan semalam hingga terbentuk 3-4 lapisan, yaitu: lapisan atas metanol sisa, lapisan tengah metil ester, dan lapisan bawah sisa minyak kedelai, gliserol, dan katalis. Produk metil ester dianalisis menggunakan GC-MS. Hasil reaksi transesterifikasi berupa konsentrasi fatty acid methyl ester (FAME) diuji dengan kromatografi gas-spektrometri massa (GC-MS) merek Shimadzu. Gambar 2. Struktur kristal katalis CaO dengan X-ray Diffraction Gambar 2. Struktur kristal katalis CaO dengan X-ray Diffraction Gambar 3. Morfologi permukaan katalis CaO dengan SEM 2.3. Pengujian Katalis untuk Reaksi Transesterifikasi dengan Katalis CaO Gambar 1 menunjukkan rangkaian alat reaksi transesterifikasi untuk pengujian katalis dalam reaktor Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 215 fluidized CSTR, yang terdiri dari labu leher tiga (1), yang dilengkapi dengan pengaduk (8), kondenser refluks (6), elbow (7), adapter (10) dan termometer (3). Pengaduk digerakkan oleh motor penggerak (4) dengan pengatur motor controller (5). Rangkaian diletakkan pada pemanas mantel (M-Top Heating Mantle MS-E) (2) yang berada di atas rangka portable (11) yang dilengkapi dengan statif dan klem (9). Selang air pendingin menghubungkan ujung bawah dan atas kondenser refluks. ini, hubungan antara berat katalis yang dibutuhkan oleh reaksi dan konversi trigliserida menjadi biodiesel dapat diketahui sehingga jumlah kebutuhan katalis untuk mencapai tingkat konversi tertentu dapat diprediksi. Lebih jauh lagi, perhitungan volume reaktor yang dibutuhkan untuk mencapai konversi reaksi tersebut dapat dilakukan. 2.4. Pengujian Kinetika Reaksi untuk Memperoleh Model Persamaan Kecepatan Reaksi Kajian kinetika reaksi meliputi: (1). analisis ketergantungan perubahan laju reaksi terhadap perubahan perbandingan masing-masing reaktan; (2) prediksi mekanisme reaksi yang sesuai dengan data eksperimen; dan (3). perumusan persamaan akhir kecepatan reaksi. Dari analisis ketergantungan kecepatan reaksi (-rA) terhadap konsentrasi reaktan diperoleh prediksi model kecepatan reaksi. Model persamaan kecepatan reaksi ini kemudian dilakukan curve fitting terhadap data-data eksperimen (hubungan antara variasi konsentrasi dengan kecepatan reaksi) menggunakan aplikasi Polymath (dipilih berdasarkan koefisien determinasi R2 yang paling besar), sehingga didapatkan besarnya parameter1-parameter model. Model persamaan kecepatan reaksi akhir yang dihasilkan dapat dipakai pada desain perancangan reaktor. Dalam tahapan Gambar 3. Morfologi permukaan katalis CaO dengan SEM 3.2. Analisis Ketergantungan Kecepatan Reaksi terhadap Reaktan pada Pengujian Transesterifikasi Reaktan minyak kedelai (trigliserida) dan metanol bereaksi melewati katalis padat CaO menghasilkan biodiesel (metil ester) dan gliserol menurut persamaan reaksi berikut: Run Rasio molar CA CB 𝐶𝐶 𝐶𝐷 -rA exp, ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡.𝑠) A B Trigliserida (mol/L) Metanol (mol/L) Biodiesel (mol/L) Gliserol (mol/L) 1 0,3 6 0,20 4,09 0,53 3,31 3,26 x 10-6 2 0,4 6 0,29 4,31 0,52 3,26 2,90 x 10-6 3 0,6 6 0,44 4,40 0,59 2,93 2,83 x 10-6 4 0,75 6 0,57 4,53 0,60 2,88 2,71 x 10-6 5 1 6 0,80 4,83 0,60 2,82 2,56 x 10-6 6 2 6 1,61 4,84 0,58 2,77 2,25 x 10-6 7 3 6 2,44 4,90 0,60 2,71 2,22 x 10-6 Kondisi pada pengujian reaktor batch dengan katalis 3% CaO yang digunakan pada penelitian ini adalah suhu reaksi 60°C dan waktu reaksi 180 menit. Tabel 1 menunjukkan bahwa pada run nomor 4, 5, 6 dan 7, konsentrasi metanol (CB) menurun dan kecepatan reaksi (-rA) juga menurun. Begitu juga pada run nomor 1, 2 dan 3, konsentrasi metanol (CB) menurun, sedangkan kecepatan reaksi (-rA) juga menurun. Penurunan kecepatan reaksi (-rA) diakibatkan oleh menurunnya konsentrasi metanol (CB) ini menunjukkan bahwa metanol teradsorpsi pada permukaan katalis pada saat reaksi berlangsung (berdasarkan pernyataan Fogler [22]). Hasil analisis dari kedua tabel menyatakan bahwa metanol teradsorpsi di permukaan katalis dan trigliserida tidak teradsorpsi di permukaan katalis, menunjukkan bahwa mekanisme reaksi katalitik yang terjadi adalah mekanisme Eley-Rideal di mana salah satu reaktan teradsorpsi pada permukaan katalis. Oleh karena itu, reaksi yang mungkin terjadi adalah metanol (B) teradsorpsi pada permukaan katalis bereaksi dengan trigliserida (A) pada fase cairnya menghasilkan metil ester (C) dan gliserol (D). Tabel 1. Data ketergantungan kecepatan reaksi (-rA) terhadap konsentrasi reaktan metanol (CB) pada konsentrasi trigliserida (CA) relatif konstan Tabel 1. 3.2. Analisis Ketergantungan Kecepatan Reaksi terhadap Reaktan pada Pengujian Transesterifikasi Pengujian katalis dalam rangka kajian kinetika reaksi transesterifikasi dilakukan pada reaktor batch, sedangkan konsentrasi produk dipantau pada saat reaksi dianggap Jurnal Kimia Sains dan A sudah mencapai keadaan tunak (steady state) pada kondisi perbandingan umpan yang berbeda-beda. Reaktan minyak kedelai (trigliserida) dan metanol bereaksi melewati katalis padat CaO menghasilkan biodiesel (metil ester) dan gliserol menurut persamaan reaksi berikut: Trigliserida + 3 Metanol 3 Metil ester + Gliserol (1) A B C D Kondisi pada pengujian reaktor batch dengan katalis 3% CaO yang digunakan pada penelitian ini adalah suhu reaksi 60°C dan waktu reaksi 180 menit. Tabel 1 menunjukkan bahwa pada run nomor 4, 5, 6 dan 7, konsentrasi metanol (CB) menurun dan kecepatan reaksi (-rA) juga menurun. Begitu juga pada run nomor 1, 2 dan 3, konsentrasi metanol (CB) menurun, sedangkan kecepatan reaksi (-rA) juga menurun. Penurunan kecepatan reaksi (-rA) diakibatkan oleh menurunnya konsentrasi metanol (CB) ini menunjukkan bahwa metanol teradsorpsi pada permukaan katalis pada saat reaksi berlangsung (berdasarkan pernyataan Fogler [22]). Tabel 1. Data ketergantungan kecepatan reaksi (-rA) terhadap konsentrasi reaktan metanol (CB) pada konsentrasi trigliserida (CA) relatif konstan Run Rasio molar CA CB 𝐶𝐶 𝐶𝐷 -rA exp, ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡.𝑠) A B Trigliserida (mol/L) Metanol (mol/L) Biodiesel (mol/L) Gliserol (mol/L) 1 1 20 1,70 34,06 1,59 6,19 9,85 x 10-6 2 1 15 1,91 28,68 1,72 5,59 9,48 x 10-6 3 1 10 2,20 21,95 1,97 4,51 9,46 x 10-6 4 1 8 2,32 18,59 2,08 4,13 9,44 x 10-6 5 1 6 2,48 14,87 2,20 3,64 9,39 x 10-6 6 1 3 2,74 8,23 2,42 2,33 9,31 x 10-6 7 1 2 2,85 5,69 2,48 2,28 9,22 x 10-6 Evaluasi yang sama dilakukan untuk mengetahui kecenderungan hasil yang diperoleh pada berbagai variasi konsentrasi trigliserida pada konsentrasi metanol yang relatif konstan. Dengan asumsi bahwa ukuran partikel katalis dalam menentukan kecepatan reaksi dikendalikan oleh reaksi permukaan, bukan adsorpsi dan desorpsi. Sehingga ukuran partikel sekecil mungkin tidak mempengaruhi hasil tersebut dalam hal ini adsorpsi dan desorpsi diabaikan. Pada Tabel 2 run nomor 1 sampai 7, konsentrasi trigliserida (CA) meningkat tidak signifikan, sedangkan konsentrasi metanol (C ) relatif konstan Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 216 Tabel 2. Data ketergantungan kecepatan reaksi (−rA) terhadap konsentrasi reaktan trigliserida (CA) pada konsentrasi metanol (CB) relatif konstan sudah mencapai keadaan tunak (steady state) pada kondisi perbandingan umpan yang berbeda-beda. 3.2. Analisis Ketergantungan Kecepatan Reaksi terhadap Reaktan pada Pengujian Transesterifikasi Data ketergantungan kecepatan reaksi (-rA) terhadap konsentrasi reaktan metanol (CB) pada konsentrasi trigliserida (CA) relatif konstan Run Rasio molar CA CB 𝐶𝐶 𝐶𝐷 -rA exp, ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡.𝑠) A B Trigliserida (mol/L) Metanol (mol/L) Biodiesel (mol/L) Gliserol (mol/L) 1 1 20 1,70 34,06 1,59 6,19 9,85 x 10-6 2 1 15 1,91 28,68 1,72 5,59 9,48 x 10-6 3 1 10 2,20 21,95 1,97 4,51 9,46 x 10-6 4 1 8 2,32 18,59 2,08 4,13 9,44 x 10-6 5 1 6 2,48 14,87 2,20 3,64 9,39 x 10-6 6 1 3 2,74 8,23 2,42 2,33 9,31 x 10-6 7 1 2 2,85 5,69 2,48 2,28 9,22 x 10-6 Mekanisme reaksi permukaan yang mungkin terjadi adalah: ✓ A fase cair + B teradsorpsi C teradsorpsi + D fase cair (tipe 1) ✓ A fase cair + B teradsorpsi C teradsorpsi + D fase cair (tipe 1) ✓ A fase cair + B teradsorpsi C fase cair + D teradsorpsi (tipe 2) ✓ A fase cair + B teradsorpsi C fase cair + D teradsorpsi (tipe 2) Model kinetika reaksi katalitiknya dapat disusun sebagai berikut: Evaluasi yang sama dilakukan untuk mengetahui kecenderungan hasil yang diperoleh pada berbagai variasi konsentrasi trigliserida pada konsentrasi metanol yang relatif konstan. Dengan asumsi bahwa ukuran partikel katalis dalam menentukan kecepatan reaksi dikendalikan oleh reaksi permukaan, bukan adsorpsi dan desorpsi. Sehingga ukuran partikel sekecil mungkin tidak mempengaruhi hasil tersebut dalam hal ini adsorpsi dan desorpsi diabaikan. Pada Tabel 2 run nomor 1 sampai 7, konsentrasi trigliserida (CA) meningkat tidak signifikan, sedangkan konsentrasi metanol (CB) relatif konstan namun kecepatan reaksi semakin menurun. Kecenderungan ini menunjukkan bahwa trigliserida tidak teradsorpsi pada permukaan katalis pernyataan oleh Fogler[22]. 1. Reaksi tipe 1: A + B•S C•S + D c. Desorpsi C pada permukaan katalis S c. Desorpsi C pada permukaan katalis S Rasio molar 𝐹𝐴0 -rA exp X W (gcat) Trigliserida A Metanol B (mol trigliserida/s) ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡. 𝑠) 1 20 7,88 x 10-5 9,85 x 10-6 0,93 7,46 1 15 8,85 x 10-5 9,48 x 10-6 0,90 8,38 1 10 1,02 x 10-4 9,46 x 10-6 0,90 9,62 1 8 1,08 x 10-4 9,44 x 10-6 0,89 10,19 1 6 1,15 x 10-4 9,39 x 10-6 0,89 10,86 1 3 1,27 x 10-4 9,31 x 10-6 0,88 12,02 1 2 1,32 x 10-4 9,22 x 10-6 0,87 12,48 Gambar 2. Hubungan antara konversi trigliserida (X) dan berat katalis (W) yang dibutuhkan pada reaktor yang beroperasi sebagaimana fluidized CSTR. Kecepatan desorpsi C: Kecepatan desorpsi C: Rasio molar 𝐹𝐴0 -rA exp X W (gcat) Trigliserida A Metanol B (mol trigliserida/s) ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡. 𝑠) 1 20 7,88 x 10-5 9,85 x 10-6 0,93 7,46 1 15 8,85 x 10-5 9,48 x 10-6 0,90 8,38 1 10 1,02 x 10-4 9,46 x 10-6 0,90 9,62 1 8 1,08 x 10-4 9,44 x 10-6 0,89 10,19 1 6 1,15 x 10-4 9,39 x 10-6 0,89 10,86 1 3 1,27 x 10-4 9,31 x 10-6 0,88 12,02 1 2 1,32 x 10-4 9,22 x 10-6 0,87 12,48 −𝑟𝐴= 𝑘𝐷( 𝐾𝐵𝐾𝑆𝐶𝐴𝐶𝐵 𝐶𝐶 − 𝐶𝐷 𝐾𝐷) 𝐾𝐵𝐶𝐵 + 𝐾𝐵𝐾𝑆𝐶𝐴𝐶𝐵 𝐶𝐶 + 1 (6) (6) Analisis kesesuaian antara fitting model kecepatan reaksi ke data eksperimen untuk menentukan parameter- parameter model kecepatan reaksi adalah: (1) dependent variable: –rA, (2) independent variable: CA, CB, CC, CD, dan (3) parameter-parameter model: kB, kS, kC, kD, KB, KS, KC, KD, di mana −rA adalah kecepatan reaksi (mol/gcat.s), secara berurutan CA, CB, CC, CD adalah konsentrasi reaktan trigliserida (mol/L), konsentrasi reaktan metanol (mol/gcat), konsentrasi produk biodiesel (mol/L), konsentrasi produk gliserol (mol/L), sedangkan kB, kS, kC, kD secara berurutan adalah konstanta kecepatan adsorpsi B (s-1), konstanta kecepatan adsorpsi S (s-1), konstanta kecepatan adsorpsi C (s-1), konstanta kecepatan adsorpsi D (s-1). Sementara itu, KB, KS, KC, dan KD secara berurutan adalah konstanta kesetimbangan desorpsi B, konstanta kesetimbangan desorpsi S, konstanta kesetimbangan desorpsi C, dan konstanta kesetimbangan desorpsi D. Dari hasil Curve fitting menggunakan aplikasi Polymath, diperoleh nilai kS= 4,7901; KB= 0,7240; KS= 101,9984; dan KC= 2,4342, dengan nilai terdekat R2= 0,8835. Hasil dari curve fitting model persamaan kecepatan reaksi (-rA) pada data-data eksperimen diperoleh model persamaan kecepatan reaksi yang sesuai dengan Persamaan (2) adalah: Gambar 2. 1. Reaksi tipe 1: A + B•S C•S + D a. Adsorpsi B pada permukaan katalis S −𝑟𝐴= 𝑘𝐵(𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐵𝐾𝐶𝐾𝑆𝐶𝐴) 𝐶𝐶𝐶𝐷 𝐾𝐶𝐾𝑆𝐶𝐴 + 𝐶𝐶 𝐾𝐶 + 1 (1) (1) b. Reaksi permukaan katalis S Kecepatan reaksi permukaan: −𝑟𝐴= 𝑘𝑆(𝐾𝐵𝐶𝐴𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐶𝐾𝑆) 𝐾𝐵𝐶𝐵 + 𝐶𝐶 𝐾𝐶 + 1 −𝑟𝐴= 𝑘𝑆(𝐾𝐵𝐶𝐴𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐶𝐾𝑆) 𝐾𝐵𝐶𝐵 + 𝐶𝐶 𝐾𝐶 + 1 (2) (2) Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 217 c. Desorpsi C pada permukaan katalis S c. Desorpsi C pada permukaan katalis S dihasilkan tersebut Persamaan (7) dapat digunakan untuk perhitungan berat katalis yang dibutuhkan pada desain reaktor skala industri yang beroperasi sebagaimana reaktor fluidized CSTR saat keadaan steady. Implementasi dari hasil model persamaan kecepatan reaksi (-rA) dapat digunakan pada perhitungan desain reaktor terutama hubungan antara berat katalis yang dibutuhkan dengan laju alir umpan dan target konversi yang ingin dicapai, sesuai dengan neraca massa sebagai berikut: Kecepatan desorpsi C: Kecepatan desorpsi C: −𝑟𝐴= 𝑘𝐶( 𝐾𝐵𝐾𝑆𝐶𝐴𝐶𝐵 𝐶𝐷 − 𝐶𝐶 𝐾𝐶) 𝐾𝐵𝐶𝐵 + 𝐾𝐵𝐾𝑆𝐶𝐴𝐶𝐵 𝐶𝐷 + 1 (3) (3) 2. Reaksi tipe 2: A + B•S C + D•S 2. Reaksi tipe 2: A + B•S C + D•S 2. Reaksi tipe 2: A + B•S C + D•S a. Adsorpsi B pada permukaan katalis S a. Adsorpsi B pada permukaan katalis S In – Out + Generation = Accumulation (8) FA0 −FA + (−rA). W = 0 (Steady) (9) W = FA0− FA −rA (10) W = FA0.X −rA (11) In – Out + Generation = Accumulation (8) FA0 −FA + (−rA). W = 0 (Steady) (9) W = FA0− FA −rA (10) W = FA0.X −rA (11) Kecepatan adsorpsi B: (8) −𝑟𝐴= 𝑘𝐵(𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐵𝐾𝐷𝐾𝑆𝐶𝐴) 𝐶𝐶𝐶𝐷 𝐾𝐷𝐾𝑆𝐶𝐴 + 𝐶𝐷 𝐾𝐷 + 1 (4) −𝑟𝐴= 𝑘𝐵(𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐵𝐾𝐷𝐾𝑆𝐶𝐴) 𝐶𝐶𝐶𝐷 𝐾𝐷𝐾𝑆𝐶𝐴 + 𝐶𝐷 𝐾𝐷 + 1 (4) (10) (11) b. Reaksi permukaan katalis S b. Reaksi permukaan katalis S Dari persamaan (11) diperoleh hubungan berat katalis (W) sebagai fungsi dari konversi trigliserida (X) dengan menggunakan data Tabel 1, yang ditunjukkan pada Tabel 3 dan Gambar 2. Kecepatan reaksi permukaan: Kecepatan reaksi permukaan: −𝑟𝐴= 𝑘𝑆(𝐾𝐵𝐶𝐴𝐶𝐵 − 𝐶𝐶𝐶𝐷 𝐾𝐷𝐾𝑆) 𝐾𝐵𝐶𝐵 + 𝐶𝐷 𝐾𝐷 + 1 (5) (5) Tabel 3. Hubungan antara konversi trigliserida menjadi biodiesel (X) sebagai fungsi berat katalis (W) 4. Kesimpulan Pada penelitian ini, mekanisme reaksi yang paling mungkin terjadi pada reaksi transesterifikasi trigliserida dan metanol menjadi biodiesel menggunakan katalis CaO adalah mekanisme Eley Rideal, di mana metanol teradsorpsi di permukaan katalis CaO yang bereaksi dengan trigliserida (minyak kedelai) pada fase cairnya untuk menghasilkan produk biodiesel dan gliserol. Bentuk model persamaan kecepatan reaksi transesterifikasi minyak kedelai dan metanol menggunakan katalis CaO yaitu: −rA = 4,7901 (0,7240 CACB − CCCD (248,2845)) 0,7240 CB + CC 2,4342 + 1 . Persamaan ini dapat digunakan [5] Masato Kouzu, Takekazu Kasuno, Masahiko Tajika, Yoshikazu Sugimoto, Shinya Yamanaka, Jusuke Hidaka, Calcium oxide as a solid base catalyst for transesterification of soybean oil and its application to biodiesel production, Fuel, 87, 12, (2008) 2798- 2806 https://doi.org/10.1016/j.fuel.2007.10.019 [6] Yongsheng Lu, Zaiwu Zhang, Yunfeng Xu, Qiang Liu, Guangren Qian, CaFeAl mixed oxide derived heterogeneous catalysts for transesterification of soybean oil to biodiesel, Bioresource Technology, 190, (2015) 438-441 https://doi.org/10.1016/j.biortech.2015.02.046 dalam perancangan reaktor, sehingga hubungan antara konversi trigliserida menjadi biodiesel dengan kebutuhan berat katalis dan volume reaktor yang diperlukan dapat diprediksi. [7] Francisco Javier Palacios-Nereo, Pilar Olivares- Carrillo, Antonia Pérez de los Ríos, Joaquín Quesada- Medina, High-yield non-catalytic supercritical transesterification of soybean oil to biodiesel induced by gradual heating in a batch reactor, The Journal of Supercritical Fluids, 111, (2016) 135-142 https://doi.org/10.1016/j.supflu.2016.01.022 Management, 52, (2016) 367-374 https://doi.org/10.1016/j.wasman.2016.03.053 [2] Nuria Sánchez, Ramiro Sánchez, José M. Encinar, Juan F. González, Gloria Martínez, Complete analysis of castor oil methanolysis to obtain biodiesel, Fuel, 147, (2015) 95-99 https://doi.org/10.1016/j.fuel.2015.01.062 [3] Ali A. Jazie, H. Pramanik, A. S. K. Sinha, Transesterification of peanut and rapeseed oils using waste of animal bone as cost effective catalyst, Materials for Renewable and Sustainable Energy, 2, 2, (2013) 11 https://doi.org/10.1007/s40243-013-0011-4 [4] Y. C. Wong, Y. P. Tan, Y. H. Taufiq-Yap, I. Ramli, H. S. Tee, Biodiesel production via transesterification of palm oil by using CaO–CeO2 mixed oxide catalysts, Fuel, 162, (2015) 288-293 https://doi.org/10.1016/j.fuel.2015.09.012 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 218 Tabel 4. Prediksi berat katalis dan volume reaktor operasi fluidized CSTR untuk berbagai variasi kebutuhan katalis. Tabel 4. Prediksi berat katalis dan volume reaktor operasi fluidized CSTR untuk berbagai variasi kebutuhan katalis. Rasio molar -rA exp X W (gcat) V reaktor (mL) Trigliserida A Metanol B ( 𝑚𝑜𝑙 𝑔𝑐𝑎𝑡. 𝑠) 1 20 9,85 x 10-6 0,93 7,46 14,92 1 15 9,48 x 10-6 0,90 8,38 16,76 1 10 9,46 x 10-6 0,90 9,62 19,24 1 8 9,44 x 10-6 0,89 10,19 20,38 1 6 9,39 x 10-6 0,89 10,86 21,72 1 3 9,31 x 10-6 0,88 12,02 24,04 1 2 9,22 x 10-6 0,87 12,48 24,96 Management, 52, (2016) 367-374 https://doi.org/10.1016/j.wasman.2016.03.053 Notasi dan keterangan A B −rA −rA ex CA CB CC CD kB kS kC kD KB KS KC KD −𝑟𝐵 ′ 𝑟𝐶 ′ ∆𝑊 𝑣0 A = trigliserida B = metanol −rA = kecepatan reaksi (mol/gcat.s) −rA exp = kecepatan reaksi eksperimen (mol/gcat.s) CA = konsentrasi reaktan trigliserida (mol/gcat) CB = konsentrasi reaktan metanol (mol/gcat) CC = konsentrasi produk biodiesel (mol/gcat) CD = konsentrasi produk gliserol (mol/gcat) kB = konstanta kecepatan adsorpsi B kS = konstanta kecepatan adsorpsi S kC = konstanta kecepatan adsorpsi C kD = konstanta kecepatan adsorpsi D KB = konstanta kesetimbangan desorpsi B KS = konstanta kesetimbangan desorpsi S KC = konstanta kesetimbangan desorpsi C KD = konstanta kesetimbangan desorpsi D −𝑟𝐵 ′ = kecepatan alir metanol 𝑟𝐶 ′ = kecepatan reaksi biodiesel ∆𝑊 = berat katalis 𝑣0 = laju alir awal [8] Boyang Wang, Shufen Li, Songjiang Tian, Rihua Feng, Yonglu Meng, A new solid base catalyst for the transesterification of rapeseed oil to biodiesel with methanol, Fuel, 104, (2013) 698-703 https://doi.org/10.1016/j.fuel.2012.08.034 [9] Milan D. Kostić, Alireza Bazargan, Olivera S. Stamenković, Vlada B. Veljković, Gordon McKay, Optimization and kinetics of sunflower oil methanolysis catalyzed by calcium oxide-based catalyst derived from palm kernel shell biochar, Fuel, 163, (2016) 304-313 https://doi.org/10.1016/j.fuel.2015.09.042 [10] Marcos Sánchez, Jorge M. Marchetti, Noureddin El Boulifi, José Aracil, Mercedes Martínez, Kinetics of Jojoba oil methanolysis using a waste from fish industry as catalyst, Chemical Engineering Journal, 262, (2015) 640-647 https://doi.org/10.1016/j.cej.2014.09.088 [11] Chawalit Ngamcharussrivichai, Pramwit Nunthasanti, Sithikorn Tanachai, Kunchana Bunyakiat, Biodiesel production through transesterification over natural calciums, Fuel Processing Technology, 91, 11, (2010) 1409-1415 https://doi.org/10.1016/j.fuproc.2010.05.014 c. Desorpsi C pada permukaan katalis S Hubungan antara konversi trigliserida (X) dan berat katalis (W) yang dibutuhkan pada reaktor yang beroperasi sebagaimana fluidized CSTR. Sebagai contoh pada desain reaktor, jika diambil asumsi bulk density dalam reaktor fluidized CSTR adalah 0,5 g/mL, maka perhitungan volume reaktor yang dibutuhkan jika berat katalis (W) 7,46 g untuk memperoleh konversi 93% adalah 14,92 mL. Lebih lengkap perhitungan berat katalis dan volume reaktor yang diperlukan ditunjukkan pada Tabel 4. Cara perhitungan desain reaktor ini dapat juga diterapkan pada skala yang lebih besar untuk menghitung kebutuhan katalis pada volume reaktor tertentu untuk mencapat konversi tertentu. −rA = 4,7901 (0,7240 CACB − CCCD (248,2845)) 0,7240 CB + CC 2,4342 + 1 (7) (7) dengan nilai koefisien determinasi R2 sebesar 0,8835 untuk katalis CaO 3% pada reaksi transesterifikasi trigliserida dan metanol menghasilkan biodiesel pada reaktor batch. Persamaan kecepatan reaksi yang Daftar Pustaka [1] Thi Tuong Vi Tran, Sunanta Kaiprommarat, Suwadee Kongparakul, Prasert Reubroycharoen, Guoqing Guan, Manh Huan Nguyen, Chanatip Samart, Green biodiesel production from waste cooking oil using an environmentally benign acid catalyst, Waste [12] Masoud Zabeti, Wan Mohd Ashri Wan Daud, Mohamed Kheireddine Aroua, Activity of solid catalysts for biodiesel production: A review, Fuel 219 Jurnal Kimia Sains dan Aplikasi 22 (5) (2019): 213–219 Processing Technology, 90, 6, (2009) 770-777 https://doi.org/10.1016/j.fuproc.2009.03.010 Processing Technology, 90, 6, (2009) 770-777 https://doi.org/10.1016/j.fuproc.2009.03.010 [13] Peng-Lim Boey, Gaanty Pragas Maniam, Shafida Abd Hamid, Performance of calcium oxide as a heterogeneous catalyst in biodiesel production: A review, Chemical Engineering Journal, 168, 1, (2011) 15-22 https://doi.org/10.1016/j.cej.2011.01.009 [14] Masato Kouzu, Jyu-suke Hidaka, Transesterification of vegetable oil into biodiesel catalyzed by CaO: A review, Fuel, 93, (2012) 1-12 https://doi.org/10.1016/j.fuel.2011.09.015 [15] Masduki, Sutijan, Arief Budiman, Kinetika Reaksi Esterifikasi Palm Fatty Acid Distilate (PFAD) menjadi Biodiesel dengan Katalis Zeolit-Zirkonia Tersulfatasi, Jurnal Rekayasa Proses, 7, 2, (2014) 58- 63 https://doi.org/10.22146/jrekpros.4953 [16] Buchori Luqman, Budi Sasongko Setia, Kinetika Transesterifikasi Biodiesel Jarak Pagar, Teknik, 33, 2, (2012) 52-57 https://doi.org/10.14710/teknik.v33i2.4383 [17] Elizabeth DC Sidabutar, M Nur Faniudin, M Said, Pengaruh Rasio Reaktan Dan Jumlah Katalis Terhadap Konversi Minyak Jagung Menjadi Metil Ester, Jurnal Teknik Kimia, 19, 1, (2013) 40-49 [18] Tuti Indah Sari, M Said, Ani Karlena Sari, Katalis Basa Heterogen Campuran CaO & SrO Pada Reaksi Transesterifikasi Minyak Kelapa Sawit, Seminar Nasional AvoER, Palembang, (2011) 482-493 [19] Isalmi Aziz, Kinetika Reaksi Transesterifikasi Minyak Goreng Bekas, Jurnal Kimia Valensi, 1, 1, (2007) 19-23 https://doi.org/10.15408/jkv.v1i1.209. [20] Nurhayati, Muhdarina, Amilia Linggawati, Sofia Anita, Tengku Ariful Amri, Preparation and Characterization of Calcium Oxide Heterogeneous Catalyst Derived from AnadaraGranosaShell for Biodiesel Synthesis, in: 1st International Conference on Science and Engineering, Pekanbaru, 2015. [21] T. N. Blanton, C. L. Barnes, D-75 Quantitative Analysis of Calcium Oxide Desiccant Conversion to Calcium Hydroxide Using X-ray Diffraction, Powder Diffraction, 19, 2, (2004) 201-201 http://doi.org/10.1154/1.1779815 [22] H. Scott Fogler, Elements of Chemical Reaction Engineering, Prentice Hall, 2016.
https://openalex.org/W4297878049	https://zenodo.org/record/3693987/files/20.pdf	English	null	Density functional theory calculations of the water interactions with ZrO2 nanoparticles Y2O3 doped	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	9,273	Journal of Physics: Conference Series Journal of Physics: Conference Series PAPER • OPEN ACCESS Density functional theory calculations of the water interactions with ZrO2 nanoparticles Y2O3 doped To cite this article: Mekhrdod Subhoni et al 2018 J. Phys.: Conf. Ser. 994 012013 Nonequilibrium chemo-electronic conversion of water on the nanosized YSZ: experiment and Molecular Dynamics modelling problem formulation A S Doroshkevich, A I Lyubchyk, A K Islamov et al. - Nonequilibrium chemo-electronic conversion of water on the nanosized YSZ: experiment and Molecular Dynamics modelling problem formulation A S Doroshkevich, A I Lyubchyk, A K Islamov et al. - Density functional theory calculations of the water interactions with ZrO2 nanoparticles Y2O3 doped p Qingwei Niu, Zili Li, Guodong Liu et al. View the article online for updates and enhancements. This content was downloaded from IP address 93.72.77.245 on 02/03/2020 at 14:57 Related content Related content Nonequilibrium chemo-electronic conversion of water on the nanosized YSZ: experiment and Molecular Dynamics modelling problem formulation A S Doroshkevich, A I Lyubchyk, A K Islamov et al. - Characterization and Properties of Ni-W- ZrO2 Composite Coating by Ultrasonic Electrodeposition Qingwei Niu, Zili Li, Guodong Liu et al. - Improving the sonocatalytic performance of good crystallinity ZrO2 nanocomposite through graphene addition Y Kristianto, A Taufik and R Saleh - This content was downloaded from IP address 93.72.77.245 on 02/03/2020 at 14:57 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” onf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 1 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd Density functional theory calculations of the water interactions with ZrO2 nanoparticles Y2O3 doped Mekhrdod Subhoni1,, Kholmirzo Kholmurodov1,2, Aleksandr Doroshkevich2, Elmar Asgerov2,4, Tomoyuki Yamamoto5, Andrei Lyubchyk6, Valer Almasan7, Afag Madadzada2,4 Mekhrdod Subhoni1,, Kholmirzo Kholmurodov1,2, Aleksandr Doroshkevich2, Elmar Asgerov2,4, Tomoyuki Yamamoto5, Andrei Lyubchyk6, Valer Almasan7, Afag Madadzada2,4 1S.U. Umarov Physical-Technical Institute, Academy of Sciences, Republic of Tajikistan. 2Joint Institute for Nuclear Research, 141980, Dubna, Moscow Region, Russian Federation. 2Joint Institute for Nuclear Research, 141980, Dubna, Moscow Region, Russian Federation. 3Dubna State University, 141980, Dubna, Moscow Region, Russian Federation. 4 3Dubna State University, 141980, Dubna, Moscow Region, Russian Feder 4National Nuclear Research Center CJSC, AZ1073, Baku, Azerbaijan. Dubna State University, 141980, Dubna, Moscow Region, Russian Fed 4National Nuclear Research Center CJSC, AZ1073, Baku, Azerbaijan. 4National Nuclear Research Center CJSC, AZ1073, Baku, Azerbaijan. 5Waseda University, 169-0051, Shinjuku, Tokyo, Japan. 6 5Waseda University, 169-0051, Shinjuku, Tokyo, Japan. 6 5Waseda University, 169-0051, Shinjuku, Tokyo, Japan. 6 6i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, New University of Lisbon and CEMOP/UNINOVA, Campus de Caparica, 2829-516 Caparica, Portugal. 6i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, New University of Lisbon and CEMOP/UNINOVA, Campus de Caparica, 2829-516 Caparica, Portugal. 7National Institute for Research and Development of Isotopic and Molecular Technologies Cluj-Napoca, Romania 400293 Cluj-Napoca România. 7National Institute for Research and Development of Isotopic and Molecular Technologies Cluj-Napoca, Romania 400293 Cluj-Napoca România. Mekhrdod Subhoni-734063, Republic of Tajikistan, Dushanbe, st. Ayni 299/1, Physical-Technical Institute -Technical Institute AS RT, E-mail: mehrdodq@gmail.com 1. Introduction The water is well known conventional source of electrical energy used by mankind. In liquid state, it is traditionally used for the electric energy conversion by an external thermostat of hydropower plants. However, direct conversion of water in gas state, (atmospheric moisture) to electric energy, remained unrealizable for centuries as a top scientific and practical aim. With the development of nanotechnology, the implementation of these ideas becomes potentially possible In this regard, a nanopowder system based on zirconia dioxide (ZrO2) is the best candidate for the direct conversion of the energy of water adsorption into electric energy [1]. It is well known [1-2] that the ZrO2 surface is an extremely nonequilibrium thermodynamically, the surface of the ZrO2 based nano- dispersed oxide systems is exist in a state of dynamic charge and adsorption equilibration. I.e., the change of quantity of adsorbates is accompanied by a changing of the total charge in the system. In the case of nanoscale powder of zirconia dioxide, on its surface are preferably water is absorbed, and certain stages of a matter exchange between the system and external environment have an exothermic behaviour. Hence, in a cyclic mode, the nanopowder system based on ZrO2 can convert the chemical energy of water molecules adsorption to an electric form [1-4]. As is known, traditional photoelectric converters use light energy to localize the electron-hole pair in the material of the so-called adsorber (Figure 1). Then the electron-hole pair is converted into a pair of free charge carriers by the heterojunction field. Figure 1. Schematic diagram of the operation of a traditional solar photocell (FEP). Figure 1. Schematic diagram of the operation of a traditional solar photocell (FEP). To obtain such a heterojunction, some amount of impurity, with a valence greater than the valence of the base material (intrinsic semiconductor), for example, phosphorus, is introduced into the top layer of silicon. As a result, a layer is formed with an excess of electrons, that is, a negative charge. At the same time, the lower layer is doped with another valence impurity with a valence less than the valence of the intrinsic semiconductor, for example, by boron, which leads to a decrease in the number of electrons, thus creating a positive charge. As a result, an electric field is formed at the junction of these semiconductors, which provides separation and transport of free charge carriers to the electrodes. E-mail: mirzo@jinr.ru Abstract. Development of a new electricity generation techniques is one of the most relevant tasks, especially nowadays under conditions of extreme growth in energy consumption. The exothermic heterogeneous electrochemical energy conversion to the electric energy through interaction of the ZrO2 based nanopowder system with atmospheric moisture is one of the ways of electric energy obtaining. The questions of conversion into the electric form of the energy of water molecules adsorption in 3 mol% Y2O3 doped ZrO2 nanopowder systems were investigated using the density functional theory calculations. The density functional theory calculations has been realized as in the Kohn-Sham formulation, where the exchange- correlation potential is approximated by a functional of the electronic density. The electronic density, total energy and band structure calculations are carried out using the all-electron, full potential, linear augmented plane wave method of the electronic density and related approximations, i.e. the local density, the generalized gradient and their hybrid approximations. 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1. Introduction The method of obtaining the energy considered in this paper is the phenomenon of injection of hot electrons (the mechanism of transfer of electric energy to the semiconductor crystal, which is realized during the course of a heterogeneous catalytic chemical reaction on its metalized surface) [5] (Figure 2). With a nanometer film thickness of <20 nm, so-called "hot electrons" with an energy of 1-3 eV ballistically, i.e. without loss of energy, reach the Schottky barrier and overcome due to its kinetic energy and, entering the semiconductor, form an electric current in the external circuit. Thus, in this case the appearance of free charge carriers is a consequence not of localization from the lattice, but of tunneling through the heterophase boundary to the outer space. 2 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 Figure 2. Scheme for the realization of the mechanism of ballistic electron transport on a metal- semiconductor transition during chemical adsorption. e V Echem φ2 Semiconductor Е Schottky barrier φ1 Н Н Metal Dissociative adsorption Dissociative adsorption Н Metal Е Schottky barrier Figure 2. Scheme for the realization of the mechanism of ballistic electron transport on a metal- semiconductor transition during chemical adsorption. Figure 2. Scheme for the realization of the mechanism of ballistic electron transport on a metal- semiconductor transition during chemical adsorption. The phenomenon of energy conversion of heterogeneous exothermic chemical reactions by analogy with photo-EMF [3] was called chemo-EMF (the prefix of chemo-, like photo-, emphasizes the nonthermic origin of the phenomenon) [6-9]. This way of converting chemical energy into an electrical form is good because the transformation from the chemical form of energy into electrical energy occurs directly, bypassing the intermediate stages. gy y yp g g There are other ways of obtaining electrical energy from moisture. For example, attempts have been made to collect a charge from jumping droplets of water mist by means of electrostatic capture or causing mechanical vibrations of piezoelectric plates [10-11]. These methods of obtaining energy assume the presence of intermediate energy forms and will have a lower efficiency than in the case of direct energy conversion. However, until now it is not clear in which act of a physic-chemical process a free electron is formed. 1. Introduction It can be assumed that when a surface of a nanoparticle interacts with a water molecule, a process similar to that realized in redox processes takes place [12]. This paper is based on the results of the DFT calculations of the structure and energy state of the surface layer of nanoparticles of a solid solution based on ZrO2 [13], The main tasks of work are: 1. To calculate the energy characteristics of the system, to predict the most probable path of realization of oxidation-reduction processes on the surface of nanoparticles of the investigated disperse system, 2. Calculate the oxidation-reduction potentials of the system, 1. To calculate the energy characteristics of the system, to predict the most probable path of realization of oxidation-reduction processes on the surface of nanoparticles of the investigated disperse system, 2. Calculate the oxidation-reduction potentials of the system, p 3. To determine the reaction pathway, 4. To find the EMF of the reaction, and the energy that can be produced when the electron formed by the interaction of the water molecule with the surface of ZrO2 - nanoparticles, at EV- / V = 0.7V; EOH / OH = 0.3V, ΔE = EMF = 0.4V. To check the scheme 1 realization. To calculate the allocated energy W = - e ΔE [14]. 4. To find the EMF of the reaction, and the energy that can be produced when the electron formed by the interaction of the water molecule with the surface of ZrO2 - nanoparticles, at EV- / V = 0.7V; EOH / OH = 0.3V, ΔE = EMF = 0.4V. To check the scheme 1 realization. To calculate the allocated energy W = - e ΔE [14]. 1.1. The nature of the adsorption of gas molecules by the surface of nanoparticles of oxides. 1.1. The nature of the adsorption of gas molecules by the surface of nanoparticles of oxides. Theoretically, any oxide nanoparticle is a spherical fragment 4-10 nm in diameter, isolated from a regular crystal lattice of an ionic crystal. According to the principle of electroneutrality, the surface of such a nanoparticle is initially electrically neutral, a violation of the periodicity of the crystal potential by the surface leads to the formation of additional eigen levels in the forbidden gap of the energy spectrum of the crystal. These are the so-called Shockley / Tamm localized states, which can be treated as unsaturated chemical bonds of atoms on the surface [15-17]. In the chemical sense, these are the so-called "active centers". Depending on whether the levels are donor or acceptor, the nature of the 3 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 adsorbed ions will depend and, subsequently, the sign of the surface charge of the particle. In [18], it was proposed to use the value of its isoelectric point: pE as an index of the chemical activity of oxides. adsorbed ions will depend and, subsequently, the sign of the surface charge of the particle. In [18], it was proposed to use the value of its isoelectric point: pE as an index of the chemical activity of oxides. Coordinative, the unsaturated surface Zr4+ cations are strong acid sites of adsorption (Lewis centers, pE <7), therefore, the localized states in ZrO2 are of a donor nature. Thus, the influx of neutral molecules into the reaction zone (the region near the surface of the nanoparticles) is provided due to the individual chemical properties of the nanoparticle material and its defectiveness. The density functional theory (DFT) calculations aimed on determining the nature of the active centers responsible for the formation of a free charge carrier in the system under study. In DFT modeling we simulate water adsorption mechanism on the surface of nanosized yttria-stabilized zirconia (YSZ) (Figure 3). For the DFT calculations we have been aimed to perform a comparative analysis of water molecule interaction with two different surfaces: Figure 3. (Color online) A model structure of the ZrO2 system doped with Y2O3. (1) ZrO2 and (2) ZrO2 + 3% mol Y2O3 (1) ZrO2 and (2) ZrO2 + 3% mol Y2O3 Below the electronic structure calculations present the electronic densities, energy bands, densities of states (DOS), X-ray spectra separately for ZrO2 and ZrO2 + 3% mol Y2O3 models. Next, the DFT calculated results are compared respectively for the systems: p p y 3) H2O / ZrO2 and (4) H2O / ZrO2 + 3% mol Y2O3. p p y y 2O / ZrO2 and (4) H2O / ZrO2 + 3% mol Y2O3. So far, the developing of computational DFT technique for the above listed molecular systems has aimed on the accurate description of their electronic structures. As one expect, the replacing of zirconium with yttrium causes a large shift in the energy gap, making the compound ZrO2 + 3% mol Y2O3 a more active target for the water absorption. 1.1. The nature of the adsorption of gas molecules by the surface of nanoparticles of oxides. The structure model (ZrO2+3% mol Y2O3) includes 15 atoms of Zr, 32 atoms O and one atom of Y. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. All calculation of charge density were performed for top layers. FIRST LAYER SECOND LAYER FIRST LAYER SECOND LAYER Figure 3. (Color online) A model structure of the ZrO2 system doped with Y2O3. The structure model (ZrO2+3% mol Y2O3) includes 15 atoms of Zr, 32 atoms O and one atom of Y. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. All calculation of charge density were performed for top layers. 1.2. The DFT technique The density functional theory (DFT) in the Kohn-Sham formulation [19] and its practical utilization by different approaches [20] is the most widely used approach for today electronic states calculations of functional materials. In the DFT method the exchange-correlation potential is approximated by a functional of the electronic density; the most common approximations are the local density approximation (LDA) [20], the generalized gradient approximation (GGA) [21], and the hybrid approximation [22]. One have to mention some peculiarities here, that while LDA and GGA provide a successful description of ground-state properties in crystals, this success does not extend to a 4 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 CMSMS17 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 description of excited states. In many systems (semiconductors, crystal lattices, etc) the LDA and GGA strongly underestimate the value of the energy gap. Improved values for the band gaps are usually obtained by using the GW method [23]. However, the high computational cost of this method limits its applicability to crystals with a small number of atoms in the unit cell. An exchange potential was recently proposed by Becke and Johnson (BJ), designed to yield the exact exchange potential in atoms [24]. Unfortunately, the use of this potential led to a slight improvement in the energy gap values for many semiconductors [25]. A simple modification of the BJ potential was proposed by Tran and Blaha (TB-mBJ method). Studies have shown that the TB-mBJ potential is generally as accurate in predicting the energy gaps of many semiconductors as the much more expensive GW method [26]. h k h l d b d l l i i d i h In the present work, the total energy and band structure calculations are carried out using the all-electron, full potential, linear augmented plane wave (FP-LAPW) method as implemented in the WIEN2k code [27]. We have utilized the WIEN2k code calculations for the 3 mol% Y2O3 doped ZrO2 interacting with H2O. Thereby, in the DFT/WIEN2k each atom is surrounded by a muffin-tin sphere, and the total space is divided into two regions. One region consists of the interior of these non- overlapping spheres, while the rest of the space constitutes the interstitial region. 2. The results summary of native experiments 2.1. The mechanism of formation of free charge carriers during the adsorption of a neutral water molecule. The potential energy of the chemical interaction of atmospheric molecules with the surface of a solid base. f roceeding from the principle of electroneutrality [28] we will consider the number of negative and positive ions in the atmosphere equal, and the atmosphere as a whole - electrically neutral. Let us consider the mechanism of inflow to the surface of a nanoparticle based on zirconia dioxide of neutral water molecules and the selection of the energy of chemical interaction. Proceeding from the principle of electroneutrality [28] we will consider the number of negative and positive ions in the atmosphere equal, and the atmosphere as a whole - electrically neutral. Let us consider the mechanism of inflow to the surface of a nanoparticle based on zirconia dioxide of neutral water molecules and the selection of the energy of chemical interaction. 1.2. The DFT technique The radii of the muffin-tin spheres are 2.1a0 for Zr, 1.9a0 for O, 2.1a0 for Y and 0.6a0 for H, where a0 is the Bohr radius. In GGA calculations, the exchange correlation potential is that proposed in reference [21]. The valence electrons’ wave functions inside the muffin-tin spheres are expanded in terms of spherical harmonics up to lmax=10. In the interstitial regions, they are expanded in terms of plane waves, with a wave vector cutoff of Kmax. Because of the small muffin-tin radius of hydrogen atoms, we set RHKmax=1.12 in ZrO2 and ZrO2+3% mol Y2O3, where RH=0.32a0 is the muffin-tin radius of the H atom. In the remaining four compounds, we set RmtKmax=2.1, where Rmt is the smallest muffin-tin radius. The charge density is Fourier expanded up to a maximum wave vector of Gmax, where Gmax=12a0−1 for ZrO2, G=12a0−1 for ZrO2+3% mol Y2O3, and G=20a0−1 for the remaining compounds. The convergence of the self consistent calculations is achieved with a total energy tolerance of 0.001mRy and a charge convergence of 0.0004e. 2.4. The formation of an adsorption layer on the surface of particles. An own localized electronic states. 2.4. The formation of an adsorption layer on the surface of particles. An own localized electronic states. Violation of the periodicity of the crystalline potential by the surface (breaking of chemical bonds) leads to the formation of eigen levels in the band gap of the energy spectrum of the crystal- Shockley/Tamm localized states, which can be treated as unsaturated chemical bonds of atoms on the surface [32]. Their concentration in the ideal case should be equal in order of magnitude to the concentration of surface atoms. Such a configuration of the surface is not energetically favorable, so the population of these states (saturation of free valence bonds) occurs mainly by adsorption of molecules and ions from the atmosphere by the surface of nanoparticles. Thus, the influx of neutral molecules into the reaction zone (the region near the surface of the nanoparticles) is provided by the individual chemical properties of the nanoparticle material and its defectiveness. 2.5. Adsorption of molecules from the gas phase in ZrO2 - 3 mol% Y2O3. If the level is acceptor, on it with a probability determined by the Fermi-Dirac function 2.5. Adsorption of molecules from the gas phase in ZrO2 - 3 mol% Y2O3. If the level is acceptor, on it with a probability determined by the Fermi-Dirac function   1 1 exp f c f E E kT         (1) (1) The electron (belonging to the nanoparticle material) is localized, additional surface states are filled at the bottom of the conduction band, and the bond formation between the surface and adsorbates is formed by the exchange interaction. As a result, a potential-forming layer of adsorption origin is formed (Helmholtz layer [33, 34], 2, Figure 5), and the neutral surface acquires a charge. In the case of ZrO2 - 3 mol% Y2O3, this charge is negative. To compensate the charge of the potential- forming layer, an external diffuse layer is formed (3, Figure 5). 2.2. The nature of the adsorption of gas molecules by the surface of nanoparticles of oxides. Figure 4. Qualitative band model of β-ZrO2 with yttrium (a), with oxygen vacancies (b), with yttrium and oxygen vacancies (c). Theoretically, any oxide nanoparticle can be represented in the form of a spherical fragment with a diameter of 4-10 nm, isolated from a regular crystal lattice of an ionic crystal. According to the principle of electroneutrality, the surface of such a nanoparticle is initially electrically neural. I is the conduction band, and II is the valence band. 5 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 CMSMS17 1234567890 ‘’“” C S S O g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” onf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 2.3. The mechanism of charge compensation of an impurity in the system ZrO2 - Y2O3. 3 2.3. The mechanism of charge compensation of an impurity in the system ZrO2 - Y2O3. In a solid substitution solution based on zirconium dioxide, the Y3+ impurity atoms have one electron less than the Zr4+ atoms and are acceptors, that is, they create in the forbidden band levels near the top of the valence band. The oxygen vacancies VO are formed to compensate the excess volume and charge of the impurity are electron donors, ie, donor levels (holes, near the bottom of the conduction band Figure 4) are created near the bottom of the conduction band. The acceptor and donor character of β-ZrO2 for Y3+ and oxygen vacancies, respectively, is confirmed by the results of [29]. As a result of thermal fluctuations, the holes give the electron to the conduction band (ionization of the hole), which by an Coulomb interaction finds an impurity atom and hits its orbital [30]. An impurity-vacancy dipole (IVD) of the Me-VO type is formed, and the corresponding ionic bond according to [31] is the stabilizing element of the lattice (T-phase). In this case, the impurity atom acquires a negative charge Y3+ (-1), and the vacancy is a positive V (+). That is, donors and acceptors co-exist in the form of V (+) - Y3+ (-)IVD type2 2 The nanoparticle as a physical object can be considered theoretically as a nano-sized fragment of an extended electrically neutral crystal lattice of a solid solution of the appropriate composition. Consequently, initially the nanoparticle is electrically neutral. where H – a radical hydrogen form. Schematic representation of the proposed process is shown in Figure 6. The formation of a radical form of hydrogen leads to the launch of a chain reaction of ionization of the molecules of the dispersion medium. As a result, by the relay mechanism the charge is transported to the region with a lower charge concentration of the corresponding sign. Unlike photocatalytic systems, the nanopowder heterophase system is thermodynamically closed, i.e., does not receive energy from outside as radiation, and as a consequence has a specific physical limit on electron production. 2.6. The structure of the reaction zone. A particle of a dispersed phase, together with a DEL, is called a micelle (Figure 5). The aggregate of the substance {[ZrO2] m} with potential-forming ions, (predominantly OH- for ZrO2 + 3 mol% Y2O3 [35, 36]) form the core of the micelle {m [ZrO2] nOH-}. Potential-forming ions are connected with the surface of a relatively strong (E> 0.3 eV [37]) chemical bond. The diffuse layer is connected with the core of the micelle physically by the forces of electrostatic interaction and consists mainly of H + 2 The nanoparticle as a physical object can be considered theoretically as a nano-sized fragment of an extended electrically neutral crystal lattice of a solid solution of the appropriate composition. Consequently, initially the nanoparticle is electrically neutral. 6 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 protons and polarized water molecules. It is the diffuse layer that is the receptor of molecules from the outer atmosphere and their supplier into the reaction zone. The system of adsorbates on the surface of ZrO2 is in dynamic equilibrium [38]. Consequently, when the external conditions change, it undergoes a change and a charge state of the near-surface layer. protons and polarized water molecules. It is the diffuse layer that is the receptor of molecules from the outer atmosphere and their supplier into the reaction zone. The system of adsorbates on the surface of ZrO2 is in dynamic equilibrium [38]. Consequently, when the external conditions change, it undergoes a change and a charge state of the near-surface layer. The real mechanism of interaction of the water molecule with the surface of the micelle is multistage and is theoretically considered in detail in [39-40]. An interesting question is how the adsorption energy is transferred from the diffuse layer to the heterophase boundary, that is, to the surface of the nanoparticles. 2.7. The physical basis of the chemoelectronic effect. Upon reaching the surface of an electrically neutral zirconium oxide nanoparticle, the neutral water molecule under dissociation of the surface force gradient dissociates into a proton of the H+ and OH- group. In the case of physical adsorption, an electrochemical process is realized such as [41]: 2 ads H O e H OH      The total charge of the surface changes, in this case, becomes negative. This leads to bending of the levels in the energy bands, and the charge exchange of electronic states localized near the surface. In particular, the localization of a part of electrons from the crystal lattice of nanoparticles near the surface and the formation of a space-charge region of an electronic type occur there with the probability determined by the Fermi-Dirac function. This region can be used as electrical capacitance in the development of ionistors. p In the case of chemical adsorption, processes with electron transfer via the phase interface are probably realized. Taking into account the acceptor character of the impurity level in the system under study, it can be assumed that the process proceeds according to the scheme [42]: 2 2 H O e H O H OH         , where H – a radical hydrogen form. 3. Simulation results 3.1. ZrO2 : crystal and energy bands structures. 3.1. ZrO2 : crystal and energy bands structures. The density of states of ZrO2 is shown in Figures 8 (top and bottom), where we see that the low-lying conduction bands are derived from O p states. On the other hand, the bands in the range -2 eV to 0 eV are dominated by oxygen-derived states. The valence band just below the Fermi energy is derived from zirconium s and p states. These observations become clear upon considering the atomic orbital character of the bands, which is presented in Figure 8 (bottom). The contribution of the chosen atomic orbital to the eigenstates at each k-point, where the CBM is derived mostly from Zr p states. The VBM also dominated by Zr, though a mixture of Zr s and O p states is clear. The anti-bonding state formed from these s and p states is pushed up in energy close to the Fermi level. The large contribution of Zr s (l = 0) states to the VBM and Zr p (l = 1) states to the CBM suggests that there are some transitions between the VBM and CBM (∆l = 1), to be useful of this material in charge conduction process. Table 2. Lattice energy per unit cell for both ZrO2 and ZrO2+3% mol Y2O3. Table 2. Lattice energy per unit cell for both ZrO2 and ZrO2+3% mol Y2O3. Lattice Energy per unit cell ZrO2 (eV) -138.6 -158.4 -163.1 -159.25 -165.2 -148.13 -84.1 Lattice Energy per unit cell ZrO2+3% mol Y2O3 (eV) -124.75 -137.88 -139.1 -137.25 -141.2 -132.75 -76.75 Volume Å3 114.79 116.58 118.58 119.35 120.13 121.43 122.46 In Figure 7 we present the calculated energy bands of ZrO2. The electronic density of states in Figure 7 have shown for high symmetry directions of the Brillouin zone (BZ). The valence band maximum (VBM) and conduction band minimum (CBM) occur at the Γ-point, the BZ center. In ZrO2, the gap occurs at point R(1/2, 1/2, 1/2). Point R of the cubic lattice BZ is zone-folded into the Γ-point of the body-centered tetragonal lattice BZ. The density of states of ZrO2 is shown in Figures 8 (top and bottom), where we see that the low-lying conduction bands are derived from O p states. On the other hand, the bands in the range -2 eV to 0 eV are dominated by oxygen-derived states. The valence band just below the Fermi energy is derived from zirconium s and p states. 3.1. ZrO2 : crystal and energy bands structures. 3.1. ZrO2 : crystal and energy bands structures. The cubic unit cell with space group F m -3 m and lattice constants a=b=c=4.938 Å was used in DFT calculation. The lattice parameters obtained (Table 1.) show excellent agreement with the experimental results and the differences is less than 3%. The calculated surface energies are presented in Table 2, which the minimum lattice energy in the 120.13 Å3 is received -165.2 eV. This volume of unit cell was chosen to all calculation experiential. Calculations were performed using Abinit code and GGA (PBE) - Fritz-Haber-Institute (FHI) pseudopotential with energy convergence of 0.0001. 7 IOP Publishing 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 ”012013 doi :10.1088/1742-6596/994/1/01201 2 1 3 OH groups The boundary of the diffusion layer 2 1 3 OH groups The boundary of the diffusion layer Figure 6. Schematic repre- sentation of a possible electrochemical process with an electron transfer through the phase-gap interface during adsorption of water molecules to the surface of nanoparticles based on ZrO2. Figure 5. Schematic representation of a ZrO2 (1) nanoparticle surrounded by an ionic atmosphere, including adsorption (2) and diffuse (3) layers. Table 1. Calculated and observed structural parameters of cubic ZrO2 (a[43]; b[44]; c[45]). Table 1. Calculated and observed structural parameters of cubic ZrO2 (a[43]; b[44]; c[45]). Parameters (exp) (calc) a=b=c (Å) 4.913a 5.07b 4.938 Lattice energy per ZrO2 (eV) -109.76c -165.2 Lattice Energy per ZrO2+3% mol Y2O3 (eV) -106.1c 141.2 Table 2. Lattice energy per unit cell for both ZrO2 and ZrO2+3% mol Y2O3. Lattice Energy per unit cell ZrO2 (eV) -138.6 -158.4 -163.1 -159.25 -165.2 -148.13 -84.1 Lattice Energy per unit cell ZrO2+3% mol Y2O3 (eV) -124.75 -137.88 -139.1 -137.25 -141.2 -132.75 -76.75 Volume Å3 114.79 116.58 118.58 119.35 120.13 121.43 122.46 In Figure 7 we present the calculated energy bands of ZrO2. The electronic density of states in Figure 7 have shown for high symmetry directions of the Brillouin zone (BZ). The valence band maximum (VBM) and conduction band minimum (CBM) occur at the Γ-point, the BZ center. In ZrO2, the gap occurs at point R(1/2, 1/2, 1/2). Point R of the cubic lattice BZ is zone-folded into the Γ-point of the body-centered tetragonal lattice BZ. 3.1. ZrO2 : crystal and energy bands structures. These observations become clear upon considering the atomic orbital character of the bands, which is presented in Figure 8 (bottom). The contribution of the chosen atomic orbital to the eigenstates at each k-point, where the CBM is derived mostly from Zr p states. The VBM also dominated by Zr, though a mixture of Zr s and O p states is clear. The anti-bonding state formed from these s and p states is pushed up in energy close to the Fermi level. The large contribution of Zr s (l = 0) states to the VBM and Zr p (l = 1) states to the CBM suggests that there are some transitions between the VBM and CBM (∆l = 1), to be useful of this material in charge conduction process. In Figure 7 we present the calculated energy bands of ZrO2. The electronic density of states in Figure 7 have shown for high symmetry directions of the Brillouin zone (BZ). The valence band maximum (VBM) and conduction band minimum (CBM) occur at the Γ-point, the BZ center. In ZrO2, the gap occurs at point R(1/2, 1/2, 1/2). Point R of the cubic lattice BZ is zone-folded into the Γ-point of the body-centered tetragonal lattice BZ. The density of states of ZrO2 is shown in Figures 8 (top and bottom), where we see that the low-lying conduction bands are derived from O p states. On the other hand, the bands in the range -2 eV to 0 eV are dominated by oxygen-derived states. The valence band just below the Fermi energy is derived from zirconium s and p states. These observations become clear upon considering the atomic orbital character of the bands, which is presented in Figure 8 (bottom). The contribution of the chosen atomic orbital to the eigenstates at each k-point, where the CBM is derived mostly from Zr p states. The VBM also dominated by Zr, though a mixture of Zr s and O p states is clear. The anti-bonding state formed from these s and p states is pushed up in energy close to the Fermi level. The large contribution of Zr s (l = 0) states to the VBM and Zr p (l = 1) states to the CBM suggests that there are some transitions between the VBM and CBM (∆l = 1), to be useful of this material in charge conduction process. 3.1. ZrO2 : crystal and energy bands structures. 8 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 CMSMS17 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 y gy A model structure of the zirconium dioxide doped with yttrium is shown in Figure 9. The structure model (ZrO2+3% mol Y2O3) includes 15 atoms of Zr, 32 atoms O and one atom of Y. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. All calculation of charge density were performed for top layers as shown in Figure 3 above. 9 Figure 7. The calculated energy bands of ZrO2. Figure 7. The calculated energy bands of ZrO2. gure 7. The calculated energy bands of ZrO2. Figure 7. The calculated energy bands of ZrO2. Figure 7. The calculated energy bands of ZrO2. 9 9 9 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 Figure 8. The calculated densities of states of ZrO2 (top and bottom). Figure 9. (Color online) A model structure of the ZrO2 system doped with Y2O3. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. In Figure 10 the calculated energy bands are presented for the system ZrO2 + 3% mol Y2O3. The electronic density of states in Figure 11 demonstrate the relatively higher densities, in comparison with ZrO2(Figure 7), and narrowing of the gap between the VBM and CBM zones. The Fermi level slightly Figure 8. The calculated densities of states of ZrO2 (top and bottom). Figure 8. The calculated densities of states of ZrO2 (top and bottom). Figure 8. The calculated densities of states of ZrO2 (top and bottom). Figure 9. (Color online) A model structure of the ZrO2 system doped with Y2O3. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. Figure 9. (Color online) A model structure of the ZrO2 system doped with Y2O3. The zirconium atom are shown in green, oxygen atoms - in red, yttrium atom - in cyan. Figure 9. 3.1. ZrO2 : crystal and energy bands structures. (Color online) A model structure of the ZrO2 system doped with Y2O3. The zirconium atoms are shown in green, oxygen atoms - in red, yttrium atom - in cyan. In Figure 10 the calculated energy bands are presented for the system ZrO2 + 3% mol Y2O3. The electronic density of states in Figure 11 demonstrate the relatively higher densities, in comparison with ZrO2 (Figure 7), and narrowing of the gap between the VBM and CBM zones. The Fermi level slightly shifted up to, so the gap between the VBM and CBM has narrowed on 1 eV. In Figure 10 the calculated energy bands are presented for the system ZrO2 + 3% mol Y2O3. The electronic density of states in Figure 11 demonstrate the relatively higher densities, in comparison with ZrO2 (Figure 7), and narrowing of the gap between the VBM and CBM zones. The Fermi level slightly shifted up to, so the gap between the VBM and CBM has narrowed on 1 eV. 10 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 Figure 10. The calculated energy bands of ZrO2+ 3% mol Y2O3. Figure 10. The calculated energy bands of ZrO2+ 3% mol Y2O3. Figures 11 (top and bottom) show the density of states for ZrO2 + 3% mol Y2O3. Now doping of yttrium to the zirconia structure causes the dominating of oxygen in the interval from -5 eV to 0 eV. At the same time, the yttrium with zirconium dominate together in the conduction zone. These observations clearly demonstrate the atomic orbital modifications of the bands, which are influenced by the yttrium doping to the zirconium dioxide. 11 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” g IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 doi :10.1088/1742-6596/994/1/012013 Figure 11. The calculated densities of states of ZrO2 + 3% mol Y2O3 (top and bottom). Figure 11. The calculated densities of states of ZrO2 + 3% mol Y2O3 (top and bottom). Figure 11. The calculated densities of states of ZrO2 + 3% mol Y2O3 (top and bottom). The calculated above electronic densities of ZrO2 + 3% mol Y2O3 should be compared with the ZrO2 model. 3.1. ZrO2 : crystal and energy bands structures. We can observe the DOS spectra peculiarities for ZrO2 + 3% mol Y2O3 that possess visibly higher amplitudes in comparison with ZrO2 ones. This observation could be a precursor for a charge re-distribution process, anticipating a charge conduction of agents as like as water molecule on the ZrO2 + 3% mol Y2O3 surface. Figure 12. (Color online) A model structure of the H2O/ZrO2. The structure model (ZrO2+H2O) includes 16 atoms of Zr and 32 atoms O of the periodic surface, and one water molecule. The zirconium atoms are shown in green, oxygen atoms - in red, hydrogen atom - in white. The distance between atom of oxygen of H2O and first layer is 6.334199 Bohr. Figure 12. (Color online) A model structure of the H2O/ZrO2. The structure model (ZrO2+H2O) includes 16 atoms of Zr and 32 atoms O of the periodic surface, and one water molecule. The zirconium atoms are shown in green, oxygen atoms - in red, hydrogen atom - in white. The distance between atom of oxygen of H2O and first layer is 6.334199 Bohr. 12 CMSMS17 CMSMS17 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 3.3. H2O/ZrO2 – system. So far, the use of DFT for the above listed molecular systems has aimed on the accurate description of their comparative electronic structures. As one expect, the replacing of zirconium with yttrium causes a large shift in the energy gap, making the compound ZrO2 + 3% mol Y2O3 a more active target for the water absorption. Next, the DFT calculated results are compared respectively for the systems: (3) H2O / ZrO2 (Figure 12) and (4) H2O / ZrO2 + 3% mol Y2O3 (Figure 14). g g In Figures 12-13 and 14-15 the configuration snapshots and calculated DOS are shown for H2O/ZrO2 and H2O/ZrO2+ 3% mol Y2O3, respectively. The important observation is that the inducing 3% mol Y2O3 doping to system H2O/ZrO2 brought to the shift of DOS amplitude from 4 eV to 0. This is obviously indicate on a charge redistribution of the water interaction with zirconia surface as a result of ittrium ion doping. Concluding the DFT results with the experimental data, it is worth noting that the idea is that, when molecule of water approaching on the surface of ZrO2 (ZrO2+3% mol Y2O3) it's kinetic energy leads to a change the DOS and the electron density of the ZrO2 (ZrO2+3% mol Y2O3). On the basis of the first Hohenberg and Kohn theorem the total energy calculated with this density of a electron system ( )r  will always be larger or equal then the ground state energy 0 E   0 E ( ) E r   (2) (2) At the kinetic energy transition of the water molecule T to above system (ZrO2) according the first Hohenberg theorem the electron density of the system should be changed:   0 E ( ) ( ) E T r r      (3) (3) Depending on the distance between water molecule and first layer of ZrO2 (ZrO2+3% mol Y2O3) the kinetic energy transition will be different. Hence, in this simulation the distances between the water molecules and the oxide surface was chosen for the 4 values. Figure 13. The calculated densities of states of H2O/ZrO2 (top, middle and bottom). The distance between atom of oxygen of H2O and first layer is 2.6205095 Bohr. Figure 13. The calculated densities of states of H2O/ZrO2 (top, middle and bottom). The distance between atom of oxygen of H2O and first layer is 2.6205095 Bohr. 3.3. H2O/ZrO2 – system. 13 CMSMS17 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 H2O/ZrO2+ 3% mol Y2O3- system 3.4. H2O/ZrO2+ 3% mol Y2O3- system 3.4. H2O/ZrO2+ 3% mol Y2O3- system y Concluding the DFT results with the experimental data, it is worth noting that the idea is that, when molecule of water approaching on the surface of ZrO2 (ZrO2+3% mol Y2O3) leads to a Figure 14. (Color online) A model structure of the H2O/ZrO2+ 3% mol Y2O3. The structure model (H2O/ZrO2+ 3% mol Y2O3) includes 15 atoms of Zr, 1 atom Y and 32 atoms O of the periodic surface, and one water molecule. The zirconium atoms are shown in green, oxygen atoms - in red, hydrogen atom - in white and yttrium atom - in cyan. The distance between atom of oxygen of H2O and first layer is 1.32077 Bohr. Figure 14. (Color online) A model structure of the H2O/ZrO2+ 3% mol Y2O3. The structure model (H2O/ZrO2+ 3% mol Y2O3) includes 15 atoms of Zr, 1 atom Y and 32 atoms O of the periodic surface, and one water molecule. The zirconium atoms are shown in green, oxygen atoms - in red, hydrogen atom - in white and yttrium atom - in cyan. The distance between atom of oxygen of H2O and first layer is 1.32077 Bohr. Figure 15. The calculated densities of states of H2O/ZrO2+ 3% mol Y2O3 (top and bottom). The distance between atom of oxygen of H2O and first layer is 2.6205095 Bohr. Figure 15. The calculated densities of states of H2O/ZrO2+ 3% mol Y2O3 (top and bottom). The distance between atom of oxygen of H2O and first layer is 2.6205095 Bohr. 14 1234567890 ‘’“” CMSMS17 IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/012013 1234567890 ‘’“” onf. Series: Journal of Physics: Conf. Series 994 (2018) 012013 doi :10.1088/1742-6596/994/1/0120 change the DOS and the electron density of the ZrO2 (ZrO2+3% mol Y2O3). On the basis of the first Hohenberg and Kohn theorem [46] the total energy calculated with this density of a electron system (r) will always be larger or equal then the ground state energy   0 0 , E ( ) E E r   , At the kinetic energy transition of the water molecule T to above system (ZrO2) according the first Hohenberg theorem the electron density of the system should be changed:   0 E ( ) ( ) E T r r      . 3.4. H2O/ZrO2+ 3% mol Y2O3- system Depending on the distance between water molecule and first layer of ZrO2 (ZrO2+3% mol Y2O3) the kinetic energy transition will be different. Hence, in this simulation the distances between the water molecules and the oxide surface was chosen for the 4 values. 4. Conclusions Density functional theory calculations have been performed on ZrO2 nanoparticles doped with Y2O3 and interacting with the water molecule to evaluate the electronic density, total energy and band structures. It is shown that the surface of ZrO2 nanoparticles can be considered as a reaction zone for electrochemical processes. The important observation is that the inducing 3% mol Y2O3 doping to system H2O/ZrO2 brought to the shift of DOS amplitude from 4 eV to 0. It’s show’s that water molecules adsorbed from the atmosphere on the surface of ZrO2-based nanoparticles leads to realization of the process of electron localization from the crystal lattice of nanoparticles and its transport outside the particles. Acknowledgments RFBR grant No.17-52-45062 ИНД_а is acknowledged. g _ g A.S. Doroshkevich, A.I. Lyubchyk acknowledges funding by the EU H2020-MSCA-RISE-2015 through the HUNTER project (grant nº 691010). A.S. Doroshkevich, A.I. Lyubchyk acknowledges funding by the EU H2020-MSCA-RISE-2015 through the HUNTER project (grant nº 691010). 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https://openalex.org/W3099574012	https://repository.ubn.ru.nl/bitstream/handle/2066/230387/1/230387.pdf	English	null	MRI-Based 3-Dimensional Visualization Workflow for the Preoperative Planning of Nephron-Sparing Surgery in Wilms’ Tumor Surgery: A Pilot Study	Journal of healthcare engineering	2,020	cc-by	5,212	MRI-Based 3-Dimensional Visualization Workflow for the Preoperative Planning of Nephron- Sparing Surgery in Wilms' Tumor Surgery: A Pilot Study Fitski, Matthijs; Meulstee, J.W.; Littooij, Annemieke S.; Ven, Cornelis P. van de; Steeg, Alida F.W. van der; Wijnen, M.H.W.A. 2020, Article / Letter to editor (Journal of Healthcare Engineering, 2020, (2020), article 8899049) Doi link to publisher: https://doi.org/10.1155/2020/8899049 Version of the following full text: Publisher’s version Downloaded from: https://hdl.handle.net/2066/230387 Download date: 2024-10-24 MatthijsFitski ,1JeneW.Meulstee ,2AnnemiekeS.Littooij ,3,4CornelisP.vandeVen,1 Alida F. W. van der Steeg,1 and Marc H. W. A. Wijnen 1 1Department of Pediatric Surgery, Princess M´axima Center for Pediatric Oncology, 3584 CS Utrecht, Netherlands 23D Lab, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands 3 Department of Radiology and Nuclear Medicine, University Medical Center Utrecht/Wilhelmina Children’s Hosp 3584 CX Utrecht, Netherlands 4Department of Radiology and Nuclear Medicine, Princess M´axima Center for Pediatric Oncology, 3584 CS Utrecht, Netherlands Correspondence should be addressed to Marc H. W. A. Wijnen; m.h.w.wijnen-5@prinsesmaximacentrum.nl Received 10 July 2020; Revised 21 October 2020; Accepted 29 October 2020; Published 9 November 2020 Academic Editor: Zhihan Lv Academic Editor: Zhihan Lv Copyright © 2020 Matthijs Fitski 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. Purpose. Due to the size and localization of Wilms’ tumor (WT), nephron-sparing surgery (NSS) is only possible in a limited number of cases. When NSS is considered, the surgeon preoperatively requires a thorough understanding of the patient-speciﬁc anatomy to prevent positive surgical margins and other complications. Trough a collaboration between the radiology and pediatric surgery departments and 3D imaging specialists, a 3D visualization workﬂow was developed to improve preoperative planning of NSS for WT patients. Methods. Te 3D visualization workﬂow combines a MRA sequence, a segmentation protocol, and augmented reality (AR) visualization, additional to in-house 3D printing. A noncontrast-enhanced MRA scan was added to the MRI protocol. MRI sequences were segmented with a segmentation protocol in an open-source software package. Te resulting 3D models were visualized in AR with a HoloLens and 3D print. Results. In a pilot study, ﬁve WTpatients eligible for NSS were preoperatively planned through the 3D visualization workﬂow. AR visualization software was fast and free to use and allowed adequate handling of the 3D holograms. Te 3D printed models were considered convenient and practical for intraoperative orientation. Te patient-friendly, fast, and low-cost 3D visualization workﬂow was easily implemented and appeared to be valuable for the preparation of NSS. Conclusion. Tis pilot study demonstrates how a strong collaboration between the pediatric surgery and radiology departments and 3D imaging specialists will help to shape the future of pediatric oncological surgery. Tis 3D visualization workﬂow aims to prepare pediatric oncological surgeons for nephron-sparing surgery in patients with Wilms’ tumors. nephrectomy for local treatment, nephron-sparing surgery (NSS) can be used for nephrogenic preservation. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Hindawi Hindawi Journal of Healthcare Engineering Volume 2020, Article ID 8899049, 6 pages https://doi.org/10.1155/2020/8899049 MatthijsFitski ,1JeneW.Meulstee ,2AnnemiekeS.Littooij ,3,4CornelisP.vandeVen,1 Alida F. W. van der Steeg,1 and Marc H. W. A. Wijnen 1 Tis helps to protect the patient from excessive functional parenchymal loss in the future [3]. NSS is a technically demanding procedure and it requires a thorough preoperative under- standing of the patient-speciﬁc renal anatomy and intra- parenchymal vasculature [4]. Patients with bilateral disease, with or without a predisposing syndrome, might be eligible for NSS depending on the tumor location, size, and inﬁl- tration. Due to the risk of a positive surgical margin with Research Article MRI-Based 3-Dimensional Visualization Workflow for the Preoperative Planning of Nephron-Sparing Surgery in Wilms’ Tumor Surgery: A Pilot Study MatthijsFitski ,1JeneW.Meulstee ,2AnnemiekeS.Littooij ,3,4CornelisP.vandeVen,1 Alida F. W. van der Steeg,1 and Marc H. W. A. Wijnen 1 1. Introduction Te standard pediatric kidney tumor MRI protocol was performed at presentation and after the neo- adjuvant chemotherapy in accordance with the SIOP-RTSG Umbrella protocol. A 1.5 Tesla system (Achieva, Philips Medical Systems, Best, Netherlands) was used for all pa- tients. Te imaging protocol consisted of coronal 3D T2- weighted (-W) sequence, fat-suppressed T1-W sequence, diﬀusion-weighted imaging (b values of at least 0, 100, and 800 s/mm2), and a fat-suppressed T2-W sequence. Before administering the contrast agent, the NC-MRA sequence was acquired. During the administration of intravenous contrast (Gadovist, Bayer Pharma, Berlin, Germany, at a dose of 0.1 mmol/kg body weight), a 4D contrast-enhanced MRA was acquired, after which a postcontrast fat-sup- pressed T1-W sequence was performed. Children were awake, sedated, or under anesthesia depending on their ability to cooperate. Hyoscine butylbromide (Buscopan, Boehringer Ingelheim Limited, Bracknell, UK) was ad- ministered at an intravenous dose of 0.4 mg/kg body weight to reduce the peristaltic artefacts. Te used NC-MRA is an inﬂow-enhanced balanced Steady State Free Precession (b-SSFP) sequence. Tis se- quence has a unique T2/T1 contrast, which has a high contrast for blood. Te sequence is scanned with a high reconstructed resolution (0.56, 0.56, 1 mm), takes 3–5 minutes to scan, is independent of direction, and does not require a contrast agent. An inversion time (TI) of 450 ms was used. If 450 ms was not possible due to a fast heart rate, the TI was lowered based on the maximal allowed TI (90% of the R-R interval). Te cardiac trigger was set on a heartbeat measured preferably with a three-lead electrocardiogram or else with a physiological pulse unit. Te transverse ﬁeld of view (FOV) was set parallel to the renal artery of the aﬀected kidney in the coronal view using the 3D T2-W sequence. Te FOV encompasses the complete intraparenchymal arterial branch. A saturation band was positioned below the lower pole of the kidneys and a fat saturation band was positioned at the ventral aspect of the abdomen. Tis allowed saturation of signal from the vena cava and abdominal fat. High-quality imaging is crucial for high-ﬁdelity anatomical models, as the imaging quality primarily determines the model quality. Preoperative magnetic resonance imaging (MRI) is already a vital part of the SIOP-RTSG Umbrella treatment protocol and can be used for 3D visualization, as Wake et al. have previously shown for renal cancer in adults [13]. 1. Introduction Wilms’ tumor (WT) is the second most common abdominal pediatric tumor in Europe, with children being diagnosed at a median age of approximately 3.5 years [1]. In accordance with the International Society for Pediatric Oncolo- gy—Renal Tumor Study Group (SIOP-RTSG) Umbrella treatment protocol, therapy generally consists of neo- adjuvant chemotherapy, followed by radical nephrectomy and adjuvant chemotherapy [2]. In contrast to a radical Journal of Healthcare Engineering 2 planning of NSS in WT patients. In this workﬂow, we addressed the limitations in usability and model quality previously described in literature. An overview of the pro- posed workﬂow is given in Figure 1. Te following sections describe the employment of the noncontrast-enhanced MRA (NC-MRA) sequence, segmentation protocol, and visualization with Augmented Reality (AR) and with 3D printing. NSS, unilateral nonsyndromic patients are treated with a radical nephrectomy. However, in order to prevent late eﬀects of a radical nephrectomy at a young age, these pa- tients might be still considered for NSS if they have a small lesion at a favorable location at the moment of diagnosis [2]. Terefore, careful selection and preoperative planning are crucial to ensure a positive oncological outcome in com- bination with low morbidity. y For the preoperative planning of NSS, patient-speciﬁc 3-dimensional (3D) anatomical models are increasingly used [5–7]. Tis is due to the improved and more ac- cessible imaging, segmentation, and visualization tech- niques. 3D printing is a visualization technique which can be used to visualize these patient-speciﬁc models. Te positive eﬀects of 3D printed anatomical models in adults have been described and include reducing blood loss, reducing intraoperative complications, and improving patient education [5]. In renal surgery for pediatric on- cology, 3D printed anatomical models are not frequently used and if so, only on a case-by-case basis [3, 8–11]. Regarding NSS, 3D printed models are mainly useful for assisting in planning the vascular dissection. However, the vasculature information in current 3D models remains poor, primarily due to low image quality [8]. Retro- spectively, personalized 3D anatomical models have shown a signiﬁcant improvement of the anatomical un- derstanding for the renal artery, vein, tumor, and urinary collecting system and may potentially help pediatric surgeons prepare for NSS [12]. Te models were limited due to low image quality and modelling techniques which are labor-intensive, require a long processing time, and are expensive. 2.1. Imaging. 1. Introduction An additional computed tomography angiography (CTA) scan can be performed for high quality arterial imaging, yet it is un- desirable due to the radiation and contrast administration. Moreover, an additional CTA scan prolongs the preoperative workup which is already considered highly stressful for pe- diatric patients. Terefore, techniques solely based on the preoperative MRI are favorable. Wake et al. reported challenges in standardized high-resolution imaging and were also limited by the manual segmentation procedure. Image processing took around 7 hours and 3D printing costs were around $1000 (US dollar) per model. To overcome these limitations, our aim is to develop a 3D visualization workﬂow in which imaging, seg- mentation, and visualization techniques are combined, to suit the speciﬁc preoperative requirements needed for planning NSS of WT in pediatric patients. 2.2. Segmentation. A selection of the MRI sequences (3D T2-W, NC-MRA, postcontrast fat-suppressed T1-W) was used to perform the segmentation in open-source software package 3D Slicer 4.10.2. A standardized protocol was de- veloped for the segmentation of the arteries, veins, urine collecting system (UCS), tumor, and kidney. Firstly, the NC- MRA sequence was used to compute the segmentation of the arteries through an intensity-based threshold technique. Te “scissor” tool was used to remove artefacts and the resulting 2.2. Segmentation. A selection of the MRI sequences (3D T2-W, NC-MRA, postcontrast fat-suppressed T1-W) was used to perform the segmentation in open-source software package 3D Slicer 4.10.2. A standardized protocol was de- veloped for the segmentation of the arteries, veins, urine collecting system (UCS), tumor, and kidney. Firstly, the NC- MRA sequence was used to compute the segmentation of the arteries through an intensity-based threshold technique. Te “scissor” tool was used to remove artefacts and the resulting 2. Materials and Methods In close collaboration with the departments of radiology and pediatric surgery, 3D imaging specialists have designed a new 3D visualization workﬂow for the preoperative 3 3 Journal of Healthcare Engineering Diagnosis Preoperative MRI Segmentation Surgery Planning Visualization Figure 1: Schematic overview of the proposed preoperative 3D visualization workﬂow. After diagnosis and neoadjuvant chemotherapy, a WT patient receives a preoperative MRI. A high-resolution noncontrast-enhanced MRA sequence is added to the protocol to allow for visualization of the intraparenchymal arteries. Te MRI scans are segmented through a standardized segmentation protocol in 3D Slicer resulting in patient-speciﬁc 3D anatomical models. Te 3D models are displayed for preoperative planning in augmented reality through a HoloLens. 3D printed models can be brought inside the operating theater. Visualization Visualization Planning Surgery Figure 1: Schematic overview of the proposed preoperative 3D visualization workﬂow. After diagnosis and neoadjuvant chemotherapy, a WT patient receives a preoperative MRI. A high-resolution noncontrast-enhanced MRA sequence is added to the protocol to allow for visualization of the intraparenchymal arteries. Te MRI scans are segmented through a standardized segmentation protocol in 3D Slicer resulting in patient-speciﬁc 3D anatomical models. Te 3D models are displayed for preoperative planning in augmented reality through a HoloLens. 3D printed models can be brought inside the operating theater. model was smoothed with a Gaussian ﬁlter. Te arterial segmentation was used as an overlay in the T2-W sequence in order to diﬀerentiate intraparenchymal arteries and veins. Subsequently, the veins and UCS were segmented with an intensity-based brush in the aforementioned sequence. Te postcontrast fat-suppressed T1-W sequence was used for the segmentation of the tumor and kidney with a Grow-Cut algorithm [14]. Tis algorithm uses labels in the area of the tumor, kidney, and background in several slices in order to compute the border between the three labels. Te results were ﬁltered with a joint-smoothing ﬁlter. Any under- or oversegmentations were manually corrected. Te resulting segmentations were exported as a stereolithography (.STL) ﬁle. could be shared on a PC through a live stream to allow for an interactive discussion by the user and observers. Tis vi- sualization technique has been free of costs after develop- ment of software and purchasing the HoloLens. To create physical anatomical models, an Ultimaker S5 dual-extrusion printer was used with a Fused Filament Fabrication technique. 2. Materials and Methods To allow visualization of the renal pelvis, the 3D modelled kidney could be bisected in Meshmixer 3.5.474 (Autodesk, Inc., San Francisco, CA, USA) with a plane cut prior to printing. Te resulting models were printed with a ﬁne layer proﬁle of 0.1 mm thick, inﬁll density of 30% (zigzag inﬁll pattern), and support overhang angle of 60°. Te models were printed with two diﬀerent colors which allowed us to improve the contrast of speciﬁc anatomical regions. 3D printed models allowed the surgeons to get a sense of the tumor size, and the model could be taken into the OR by an assistant to help the surgeon navigate during the procedure. 2.3.Visualization. In order to visualize the 3D models in the .STL format, the T2-W images (DICOM format) were added to AR software developed in Unity 5.5.2 (Unity Technolo- gies, San Francisco, CA, USA). AR software ensured that the 3D models and MRI images were spatially aligned. Subse- quently, AR software computed a patient-speciﬁc container which could be uploaded to a visualization library installed on the head-mounted display. 3. Results Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Gender (M/F) F F M F F Age (Y) 8 2 4 5 3 Disease Unifocal right Unifocal right, bilateral nephroblastomatosis Unifocal left Bilateral, multifocal Bilateral, nephroblastomatosis Syndrome — Beckwith–Wiedemann syndrome 16p12.2 deletion — WT-1 mutation Procedure NSS NSS NSS NSS NSS NC-MRA scanning duration (min: sec) 04:50 03:55 03:55 04:50 03:28 Segmentation time (min) 29 41 34 73 28 Volume tumor segmentation (ml) 1.4 4.0 69.2 24; 17; 1.2; 0.3; 3.4; 11.6; 0.12 2.3 3D printing time (hours:min) 18:39 15:41 16:39 31:26 18:10 3D printing cost (€) 3.58 2.57 3.06 5.80 3.27 NSS nephron-sparing surgery. display costs 3000 euros, and all software packages are open- source or self-developed. Limitations in previous studies on 3D modelling for WT included low image quality, long segmentation, and long visualization processing. Wake et al. reported a 3D printing cost per model of ±$1000 (US dollars), a segmentation time of ±7 hours, and 3D printing time of ±10 hours [13]. Wellens et al. reported an average cost of $400 USD per printed model and a segmentation and 3D printing manufacturing time of 4 to 5 days [12]. In this pilot study, we have addressed these limitations through the development of our own innovative workﬂow in collabo- ration with the departments of pediatric surgery, radiology, and 3D imaging specialists. Te segmentation protocol of the workﬂow allowed for fast 3D modelling and the Hol- oLens proved to be a fast and useful visualization tool. Our 3D printing technique was slow (±20 hours) because of the ﬁne layer proﬁle (0.1 mm thickness). Increasing the layer proﬁle would decrease the printing time signiﬁcantly. Our in-house 3D printer is limited in color and materials, yet it was of suﬃcient quality, and with an average price of ±3.50 euros and machine cost of 5500 euros, it was considered a sustainable technique. Manual removal of supporting structures during the post- processing took about half an hour. Te MRA sequence added to the preoperative MRI was successful in all patients. Te segmentation and visualization in AR were performed within a day after the preoperative MRI. Figure 2 shows a 2D rendering in 3D Slicer, a 3D hologram visualized with the head-mounted display, and a 3D print of a 3D anatomical model, all of the same patient. 3. Results Between May 2019 and August 2019, a pilot study was performed. Five patients were considered for NSS, and their surgeries were preoperatively planned with the 3D visuali- zation workﬂow in addition to the standard protocol. Te patients’ ages ranged from 2 to 8 years (mean 5.2 ± 1.4 years). Patient demographics, tumor characteristics, and relevant technical outcomes for each patient are described in Table 1. T dd l d f h Between May 2019 and August 2019, a pilot study was performed. Five patients were considered for NSS, and their surgeries were preoperatively planned with the 3D visuali- zation workﬂow in addition to the standard protocol. Te patients’ ages ranged from 2 to 8 years (mean 5.2 ± 1.4 years). Patient demographics, tumor characteristics, and relevant technical outcomes for each patient are described in Table 1. Te mean additional scanning time required for the NC- MRA sequence was 04:12 ± 00:36 minutes. Te mean seg- mentation time was 41 ± 19 minutes, which seemed closely correlated to the number and size of the lesions. Te mean volume of the resulting tumor 3D model was 12.2 ± 20.5 ml per lesion. Average 3D printing time was roughly 20 hours. Preoperatively, the surgeon reviewed the patient-speciﬁc AR hologram in a real-world setting to prepare for surgery. Te surgeon had full control over the hologram: the surgeon could translate, rotate, and scale the hologram. Additionally, the transparency of the individual anatomical models could be adjusted, and the individual models could be removed. It was possible to look at the T2-W MRI in three diﬀerent planes (transverse, sagittal, and coronal) in order to correlate the orientation of the 3D models with the more commonly known MRI images. Te AR-display from the HoloLens Te mean additional scanning time required for the NC- MRA sequence was 04:12 ± 00:36 minutes. Te mean seg- mentation time was 41 ± 19 minutes, which seemed closely correlated to the number and size of the lesions. Te mean volume of the resulting tumor 3D model was 12.2 ± 20.5 ml per lesion. Average 3D printing time was roughly 20 hours. Journal of Healthcare Engineering 4 Table 1: Preoperative patient demographics, tumor characteristics, and pathologic outcomes for each patient together with the noncontrast- enhanced MRA (NC-MRA) scanning duration, the duration of the complete segmentation, the tumor volume derived through 3D Slicer, and 3D printing time and costs. 3. Results Te 3D printers generally printed the models overnight allowing ample time for the surgical team to assess the 3D models. Te arterial models visualized the intraparenchymal arteries up to the second or third segmental arterial branch. Te level of detail of the vein and UCS 3D models was noticeably less than the level of detail of the arterial models. However, surgeons considered the vein and UCS 3D models supportive and additional to the MRI imaging. Te AR visualization allowed the surgeons to assess the depth of resection with regard to renal arteries, veins, and UCS. Being able to scale, move, rotate, and walk around the hologram was very useful for the understanding of the patient-speciﬁc anatomy. Intraoperatively, an assistant showed the 3D printed models to visualize the location and rotation of the tumor in relation to the renal parenchyma. Tis proved to be mainly useful in the patients with multiple lesions. It is diﬃcult to quantify the advantages of 3D anatomical models for the preoperative planning of NSS for WT. An increase in surgical conﬁdence for NSS for WT has been shown retrospectively [12], but quantifying the advantage remains subjective [12]. In adults, 3D printed renal models based on preoperative MRI scans could help during surgical decision-making [6]. Moreover, 3D printed models did allow adult surgeons to improve their translation from 2D CT and MRI data into 3D anatomical relationships, which appeared more relevant in smaller lesions [15]. Although the results from these studies may not be directly applicable to children because image quality is generally superior in adults [16], we expect that 3D printed models could help in the preparation of pediatric oncologic surgery. Te clinical advantage of the described 3D visualization workﬂow for children may be quantiﬁed in the future. We need to 4. Discussion Previously, no signiﬁcant diﬀerence between 3D visualization techniques (AR or 3D printing) was found for WT patient-speciﬁc models [12]. However, we currently believe AR is the most desirable visualization technique due to the opportunity to develop Mixed Reality concepts. In the future, the HoloLens could allow intraoperative kidney-model matching. Mixed Reality models are used in adult laparoscopic renal surgery through registration of the 3D model with the laparoscopic image [7]. With Mixed Reality, rigid matching through an anatomical landmark registration has been described for open visceral surgery [20]. However, to the best of our knowledge, Mixed Reality for open renal surgery has not been described yet. In the context of NSS, this would allow the surgeon to get a sense of depth and inﬁltration of the tumor, and super- imposing vasculature could assist surgeons to determine the resection margins. patients. Te proposed workﬂow appears useful during the preparation of NSS for bilateral or syndromic WT patients. In unilateral nonsyndromic patients, NSS is only performed in a very speciﬁc group of patients as controversies arise due to the inherent increased risk of positive surgical margins [3]. Current ﬁgures report a positive surgical margin of the sparingly removed tumor masses (treated with the Chil- dren’s Oncology Group protocol) between 15.7% and 31% [17, 18]. In accordance with the SIOP-RTSG Umbrella protocol, in the case of a positive surgical margin these young patients will need additional chemotherapy and possibly radiotherapy. In most cases, it is unknown how these positive margins occurred. Te 3D visualization workﬂow may help surgeons to better understand compli- cated pathologic and anatomic regions and give an improved insight on where and how these positive margins occur. Additionally, 3D modelling may assist during the diﬃcult patient selection for NSS through an increased under- standing of the patient’s anatomy. Tis twofold advantage might lead to fewer positive surgical margins and improved oncological outcomes. g In order to achieve improved clinical outcomes, all anatomical structures require a high-ﬁdelity 3D model. However, the segmentation of the veins and UCS remains a manual and problematic task. Te overlay of the arterial segmentation helps to diﬀerentiate between arterial and venous vasculature. Currently, this technique is insuﬃcient for the accurate segmentation of intraparenchymal veins, likely due to intrasequential movement. Te UCS was dif- ﬁcult to segment, as the full extent of the UCS is generally diﬃcult to appreciate with standard imaging. 4. Discussion A combined imaging, protocolled segmentation, and visu- alization workﬂow resulted in patient-speciﬁc 3D anatom- ical models for the preoperative planning of WT patients. Te 3D visualization workﬂow aimed to help pediatric surgeons improve their understanding of anatomical rela- tionships and orientation. Because of the preparation time of about 1 hour and a printing time of 20 hours, this 3D vi- sualization workﬂow can be completed within a limited timeframe of two days between the preoperative MRI and surgery. Additionally, it is inexpensive as the head-mounted 5 Journal of Healthcare Engineering (a) (b) (c) Figure 2: Tree diﬀerent visualization techniques for the MRI-based 3D models of patient 3. Te patient has a transposition of the inferior vena cava below the superior mesenteric artery. (a) 2D screenshot of 3D rendering in 3D Slicer with a coronal T2-weighted MRI slice. (b) Augmented reality through the head-mounted display (HoloLens), holding the blue “cube” which allows for translation of the hologram. (c) 3D printed model printed with polylactic acid in Ultimaker S5, and the kidney is bisected. (b) (c) (a) (a) (b) (c) Figure 2: Tree diﬀerent visualization techniques for the MRI-based 3D models of patient 3. Te patient has a transposition of the inferior vena cava below the superior mesenteric artery. (a) 2D screenshot of 3D rendering in 3D Slicer with a coronal T2-weighted MRI slice. (b) Augmented reality through the head-mounted display (HoloLens), holding the blue “cube” which allows for translation of the hologram. (c) 3D printed model printed with polylactic acid in Ultimaker S5, and the kidney is bisected. [4]. For this reason, despite the low model quality of the veins and UCS, the overall models were considered to be of suﬃcient value and usability. dd h l understand how 3D modelling for preoperative planning inﬂuences pediatric surgical decision-making and conﬁ- dence in order to understand how these models beneﬁt our patients. In addition to accurate segmentations, the visualization of patient-speciﬁc 3D models is paramount to how the models are understood. Augmented reality visualization through a HoloLens oﬀered a viable and fast technique for the visualization. A hologram is computed more easily and less costly in comparison to 3D printing. However, there is no consensus on whether there is a signiﬁcant clinical ad- vantage to the use of AR instead of other visualization techniques such as 3D printing, 2D rendering on a computer monitor, or volume-rendering. Data Availability Te .STL data used for the 3D models for this study are available from the corresponding author upon request. [13] N. Wake, T. Rude, S. K. Kang et al., “3D printed renal cancer models derived from MRI data: application in pre-surgical planning,” Abdominal Radiology, vol. 42, no. 5, pp.1501–1509, 2017. 5. Conclusions [9] ´A. S´anchez-S´anchez, ´O. Gir´on-Vallejo, R. Ruiz-Pruneda et al., “Tree-dimensional printed model and virtual reconstruc- tion: an extra tool for pediatric solid tumors surgery,” Eu- ropean Journal of Pediatric Surgery Reports, vol. 6, no. 1, pp. e70–e76, 2018. Tis pilot study demonstrates how a strong collaboration between the pediatric surgery and radiology departments and 3D imaging specialists will help to shape the future of pediatric oncological surgery. A combination of speciﬁc high-resolution MRI sequences, protocolled segmentation techniques, and AR visualization improved the visualization for the preoperative planning of pediatric renal tumors. Tis designed 3D visualization workﬂow is an easily imple- mentable technique to help pediatric oncological surgeons prepare for nephron-sparing surgery in patients with Wilms’ tumors. [10] J. Schenk, K.-L. Waag, N. Graf et al., “3D-visualization by MRI for surgical planning of Wilms tumors,” R¨oFo-Fortschritte auf dem Gebiet der R¨ontgenstrahlen und der Bildgebenden Ver- fahren, vol. 176, no. 10, pp. 1447–1452, 2004. [11] D. Zhang, G. Zeng, Y. Zhang et al., “3D reconstruction computed tomography scan in diagnosis of bilateral Wilm’s tumor with its embolus in right atrium,” Journal of X-Ray Science and Technology, vol. 24, no. 5, pp. 657–660, 2016. [12] L. M. Wellens, J. Meulstee, C. P. van de Ven et al., “Com- parison of 3-dimensional and augmented reality kidney models with conventional imaging data in the preoperative assessment of children with wilms tumors,” JAMA Network Open, vol. 2, no. 4, Article ID e192633, 2019. Journal of Healthcare Engineering Journal of Healthcare Engineering 6 review,” World Journal of Urology, vol. 38, no. 4, pp. 869–881, 2019. we aim to develop more automated segmentation proce- dures for WT patients and work towards the use of intra- operative holograms through Mixed Reality. [8] [8] ´O. Gir´on-Vallejo, D. Garc´ıa-Calder´on, R. Ruiz-Pruneda et al., “Tree-dimensional printed model of bilateral Wilms tumor: a useful tool for planning nephron sparing surgery,” Pediatric Blood Cancer, vol. 65, Article ID e26894, 2018. ´ ´ Te authors report no conﬂicts of interest. [14] V. Vezhnevets and V. Konouchine, “GrowCut: interactive multi-label ND image segmentation by cellular automata,” in Proceedings of the Graphicon, pp. 150–156, Novosibirsk, Russia, June 2005. Acknowledgments Te authors would like to acknowledge CARS 2020 for publishing this abstract in the Computer Assisted Radiology and Surgery Proceedings of the 34th International Congress and Exhibition, Munich, Germany, June 23–27, 2020. [15] N. Wake, J. S. Wysock, M. A. Bjurlin et al., ““Pin the tumor on the kidney:” an evaluation of how surgeons translate CT and MRI data to 3D models,” Urology, vol. 131, pp. 255–261, 2019. [16] D. D. Laganosky, C. P. Filson, and V. A. Master, “Surgical margins in nephron-sparing surgery for renal cell carcinoma,” Current Urology Reports, vol. 18, p. 8, 2017. Current Urology Reports, vol. 18, p. 8, 2017. Conflicts of Interest Te authors report no conﬂicts of interest. 4. Discussion More speciﬁc noncontrast imaging techniques such as noncontrast-en- hanced MRVenography based on b-SSFP and MRUrog- raphy may help further improve the model quality and may speed up the segmentation. NC-MRV has the additional potential to allow for assessment of venous tumor thrombi [19]. Nevertheless, the arterial model is considered to have the highest surgical value as this is the most relevant for NSS Our pilot study is limited by the lack of evaluation of these anatomical models. A subjective analysis through questionnaires was not performed. However, we aimed to use novel imaging and visualization techniques and im- plement them into clinical care. Te technique has been improved and should be further evaluated through the prospective use of the aforementioned questionnaires. Ad- ditionally, the 3D models should be compared with the pathology specimens in order to quantify the accuracy of the 3D models. In the future, we aim to implement and evaluate 3D imaging technology in pediatric oncologic surgery as the standard of care and evaluate the added value. Additionally, References [17] M. K. Richards, A. B. Goldin, P. F. Ehrlich et al., “Partial nephrectomy for nephroblastoma: a national cancer data base review,” Te American Surgeon, vol. 84, no. 3, pp. 338–343, 2018. [1] N. Breslow, A. Olshan, J. B. Beckwith, and D. M. Green, “Epidemiology of Wilms tumor,” Medical and Pediatric Oncology, vol. 21, no. 3, pp. 172–181, 1993. [18] A. M. Davidoﬀ, R. B. Interiano, L. Wynn et al., “Overall survival and renal function of patients with synchronous bilateral Wilms tumor undergoing surgery at a single insti- tution,” Annals of Surgery, vol. 262, no. 4, pp. 570–576, 2015. [2] M. M. Van Den Heuvel-Eibrink, J. A. Hol, K. Pritchard-Jones et al., “Position paper: rationale for the treatment of Wilms tumour in the UMBRELLA SIOP–RTSG 2016 protocol,” Nature Reviews Urology, vol. 14, no. 12, p. 743, 2017. f g y [19] L. Adams, B. Ralla, G. Engel et al., “Assessing venous thrombus in renal cell carcinoma: preliminary results for unenhanced 3D-SSFP MRI,” Clinical Radiology, vol. 73, no. 8, pp. 757.e9–757.e19, 2018. [3] J. C. H. Wilde, D. C. Aronson, B. Sznajder et al., “Nephron sparing surgery (NSS) for unilateral Wilms tumor (UWT): the SIOP 2001 experience,” Pediatric Blood & Cancer, vol. 61, no. 12, pp. 2175–2179, 2014. [20] R. Tang, L. Ma, C. Xiang et al., “Augmented reality navigation in open surgery for hilar cholangiocarcinoma resection with hemihepatectomy using video-based in situ three-dimen- sional anatomical modeling: a case report,” Medicine (Balti- more), vol. 96, no. 37, Article ID e8083, 2017. [4] T. Klatte, V. Ficarra, C. Gratzke et al., “A literature review of renal surgical anatomy and surgical strategies for partial nephrectomy,” European Urology, vol. 68, no. 6, pp. 980–992, 2015. [5] C. Lupulescu and Z. Sun, “A systematic review of the clinical value and applications of three-dimensional printing in renal surgery,” Journal of Clinical Medicine, vol. 8, no. 7, p. 990, 2019. [6] N. Wake, M. A. Bjurlin, P. Rostami, H. Chandarana, and W. C. Huang, “Tree-dimensional printing and augmented reality: enhanced precision for robotic assisted partial ne- phrectomy,” Urology, vol. 116, pp. 227-228, 2018. [7] E. Checcucci, D. Amparore, C. Fiori et al., “3D imaging applications for robotic urologic surgery: an ESUT YAUWP
https://openalex.org/W2916260299	https://erp.bioscientifica.com/downloadpdf/journals/echo/5/1/ERP-17-0062.pdf	English	null	Echocardiographic assessment of left to right shunts: atrial septal defect, ventricular septal defect, atrioventricular septal defect, patent arterial duct	Echo Research and Practice	2,018	cc-by	12,110	Introduction lesions; atrial septal defect, ventricular septal defect, atrioventricular septal defect and patent arterial duct. Congenital heart disease affects 8–12 infants per 1000 live births (1). In the United Kingdom, we perform around 10,000 surgical and interventional procedures for congenital heart defects every year. Congenital heart lesions are often detected antenatally or early in life, but diagnosis may be delayed until adulthood. Echocardiography is the perfect imaging tool for congenital heart defects; it is non-invasive, easily reproducible, relatively cheap and quick and gives excellent representation of the structural abnormalities faced. Other imaging modalities can provide complementary information, but transthoracic echocardiography is the workhorse of the congenital heart disease world, from initial diagnosis to long-term follow-up. Abstract This review article will guide the reader through the basics of echocardiographic assessment of congenital left to right shunts in both paediatric and adult age groups. After reading this article, the reader will understand the pathology and clinical presentation of atrial septal defects (ASDs), ventricular septal defects (VSDs), atrioventricular septal defects (AVSDs) and patent arterial duct. Echocardiography is the mainstay in diagnosis and follow-up assessment of patients with congenital heart disease. This article will therefore describe the echocardiographic appearances of each lesion, and point the reader towards specific features to look for echocardiographically. EDUCATIONAL SERIES IN CONGENITAL HEART DISEASE Echocardiographic assessment of left to right shunts: atrial septal defect, ventricular septal defect, atrioventricular septal defect, patent arterial duct EDUCATIONAL SERIES IN CONGENITAL HEART DISEASE Echocardiographic assessment of left to right shunts: atrial septal defect, ventricular septal defect, atrioventricular septal defect, patent arterial duct Antigoni Deri MD and Kate English MbChB PhD Yorkshire Heart Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK Correspondence should be addressed to A Deri: a.deri@nhs.net Shunt lesions Shunt lesions 5:1 REVIEW Clinical manifestations of left to right shunts •• Qp:Qs: The left to right shunt can be expressed as pulmonary (Qp) to systemic (Qs) blood flow ratio and calculated using the continuity equation (10): •• Qp:Qs: The left to right shunt can be expressed as pulmonary (Qp) to systemic (Qs) blood flow ratio and calculated using the continuity equation (10): Clinical manifestations of left to right shunts Guidelines have been published regarding the RV morphometric evaluation for children (3) and adults (4). In children, this can be challenging with two- dimensional (2D) echocardiography as measurements are underestimated when compared with MRI in children (5) as opposed to adults (6). 3D echocardiography (3DE) offers more reproducible and comparable measurements (7, 8, 9). In children, the LV dimensions should be indexed to body surface area. Guidelines have been published regarding the RV morphometric evaluation for children (3) and adults (4). In children, this can be challenging with two- dimensional (2D) echocardiography as measurements are underestimated when compared with MRI in children (5) as opposed to adults (6). 3D echocardiography (3DE) offers more reproducible and comparable measurements (7, 8, 9). In children, the LV dimensions should be indexed to body surface area. •• Elevated pulmonary arterial pressure: Large left to right shunts result in elevated pulmonary arterial pressure and resistance. The systolic RV pressure can be estimated by the peak velocity of the tricuspid regurgitation (TR) or by continuous-wave Doppler interrogation of the flow across the VSD (Fig. 1). Diastolic pulmonary pressure can be calculated from the peak and end-diastolic velocities of pulmonary regurgitation, if present. •• Elevated pulmonary arterial pressure: Large left to right shunts result in elevated pulmonary arterial pressure and resistance. The systolic RV pressure can be estimated by the peak velocity of the tricuspid regurgitation (TR) or by continuous-wave Doppler interrogation of the flow across the VSD (Fig. 1). Diastolic pulmonary pressure can be calculated from the peak and end-diastolic velocities of pulmonary regurgitation, if present. Large ducts in preterm babies that do not respond to medical therapy will in some cases render the infant ventilator dependent until the duct is closed. Small ducts may present with endocarditis, incidental murmur or abnormal calcification on the chest X-ray. Patients with sizeable left to right shunt that have been left untreated may develop pulmonary vascular disease and progressive increase in the pulmonary vascular resistance. When the latter exceeds the systemic vascular resistance the direction of shunt reverses and becomes right to left, a condition known as Eisenmenger’s syndrome. •• The shape of the interventricular septum (IVS): In RV volume overload, the IVS is displaced towards the LV in diastole with subsequent septal flattening. Presence of pulmonary hypertension will lead to systolic septal flattening. Clinical manifestations of left to right shunts Shunt at the atrial level: A murmur at the pulmonary area, breathlessness and fatigue on exertion are the most common early symptoms, although usually not until later on in childhood. Rarely infants might present with congestive heart failure. It is not uncommon, though, for children to remain entirely asymptomatic until adulthood. Adults might complain of mild breathlessness or palpitations due to atrial arrhythmia as a result of right This review article aims to describe the echocardiographic features of left to right shunt www.echorespract.com © 2018 The authors https://doi.org/10.1530/ERP-17-0062 Published by Bioscientifica Ltd © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English 5:1 R2 setting of significant pulmonary hypertension or impaired right ventricular (RV) compliance. atrial enlargement. ASDs can be an incidental finding due a systolic murmur, an abnormal chest X-ray or ECG while the patient is being assessed for other problems. •• Chamber dilatation: Left to right shunts lead to volume overload of the cardiac chambers; atrial shunts cause dilatation of the right atrium (RA) and RV. Shunts at the ventricular level and ducts cause left heart dilatation. •• Chamber dilatation: Left to right shunts lead to volume overload of the cardiac chambers; atrial shunts cause dilatation of the right atrium (RA) and RV. Shunts at the ventricular level and ducts cause left heart dilatation. •• Chamber dilatation: Left to right shunts lead to volume overload of the cardiac chambers; atrial shunts cause dilatation of the right atrium (RA) and RV. Shunts at the ventricular level and ducts cause left heart dilatation. Shunt at the ventricular level or the duct: These present early on in life with increased work of breathing, difficulty with feeding and poor weight gain. A murmur might not be audible immediately after birth due to the elevated pulmonary vascular resistance. As this falls in the subsequent weeks, a large defect will cause increased pulmonary blood flow leading to congestive heart failure, and the infant will be more susceptible to respiratory infections. Small ventricular defects can be detected incidentally due to a heart murmur. They are usually managed conservatively but patients might present with secondary problems such as endocarditis, aortic regurgitation, subaortic obstruction and double- chambered right ventricle. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ Qp Qs RVOT VTI RVOT area LVOT VTI LVOT area / / = × × The Bernoulli equation allows the calculation of the pressure gradient (PG) between the right and left ventricles, in this case PG = 4 × 4.72 = 88.36 mmHg. The left ventricular systolic pressure is assumed to be equal to the cuff-measured systolic pressure provided, there is no left ventricular outflow tract obstruction. In this case, the systolic blood pressure of the patient was 115 mmHg. The right ventricular pressure will be the difference of the LV systolic pressure and the PG between the two ventricles = 115−88.36 = 26.64 mmHg. A Deri and K English Shunt lesions R3 5:1 5:1 R3 •• Secondary effects: For example, a significant VSD might lead to dilatation of the mitral valve annulus and subsequent mitral regurgitation. •• Secondary effects: For example, a significant VSD might lead to dilatation of the mitral valve annulus and subsequent mitral regurgitation. Ao * TV RV A B B •• Association with other congenital cardiac malformations: The echocardiographic investigation should be thorough, following a sequential segmental approach. Some of those malformations might be unmasked only after the shunt lesion is closed e.g. the severity of a mitral stenosis may become apparent after closure of a large ASD. •• Association with other congenital cardiac malformations: The echocardiographic investigation should be thorough, following a sequential segmental approach. Some of those malformations might be unmasked only after the shunt lesion is closed e.g. the severity of a mitral stenosis may become apparent after closure of a large ASD. Atrial septal defects (ASDs) C Types of interatrial communications The ostium primum defect is a type of atrioventricular septal defect characterised by the presence of a common atrioventricular (AV) junction without a ventricular component to the defect. This will be described in the AVSD section (Fig. 2). Ostium secundum defects are true deficiencies in the primary septum and the most common type of interatrial communication. They vary in shape and size and can be single or multiple. Sinus venosus defects are not true ASDs. These are venous abnormalities characterised by the anomalous insertion of right pulmonary veins into the wall of the superior vena cava (SVC). The SVC enters the atrial mass so that it ‘over-rides’ the atrial septum permitting flow Qp Qs RVOT VTI RVOT area LVOT VTI LVOT area / / = × × Left to right shunts share common haemodynamic features, which should be assessed by 2D imaging, colour flow Doppler mapping and spectral Doppler. We will review them before discussing each lesion individually: RVOT = right ventricular outflow tract, LVOT = left ventricular outflow tract, VTI = velocity time integral. Paradoxically, in patients with a persistent duct, the area of the RVOT and corresponding VTI are used to calculate the systemic blood flow and the pulmonary blood flow is calculated using the LVOT area and VTI. Qp: Qs should be used cautiously as there are potentially sources of error. The calculation of the RVOT and LVOT area is based on the measurement of the diameter of these outflow tracts, which is then squared in order to calculate the corresponding area. This renders the estimation of Qp:Qs highly sensitive to errors as small differences in the measurement will lead to significant discrepancies in the final result. Moreover, the measurement of the RVOT •• Size of the shunt: The size of the shunt is proportional to the size of the defect and the relative resistances of the systemic and pulmonary vascular beds. •• Direction of flow: This is usually left to right. The flow across the ASD is phasic occurring predominantly in late ventricular systole and early diastole with augmentation of the shunt during atrial contraction (2). The shunt across the VSD is present throughout the cardiac cycle and provides increased blood flow to the pulmonary circulation, resulting in left heart dilatation. Right to left or bidirectional shunt can be seen in the •• Direction of flow: This is usually left to right. The flow across the ASD is phasic occurring predominantly in late ventricular systole and early diastole with augmentation of the shunt during atrial contraction (2). The shunt across the VSD is present throughout the cardiac cycle and provides increased blood flow to the pulmonary circulation, resulting in left heart dilatation. Right to left or bidirectional shunt can be seen in the © 2018 The authors Published by Bioscientifica Ltd © 2018 The authors Published by Bioscientifica Ltd A Deri and K English can be challenging given the sometimes challenging or Ao * TV RV A B C Figure 1 Parasternal short-axis view showing a perimembranous VSD () partially covered by aneurysmal tricuspid valve tissue in 2D (A) and colour Doppler (B). (C) Shows the Continuous Doppler trace across the defect. Figure 1 Parasternal short-axis view showing a perimembranous VSD () partially covered by aneurysmal tricuspid valve tissue in 2D (A) and colour Doppler (B). (C) Shows the Continuous Doppler trace across the defect. The Bernoulli equation allows the calculation of the pressure gradient (PG) between the right and left ventricles, in this case PG = 4 × 4.72 = 88.36 mmHg. The left ventricular systolic pressure is assumed to be equal to the cuff-measured systolic pressure provided, there is no left ventricular outflow tract obstruction. In this case, the systolic blood pressure of the patient was 115 mmHg. The right ventricular pressure will be the difference of the LV systolic pressure and the PG between the two ventricles = 115−88.36 = 26.64 mmHg. Superior sinus venosus defect Inferior sinus venosus defect ASD within Oval Fossa Ostium primum ASD (AVSD) Coronary sinus defect Figure 2 Different types of interatrial communications. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45 via Open Access. This work is licensed under a Creative Co Attribution-NonCommercial 4.0 International L http://creativecommons.org/licenses/by- Superior sinus venosus defect PG = 4 × 4.72 = 88.36 mmHg. The left ventricular systolic pressure is assumed to be equal to the cuff-measured systolic pressure provided, there is no left ventricular outflow tract obstruction. In this case, the systolic blood pressure of the patient was 115 mmHg. The right ventricular pressure will be the difference of the LV systolic pressure and the PG between the two ventricles = 115−88.36 = 26.64 mmHg. ASD within Oval Fossa can be challenging given the sometimes challenging or inadequate windows. Cardiac catheterisation and cardiac MRI allow a quantitative approach in the calculation of pulmonary to systemic flows. •• Ventricular function: RV function can be affected particularly in the context of pulmonary hypertension. Older patients with significant atrial shunt can have LV diastolic dysfunction that becomes unmasked following closure of the ASD (11). Coronary sinus defect Inferior sinus venosus defect •• Effect on outflow tracts: A large shunt can exaggerate the pressure gradient across the outflow tracts e.g. a large atrial shunt can increase the pressure gradient across the pulmonary outflow tract. Figure 2 Figure 2 Different types of interatrial communications. © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Figure 1 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English 5:1 R4 SVC RPA RA LA S R * A B Figure 4 Superior sinus venosus defect () demonstrated from subcostal sagittal view on 2D (A) and Colour Doppler (B). SVC RPA RA LA S R A B from the left atrium to the right atrium. A sinus venosus defect at the orifice of the inferior vena cava will create an inferior interatrial communication. Coronary sinus (CS) defects are a type of communication between the atriums through the mouth of the sinus. The deficiency between the adjacent walls of the CS and the left atrium (LA) may vary from small fenestrations to complete absence resulting in partial or complete unroofing of the CS. The latter is associated with dilatation of the CS ostium due to the large left to right shunt and drainage of a persistent left SVC to the LA roof (12). Rarely all components of the atrial septum are absent resulting in a common atrium (13). Figure 4 Superior sinus venosus defect () demonstrated from subcostal sagittal view on 2D (A) and Colour Doppler (B). Apical four chamber In this view, the ultrasound beam is parallel to the atrial septum and measurements should be avoided, as there is risk of an artificial dropout. The four-chamber view allows haemodynamic assessment of the ASD (RA and RV dilation, Fig. 6) and estimation of the RV pressure based on the TR jet velocity. Echocardiographic assessment The atrial septum should be assessed from subcostal, apical and parasternal views: Subcostal The preferred acoustic window. The atrial septum is seen in the anterior-posterior (four-chamber view, Fig. 3) and superior-inferior axis (sagittal view). The latter allows assessment of the margins to the SVC and IVC and is the optimal window for imaging a sinus venosus defect (Fig. 4). The left anterior oblique (LAO) view shows the length of the atrial septum and demonstrates the ostium primum defect (Fig. 5) as well as dilatation of the CS if present. Partially or completely unroofed CS can be seen from this view. Colour Doppler interrogation and contrast studies can be performed. Parasternal short axis (PSAX) The atrial septum is seen posterior to the aortic root and thus the aortic rim of the defect can be identified (Fig. 7). Sinus venosus and postero-inferior defects can also be seen in this view but measurements of the defect are not recommended due to the risk of drop out. A B LA LA RA RA LV LV Figure 3 Subcostal four-chamber view shows a large ostium secundum ASD on 2D (A) and colour Doppler (B). L RA * Figure 5 Subcostal view showing a primum atrial septal defect (). The bridging leaflets of the common valve are attached to the crest of the septum () allowing shunt only at the atrial level. This is a partial AVSD. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. www.echorespract.com © 2018 The authors Published by Bioscientifica Ltd https://doi.org/10.1530/ERP-17-0062 Downloaded from Bioscientifica.com at 10/24/2024 04 via Open Access. This work is licensed under a Creative Attribution-NonCommercial 4.0 Internationa http://creativecommons.org/licenses/ L RA * Figure 5 Subcostal view showing a primum atrial septal defect (). The bridging leaflets of the common valve are attached to the crest of the septum () allowing shunt only at the atrial level. This is a partial AVSD. A B LA LA RA RA LV LV A B LA LA RA RA LV LV Figure 3 Subcostal four-chamber view shows a large ostium secundum ASD on 2D (A) and colour Doppler (B). L RA * Figure 3 Figure 5 Figure 5 Subcostal view showing a primum atrial septal defect (). The bridging leaflets of the common valve are attached to the crest of the septum () allowing shunt only at the atrial level. This is a partial AVSD. g Subcostal view showing a primum atrial septal defect (). The bridging leaflets of the common valve are attached to the crest of the septum (*) allowing shunt only at the atrial level. This is a partial AVSD. g Subcostal four-chamber view shows a large ostium secundum ASD on 2D (A) and colour Doppler (B). Subcostal four-chamber view shows a large ostium secundum ASD on 2D (A) and colour Doppler (B). © 2018 The authors Published by Bioscientifica Ltd www.echorespract.com https://doi.org/10.1530/ERP-17-0062 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English Shunt lesions R5 5:1 RA L A RV L V Figure 6 Dilated right atrium and right ventricle in an adult with a large secundum ASD (). RA LA RV A B Figure 8 Four-chamber TOE view (mid-oesophageal, 0°) on 2D (A) and colour Doppler (B). The maximum dimension of the ASD is measured and its relationship with the AV valves assessed. A Deri and K English Shunt lesions R5 5:1 A Deri and K English RA L A RV L V * Figure 6 Dilated right atrium and right ventricle in an adult with a large secundum ASD (). Shunt lesions R5 5:1 RA LA RV A B Figure 8 Four-chamber TOE view (mid-oesophageal, 0°) on 2D (A) and colour Doppler (B). The maximum dimension of the ASD is measured and its relationship with the AV valves assessed. R5 RA L A RV L V * RA LA RV A B * Figure 8 Four-chamber TOE view (mid-oesophageal, 0°) on 2D (A) and colour Doppler (B). The maximum dimension of the ASD is measured and its relationship with the AV valves assessed. Figure 5 B RA LA RV A * B RA Figure 6 Figure 8 Four-chamber TOE view (mid-oesophageal, 0°) on 2D (A) and colour Doppler (B). The maximum dimension of the ASD is measured and its relationship with the AV valves assessed. Figure 6 Dilated right atrium and right ventricle in an adult with a large secundum ASD (). High right parasternal in patients considered for a percutaneous device closure. The American Society of Echocardiography has published comprehensive guidelines for the TOE examination (14) and for the assessment and percutaneous closure of ASDs with transthoracic, transoesophageal and intracardiac echocardiography (15). The patient is positioned in the right lateral decubitus position with the probe in a superior-inferior orientation. This view is ideal for detection of sinus venosus defects. In young children TTE will usually allow a full diagnostic study. In older children and adults, in particular, those considered for a transcatheter device closure, transoesophageal echocardiography (TOE) offers additional valuable information (Figs 8, 9, 10, 11 and 12). Table 1 summarises the basic TOE views for the assessment of ASDs. As with TTE, the location, size and number of defects should be identified as well as the relationship with the surrounding structures, in particular In patients with poor acoustic windows, contrast echocardiography is another alternative in selected patients (15). Intravenous injection of agitated saline in the left arm is very useful in the investigation of a left SVC in the context of a completely unroofed CS; imaging from the apical four-chamber view will reveal the presence of contrast in the left upper corner of the LA and subsequently in the RA. Aorta R LA * A B Figure 7 Parasternal short-axis view on 2D (A) and colour Doppler (B) showing a large secundum ASD (). Note the dilated right atrium (RA). Aorta R LA * A B RA LA Ao * A B Figure 9 Transoesophageal view (mid-oesophageal, approximately 45°) of a large ASD on 2D (A) and colour Doppler (B). The aortic rim is deficient. RA LA Ao * A B B B B Figure 7 www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Perimembranous defects Perimembranous defects are located in the membranous septum. When viewed from the right ventricle, these lesions are located towards the inner curvature of the heart. Their location leads to continuity between the tricuspid and aortic valves. This type may extend into the inlet or outlet part of the right ventricle or may be large enough to shunt towards all RV components. A B RA LA * IVC rim C SVC Figure 11 Assessment of ASD () for device closure. The IVC rim is seen from the transgastric bicaval view (116°) on 2D (A) and colour Doppler (B). B Perimembranous defects may close due to the proximity of the septal leaflet of the tricuspid valve to these defects; aneurysmal tissue from the underside of the leaflet might partially or completely cover the hole. Alternatively, the right coronary cusp and sometimes the non-coronary cusp may prolapse through the defect with subsequent reduction of the size of the defect or even complete occlusion, often at the expense of aortic regurgitation. Epidemiology and morphology Ventricular septal defects are the most common congenital heart lesion, occurring in approximately 33% of patients with congenital heart disease (19). They may occur in isolation or as part of more complex congenital heart disease. Depending on their right ventricular borders they can fall into one of three categories: Ventricular septal defects (VSDs) Large ASDs or those associated with paradoxical embolism or platypnoea/orthodeoxia should be closed. Irreversible pulmonary hypertension is the only absolute contraindication for closure. Secundum type defects with adequate rims can be closed percutaneously (Fig. 13). Following device closure, the position of the device, the relationship to surrounding structures, any residual shunts should be carefully evaluated. Follow-up continues lifelong every 1–2 years due to risk of erosion (16). Patients post-surgical closure with no other cardiac abnormalities are usually discharged after a year although a small but definite incidence of atrial arrhythmias has been described even years after the repair (17, 18). Figure 10 Figure 12 TOE 3D view of the atrial septum from the RA side. The ASD is noted with . g Large atrial shunt secondary to multiple defects seen from the bicaval TOE view (mid-oesophageal, 90°) on 2D (A) and colour Doppler (B). Large atrial shunt secondary to multiple defects seen from the bicaval TOE view (mid-oesophageal, 90°) on 2D (A) and colour Doppler (B). www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Figure 9 g Parasternal short-axis view on 2D (A) and colour Doppler (B) showing a large secundum ASD (). Note the dilated right atrium (RA). g Transoesophageal view (mid-oesophageal, approximately 45°) of a large ASD on 2D (A) and colour Doppler (B). The aortic rim is deficient. Transoesophageal view (mid-oesophageal, approximately 45°) of a large ASD on 2D (A) and colour Doppler (B). The aortic rim is deficient. Parasternal short-axis view on 2D (A) and colour Doppler (B) showing a large secundum ASD (). Note the dilated right atrium (RA). This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ www.echorespract.com https://doi.org/10.1530/ERP-17-0062 © 2018 The authors Published by Bioscientifica Ltd © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27A via Open Access. This work is licensed under a Creative Commo Attribution-NonCommercial 4.0 International Licens http://creativecommons.org/licenses/by-nc/4 A Deri and K English Shunt lesions R6 5:1 LA RA SVC * A B Figure 10 Large atrial shunt secondary to multiple defects seen from the bicaval TOE view (mid-oesophageal, 90°) on 2D (A) and colour Doppler (B). SVC * Ao C IVC Figure 12 TOE 3D view of the atrial septum from the RA side. The ASD is noted with . R6 Shunt lesions R 5:1 SVC Ao C IVC Figure 12 TOE 3D view of the atrial septum from the RA side. The ASD is noted with . LA RA SVC A B Figure 10 Large atrial shunt secondary to multiple defects seen from the bicaval TOE view (mid-oesophageal, 90°) on 2D (A) and colour Doppler (B). LA RA SVC * A B SVC * Ao C IVC B This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ Juxtaarterial and doubly committed defects Juxtaarterial and doubly committed defects are characterised by fibrous continuity between the adjacent leaflets of the aortic and pulmonary valves. The outlet septum and septal component of the subpulmonary infundibulum are absent. The aortic sinus may prolapse into the defect resulting in partial or complete closure at the cost of aortic regurgitation (20). Muscular defects Muscular defects have completely muscular borders, they can be single or multiple and located anywhere within the septum. Figure 11 Assessment of ASD () for device closure. The IVC rim is seen from the transgastric bicaval view (116°) on 2D (A) and colour Doppler (B). Assessment of ASD () for device closure. The IVC rim is seen from the transgastric bicaval view (116°) on 2D (A) and colour Doppler (B). © 2018 The authors Published by Bioscientifica Ltd © 2018 The authors Published by Bioscientifica Ltd www.echorespract.com https://doi.org/10.1530/ERP-17-0062 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English Shunt lesions R7 5:1 Table 1 Transoesophageal views for the assessment of ASD. View Position in the oesophagus Multiplane angles Anatomical features Four chamber Mid-oesophagus 0°, 15°, 30° Relationship to the AV valves Posterior rims ASD diameter Upper oesophageal short axis Mid- to upper oesophagus 0°, 15°, 30°, 45° SVC rim Right upper pulmonary vein Superior aortic rim Detection of sinus venosus defect Mid-oesophageal aortic valve short axis Mid-oesophagus 30°, 45°, 60°, 75° Posterior and aortic rims PFO ASD diameter Bicaval view Mid-oesophagus 90°, 105°, 120° IVC and SVC rims Anomalous pulmonary venous drainage ASD diameter Long axis Mid- to upper oesophagus 120°, 135°, 150° Roof of the LA Left pulmonary veins AV, atrioventricular; IVC, inferior vena cava; LA, left atrium; SVC, superior vena cava. AV, atrioventricular; IVC, inferior vena cava; LA, left atrium; SVC, superior vena cava. ventricular septum and classifies VSDs as central, inlet, outlet and muscular. Understandably, there is an on-going debate and effort to achieve an international consensus for VSD definition and categorisation (21, 22). Figure 14 Figure 14 Echocardiographic assessment Multiple windows, planes and sweeps should be used to assess the ventricular septum. This classification of VSDs follows the European approach and focuses on the anatomic features immediately adjacent to the defect, the so-called border approach. An alternative approach is geographical, which focuses primarily on the position of the hole within the The perimembranous VSD can be demonstrated on the PSAX (Fig. 14) and apical four-chamber views (Fig. 15). Its relationship to the aortic valve can be seen from apical long axis and PLAX views. Occasionally, these defects are covered by apposition of the septal leaflet of the tricuspid valve and may close spontaneously, either partially or completely. Associated findings such as double-chambered RA LA * Figure 13 TOE mid-oesophageal view at 0° showing an ASD device in situ following percutaneous ASD closure. RA LA * A Ao! TV! ! LA! RA! RV! B Figure 14 A perimembranous VSD is shown in the PSAX TTE view in 2D (A) and colour Doppler (B). Note the tricuspid-aortic valve fibrous continuity. A Ao! TV! ! LA! RA! RV! B B RA RA LA! www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Location of defect(s) RV, a Gerbode defect or subaortic ridge can be seen with perimembranous VSDs. The right or non-coronary cusp of the aortic valve can prolapse into the defect leading to a new onset aortic regurgitation. The PSAX view is very useful in identifying the location of the VSD(s). A cut at the level of the aortic valve will show a perimembranous VSD between 9 o’clock and 11 o’clock positions (Fig. 14). Occasionally, these defects are partially covered by tricuspid valve aneurysmal tissue and their size can be underestimated on 2D, but colour flow Doppler will reveal the magnitude of flow. Anterior extension of these defects will be seen between 11 and 12 o’clock. Doubly committed defects are identified between 12 and 2 o’clock (pulmonary valve hinge point) (Fig. 17). A sweep performed from the PSAX view beginning from the aortic valve towards the apex can profile muscular VSDs. Anterior muscular VSDs will be seen between Muscular VSDs (Fig. 16) are located at either the inlet, outlet or trabecular portion of the septum and can be singular or multiple. The septum should be interrogated thoroughly at its entire length by sweeping from subcostal, apical and parasternal views (Video 1). Colour flow Doppler can identify small VSDs that are not visible on 2D. The sensitivity of this is reduced in newborns with persistent fetal circulation or patients of any age with elevated RV pressure. Figure 13 Figure 13 g A perimembranous VSD is shown in the PSAX TTE view in 2D (A) and colour Doppler (B). Note the tricuspid-aortic valve fibrous continuity. TOE mid-oesophageal view at 0° showing an ASD device in situ following percutaneous ASD closure. A perimembranous VSD is shown in the PSAX TTE view in 2D (A) and colour Doppler (B). Note the tricuspid-aortic valve fibrous continuity. A perimembranous VSD is shown in the PSAX TTE view in 2D (A) and colour Doppler (B). Note the tricuspid-aortic valve fibrous continuity. TOE mid-oesophageal view at 0° showing an ASD device in situ following percutaneous ASD closure. © 2018 The authors Published by Bioscientifica Ltd www.echorespract.com https://doi.org/10.1530/ERP-17-0062 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English Shunt lesions R8 5:1 RA LA LV RV * A B Figure 15 Perimembranous VSD partially covered by tricuspid valve tissue. LA RA LV RV * Figure 16 Large muscular VSD (). The LA and LV are significantly dilated. A Deri and K English Shunt lesions R8 5:1 R8 LA RA LV RV Figure 16 Large muscular VSD (). The LA and LV are significantly dilated. RA LA LV RV A B Figure 15 Perimembranous VSD partially covered by tricuspid valve tissue. RA LA LV RV * A B Figure 15 Perimembranous VSD partially covered by tricuspid valve tissue. LA RA LV RV * Figure 16 Large muscular VSD (). The LA and LV are significantly dilated. LV LV RV RV Figure 15 Figure 16 Large muscular VSD (). The LA and LV are significantly dilated. g Large muscular VSD (). The LA and LV are significantly dilated. g Perimembranous VSD partially covered by tricuspid valve tissue. Multiple VSDs sweep. View Video 1 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-1. Multiple VSDs sweep. View Video 1 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-1. Ao TV PA Figure 17 Parasternal short-axis view showing a doubly committed VSD (). The right coronary cusp of the aortic valve has a mildly elongated appearance as it is prolapsing through the defect, partially covering it. The aortic and pulmonary valves are in fibrous continuity. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04 via Open Access. This work is licensed under a Creative Attribution-NonCommercial 4.0 Internation http://creativecommons org/licenses Ao TV PA Doubly committed and juxtaarterial VSDs are best seen from the PSAX view (Fig. 17, Video 2) as well as the PLAX with slight counter clockwise rotation of the transducer. The unsupported right coronary cusp of the aortic valve can potentially prolapse into the defect leading to distortion of the leaflet and variable degree of aortic insufficiency (Video 3). Figure 17 g Parasternal short-axis view showing a doubly committed VSD (). The right coronary cusp of the aortic valve has a mildly elongated appearance as it is prolapsing through the defect, partially covering it. The aortic and pulmonary valves are in fibrous continuity. g Parasternal short-axis view showing a doubly committed VSD (). The right coronary cusp of the aortic valve has a mildly elongated appearance as it is prolapsing through the defect, partially covering it. The aortic and pulmonary valves are in fibrous continuity. g Parasternal short-axis view showing a doubly committed VSD (). The right coronary cusp of the aortic valve has a mildly elongated appearance as it is prolapsing through the defect, partially covering it. The aortic and pulmonary valves are in fibrous continuity. Video 2 DCVSD short axis. View Video 2 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-2. Figure 19 g TOE in a patient with previous surgical closure of a large VSD ( in image A is in the position of the patch) with a fenestration allowing a residual shunt as shown with colour Doppler (arrow in image B). (C) Post percutaneous device closure of the defect (*). Large malalignment VSD () as seen from the apical four-chamber view with anterior angulation to open the left ventricular outflow tract (A) and parasternal long axis view (B). The outlet septum is posteriorly deviated (arrow) creating a very narrow outflow tract. This newborn also had a small, bicuspid aortic valve and interruption of the aortic arch. 12 and 2 o’clock, mid-muscular between 10 and 12 o’clock and posterior muscular between 7 and 10 o’clock (23). treatment. Indication for closure in these patients will be the development of complications (aortic cusp prolapse, subaortic ridge, double-chambered RV, endocarditis). Infants with a significant shunt will develop congestive heart failure and will be referred for surgery. Percutaneous device closure is an alternative for a small and carefully selected population, usually older children with muscular or perimembranous defects with sufficient rims. The echocardiographer should look for malalignment of the outlet septum (Fig. 18), straddling AV valves, aortic cusp prolapse and other associated abnormalities and also measure the defect margins for consideration of transcatheter closure. Newborns and young children usually have excellent acoustic windows that will allow a thorough transthoracic echocardiographic assessment. In those patients in whom TTE imaging is limited the TOE is an excellent alternative particularly when there are haemodynamically associated lesions or the defect is considered for percutaneous closure (Fig. 19). Epidemiology and morphology Epidemiology and morphology Atrioventricular septal defects make up 4–5% of all congenital heart defects, occurring in 34.8 per 100,000 live births (24). There is a very strong association between AVSD and Down’s syndrome. Bergström and coworkers described up to 42% of patients with Trisomy 21 and congenital heart disease having an AVSD (25). Video 3 C Figure 19 TOE in a patient with previous surgical closure of a large VSD (* in image A is in the position of the patch) with a fenestration allowing a residual shunt as shown with colour Doppler (arrow in image B). (C) Post percutaneous device closure of the defect (*). Ao LV LA B Figure 18 Large malalignment VSD () as seen from the apical four-chamber view with anterior angulation to open the left ventricular outflow tract (A) and parasternal long axis view (B). The outlet septum is posteriorly deviated (arrow) creating a very narrow outflow tract. This newborn also had a small, bicuspid aortic valve and interruption of the aortic arch. LV! Ao! RV! * ! C Ao * LV LA B B LV! Figure 18 Figure 18 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24 via Open Access. This work is licensed under a Attribution-NonCommercial 4 0 Int www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Video 3 DCVSD Ao prolapse View Video 3 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-3. www.echorespract.com https://doi.org/10.1530/ERP-17-0062 © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English Shunt lesions R9 5:1 A Deri and K English Shunt lesions R9 5:1 LV RV Ao * Ao * LV LA A B Figure 18 Large malalignment VSD () as seen from the apical four-chamber view with anterior angulation to open the left ventricular outflow tract (A) and parasternal long axis view (B). The outlet septum is posteriorly deviated (arrow) creating a very narrow outflow tract. This newborn also had a small bicuspid aortic valve and interruption of the aortic arch LV RV Ao LV! Ao! RV! ** ! A B C Figure 19 TOE in a patient with previous surgical closure of a large VSD (* in image A is in the position of the patch) with a fenestration allowing a residual shunt as shown with colour Doppler (arrow in image B). (C) Post percutaneous device closure of the defect (*). Shunt lesions R9 5:1 LV RV Ao LV! Ao! RV! ** ! A B C Figure 19 TOE in a patient with previous surgical closure of a large VSD (* in image A is in the position of the patch) with a fenestration allowing a residual shunt as shown with colour Doppler (arrow in image B). (C) Post percutaneous device closure of the defect (*). R9 LV RV Ao LV! Ao! RV! ** ! A B C Figure 19 TOE in a patient with previous surgical closure of a large VSD (* in image A is in the position of the patch) with a fenestration allowing a residual shunt as shown with colour Doppler (arrow in image B). (C) Post percutaneous device closure of the defect (*). LV RV Ao A B LV RV Ao * A B LV LV! Ao! RV! ** ! Management Asymptomatic infants with a restrictive VSD and no evidence of pulmonary hypertension will not require www.echorespract.com https://doi.org/10.1530/ERP-17-0062 © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ © 2018 The authors Published by Bioscientifica Ltd A Deri and K English Shunt lesions R10 5:1 component of the bridging leaflets serves as a commissure and is often described incorrectly as a ‘cleft’. The cardinal feature of all AVSDs is the presence of a common AV junction rather than separate right and left AV junctions. This common junction is guarded by a distinctive AV valve (Fig. 20), which in the majority of cases has a common AV orifice and consists of five leaflets. Occasionally, the common valve may be divided into two orifices (right and left) under the conditions of dense attachments to the ventricular septum, dense attachments to the atrial septum (AVSD with no primum defect) or divided by a tongue of tissue. Regardless of common or separate orifices, the left component of the common AV valve has three leaflets that have no counterparts in the mitral valve of the normal heart. The left mural leaflet is exclusively in the LV. The other two are the bridging leaflets and are attached to variable extent with tension apparatus into both ventricles. The left ventricular AVSDs can be classified depending on the level of shunting across the defect. It is this anatomical feature that influences the clinical presentation. •• When neither of the bridging leaflets is attached to the septal components, there is obligatory shunt at both atrial and ventricular levels. The degree of ventricular shunt will depend on the proximity of the bridging leaflets to the crest of the ventricular septum. •• When leaflets are attached to the ventricular crest, the shunt will be at the atrial level. These are the so-called partial AVSDs or ostium primum defects and have no ventricular component. In partial AVSDs, the common valve has two orifices. •• When leaflets are fused to the underside of the atrial septum, the shunt will be at the ventricular level. The AVSDs may be either balanced or unbalanced at the ventricular level. Subcostal views Aorta The long axis and LAO view demonstrate the primum defect as well as any additional atrial communications (Fig. 5). The subcostal short axis and LAO views show the abnormal elongation of the left ventricular outflow tract due to unwedging of the aortic outflow gives the appearance of ‘goose-neck deformity’ (Fig. 21). The subcostal LAO allows the view of the common AV valve en face and can be used to determine the balance of the valve over the ventricles (Video 4). The subcostal short- axis view shows the anatomy of the AV valves and helps differentiate them from normal mitral and tricuspid valves. The size of the mural leaflet and the papillary muscles of the left AV valve can be identified in this view. A small mural leaflet and single papillary muscle have been associated with imbalance of the left AV valve and worse prognosis (27). Superior bridging leaflet Left mural leaflet Inferior bridging leaflet Echocardiographic assessment TTE provides excellent visualisation of the anatomy of AVSD and along with colour and spectral Doppler allows a comprehensive evaluation of the defect. Mitral Valve Mitral Valve Tricuspid Valve Tricuspid Valve Tricuspid Valve Ao Sup Inf Left Right B B Aorta Pulmonary Valve Sup Inf Left Right B Superior bridging leaflet Inferior bridging leaflet Left mural leaflet Pulmonary Valve Management In the latter, the common AV valve opens predominantly to one ventricle, and there is associated hypoplasia of the contralateral ventricle. Unbalance might also be noted at the atrial level; malaligned atrial septum as well as double outlet atrium have been described (26). Mitral Valve Tricuspid Valve Aorta Pulmonary Valve Sup Inf Left Right A Aorta Pulmonary Valve Sup Inf Left Right B Superior bridging leaflet Inferior bridging leaflet Left mural leaflet Figure 20 (A) Normal arrangement of atrioventricular and semilunar valves. In the normal heart the aortic and mitral valves are in fibrous continuity. (B) Common AV valve instead of normal Tricuspid and Mitral valves in AVSD. Note the ‘unwedged’ position of the Aorta in the AVSD setting. The Aorta maintains its fibrous continuity with the common AV valve. Tricuspid Valve Aorta Sup Inf Left Right A Mitral Valve Tricuspid Valve Aorta Pulmonary Valve Sup Inf Left Right A Mitral Valve Pulmonary Valve Aorta This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ PLAX view The AV valve regurgitation is further assessed from this view. Abnormal chordal attachments or a subaortic membrane may be identified (Fig. 23). Figure 20 g (A) Normal arrangement of atrioventricular and semilunar valves. In the normal heart the aortic and mitral valves are in fibrous continuity. (B) Common AV valve instead of normal Tricuspid and Mitral valves in AVSD. Note the ‘unwedged’ position of the Aorta in the AVSD setting. The Aorta maintains its fibrous continuity with the common AV valve. (A) Normal arrangement of atrioventricular and semilunar valves. In the normal heart the aortic and mitral valves are in fibrous continuity. (B) Common AV valve instead of normal Tricuspid and Mitral valves in AVSD. Note the ‘unwedged’ position of the Aorta in the AVSD setting. The Aorta maintains its fibrous continuity with the common AV valve. www.echorespract.com https://doi.org/10.1530/ERP-17-0062 © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ R11 5:1 RA LV RV Ao A B Figure 21 Subcostal left anterior oblique view of an infant with AVSD showing the ‘goose-neck deformity’ of the left ventricular outflow tract. En face view of AV valve. View Video 4 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-4. En face view of AV valve. View Video 4 at http://movie-usa. glencoesoftware.com/video/10.1530/ERP-17-0062/video-4. PSAX view This will demonstrate the VSD and identify additional ventricular defects. The AV valves can be seen in the short axis and the origin of regurgitation was assessed. AVSDs may be associated with other congenital defects such as Tetralogy of Fallot, isomerism of the atrial appendages, anomalous pulmonary venous drainage Figure 21 LA Ao LV * RV * ** A B C D E F Figure 23 Patient several years post repair of AVSD with significant subaortic obstruction. (A) PLAX reveals a subaortic membrane () but colour Doppler (B) demonstrates presence of turbulence below the level of the membrane. A 3D volume was acquired from the PLAX window. MPR showed chordal attachments to the LVOT () in addition to the subaortic membrane () (C and F). The excursion of the aortic leaflets is limited secondary to the membrane (D). This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International Downloaded from Bioscientifica.com at 10/24/2024 04 via Open Access This work is licensed under a Creative LA Ao LV * RV A B Subcostal left anterior oblique view of an infant with AVSD showing the ‘goose-neck deformity’ of the left ventricular outflow tract. B Apical view Both AV valves as well as the relationship of the common AV valve within the defect are seen in this view (Fig. 22). Posterior to anterior sweep will reveal the VSD and can demonstrate chordal attachments to the ventricular septum. Colour Doppler will show the AV valve inflow and regurgitation. The LVOT can be assessed from the apical 5-chamber view. Obstruction of the LVOT can be seen either before or after surgical repair and could be due to chordal attachments to the LV side of the septum, subaortic membrane, septal hypertrophy, anomalous or prominent papillary muscle. * ** C D E F C D Primum ASD D VSD A B Figure 22 AVSD with large atrial and ventricular components seen from the apical four-chamber view on 2D (A). Colour Doppler (B) shows a mild degree of central AV valve regurgitation. Primum ASD D VSD A B B Figure 23 Patient several years post repair of AVSD with significant subaortic obstruction. (A) PLAX reveals a subaortic membrane () but colour Doppler (B) demonstrates presence of turbulence below the level of the membrane. A 3D volume was acquired from the PLAX window. MPR showed chordal attachments to the LVOT () in addition to the subaortic membrane () (C and F). The excursion of the aortic leaflets is limited secondary to the membrane (D). Patient several years post repair of AVSD with significant subaortic obstruction. (A) PLAX reveals a subaortic membrane () but colour Doppler (B) demonstrates presence of turbulence below the level of the membrane. A 3D volume was acquired from the PLAX window. MPR showed chordal attachments to the LVOT () in addition to the subaortic membrane () (C and F). The excursion of the aortic leaflets is limited secondary to the membrane (D). Figure 22 Figure 22 g AVSD with large atrial and ventricular components seen from the apical four-chamber view on 2D (A). Colour Doppler (B) shows a mild degree of central AV valve regurgitation. © 2018 The authors Published by Bioscientifica Ltd www.echorespract.com https://doi.org/10.1530/ERP-17-0062 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ A Deri and K English Shunt lesions R12 5:1 Shunt lesions R12 5:1 A Deri and K English Patent arterial duct Primum ASD VSD RV LV RV LV A B C D Figure 24 (A) AVSD with a degree of unbalance to the left. (B) The common valve is seen from the subcostal LAO view and the area of the left AV valve to the common AV valve is calculated (AV valve index). (C and D) The inflows are assessed on colour Doppler. Note the smaller size of the LV inflow in comparison to that on the right. Primum ASD VSD RV LV A and the echocardiographer should perform a careful assessment in the usual sequential segmental approach. A Figure 24 (A) AVSD with a degree of unbalance to the left. (B) The common valve is seen from the subcostal LAO view and the area of the left AV valve to the common AV valve is calculated (AV valve index). (C and D) The inflows are assessed on colour Doppler. Note the smaller size of the LV inflow in comparison to that on the right. Management and follow-up Patients with AVSD will require surgery with timing depending on the degree and location of shunt. Those with large atrial and ventricular components are usually repaired between the age of 3 and 6 months. If there is shunting only at the atrial level, the natural history is similar to that observed in patients with atrial septal defects. The age of repair varies between institutions with some preferring to repair between 1 and 2 years of age (30) and other centres waiting up until 8 years (31). A significant degree of AV valve regurgitation may, however, necessitate an earlier intervention. A small number of patients with atrial shunting only are diagnosed in adolescent or adult life and are therefore repaired later. Unbalanced AVSD Unbalance of the atria should be assessed from the apical four-chamber view. The atrial and AV valve inflows are evaluated by colour, pulse-wave and continuous-wave Doppler to look for presence of obstruction and assess its severity. Ventricular imbalance is assessed from several views and various techniques have been proposed: En face view of the AV valve from the subcostal LAO allows the calculation of the modified AV valve index (AVVI = Left AV valve area: Total AV valve area) (28) (Fig. 24). AVVI between 0.4 and 0.6 is suggestive of balanced AVSD. Other parameters such as RV:LV inflow angle, the LA overriding the left AV valve, size parameters of the LV inflow (29), the direction of flow at the VSD in systole and in the transverse arch as well as the presence of any obstruction are felt to be likely surrogates of LV adequacy. B As with all other lesions, TOE offers valuable information in the definition of anatomy, assessment of regurgitation jets and shunt in patients with AVSD (Fig. 25). It is particularly useful in older children and adults where the acoustic windows for TTE are limited and also in the perioperative assessment of residual lesions, stenosis or regurgitation of the AV valves as well as the estimation of the RV pressure. RV LV C D High left parasternal transverse view significant shunt. The duct in a right arch can originate either from the proximal descending aorta (right duct) or the base of the left subclavian (left duct). A left duct High left parasternal transverse view shows the pulmonary artery bifurcation and the duct. Echocardiographic assessment Echocardiography is the investigation of choice in the diagnosis and assessment of the patent duct. In neonates and infants the ductal flow can be demonstrated from several imaging views using colour Doppler but is best seen from the parasternal and suprasternal windows. Figure 25 TOE in an adult with primum ASD (). Colour Doppler interrogation did not reveal shunt at the ventricular level. TOE in an adult with primum ASD (). Colour Doppler interrogation did not reveal shunt at the ventricular level. Figure 26 Figure 26 (A) Parasternal short-axis in a newborn with pulmonary hypertension. There is bidirectional flow across the duct, confirmed on spectral Doppler (B). Note the low velocity flow in keeping with elevated pulmonary arterial pressure. The duct is seen along the left lateral border of the MPA (Fig. 26). High left parasternal sagittal view (‘ductal view’) A B PDA RPA LPA DAo Figure 27 (A) High parasternal view on 2D shows the bifurcation of the main pulmonary artery and a large PDA with large left to right shunt (B). A B PDA RPA LPA DAo This demonstrates the long axis of the duct, between the descending aorta and the LPA (Fig. 27). B Parasternal long axis view This view allows assessment of the LV size and LA: Ao root ratio with MMode. Ratio ≥1.4 is suggestive of at least moderate shunt (33). The size of the LV is also assessed from this view and indexed to the infant’s body surface area. Suprasternal view From the suprasternal sagittal view the arch sidedness, its branching pattern and size should be assessed and coarctation of the aorta excluded. Holodiastolic flow reversal in the descending aorta is suggestive of a Epidemiology and morphology Echocardiography (TTE, TOE, 3D) has a key role in the follow-up of these patients; residual shunts, the degree of AV valve regurgitation or narrowing and development of subaortic stenosis are the most common reasons for re-intervention. The reported incidence of a persistent duct in term neonates is only 1 in 2000 births, accounting for 5%–10% of all congenital heart disease (32). In preterm newborns the incidence is significantly higher. © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ www.echorespract.com https://doi.org/10.1530/ERP-17-0062 LV RV RA LA * A B Figure 25 TOE in an adult with primum ASD (). Colour Doppler interrogation did not reveal shunt at the ventricular level. LV RV RA LA A B PDA MPA Ao A B LV B Video 5 MPR with 3D of muscular VSDs. View Video 5 at http:// movie-usa.glencoesoftware.com/video/10.1530/ERP-17- 0062/video-5. A complete echocardiographic assessment should be performed to exclude other cardiac malformations, particularly duct dependent lesions, such as pulmonary atresia or coarctation of the aorta. 3DE is outside the scope of this review. For more information please refer to the EAE/ASE recommendations for image acquisition and display using three-dimensional echocardiography (34) and the Consensus on 3DE in Congenital Heart Disease from the European Association of Cardiovascular Imaging and the ASE (35). 3DE is outside the scope of this review. For more information please refer to the EAE/ASE recommendations for image acquisition and display using three-dimensional echocardiography (34) and the Consensus on 3DE in Congenital Heart Disease from the European Association of Cardiovascular Imaging and the ASE (35). Colour Doppler is useful in the diagnosis of small ducts, not easily seen on 2D, particularly in adults with suboptimal echocardiographic windows. The flow across the duct during the cardiac cycle offers valuable information regarding the pulmonary pressure (Fig. 26). Funding g This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Author contribution statement 3D echocardiography is extremely useful and strongly recommended for the diagnosis, management and follow-up of patients with these lesions. The anatomy of ASDs (Fig. 12) and VSDs can be assessed with accuracy (34, 35, 36). It provides information regarding the location, shape and size of the defect (s) and the relationship with surrounding structures (Video 5). Its role is essential in planning and performing a percutaneous closure as well as in perioperative imaging. Multiplanar reformat (MPR) allows accurate measurements of the defects and facilitates the understanding of complex anatomies. K E wrote the introduction, clinical manifestations, management and follow-up. A D wrote the morphology and echocardiographic assessment. Pitfalls in the imaging of ducts •• A large duct with right to left shunt might be difficult to identify, as the flow across it resembles the flow in the descending aorta or the LPA. Other findings suggestive of pulmonary hypertension (significant TR pressure gradient, flattening of the IVS in systole) will assist in the diagnosis. Contrast echocardiography may also be helpful; intravenous injection of agitated saline will demonstrate micro bubbles in the descending but not the ascending aorta in the presence of right to left ductal shunt. •• A large duct with right to left shunt might be difficult to identify, as the flow across it resembles the flow in the descending aorta or the LPA. Other findings suggestive of pulmonary hypertension (significant TR pressure gradient, flattening of the IVS in systole) will assist in the diagnosis. Contrast echocardiography may also be helpful; intravenous injection of agitated saline will demonstrate micro bubbles in the descending but not the ascending aorta in the presence of right to left ductal shunt. Conclusion Echocardiography has a key role in the diagnosis, management and long-term follow-up of patients with left to right shunts. TTE is the investigation of choice and offers valuable information in infants and older children and can be enhanced by TOE in children and adults with suboptimal acoustic windows. 3D echocardiography is highly recommended for the examination and management of ASDs, VSDs and AVSDs. Large left to right shunts that are left untreated have a catastrophic outcome and therefore early diagnosis and careful haemodynamic assessment are paramount. •• In some cases, a red signal along the medial border of the MPA is seen in early systole that can be confused with a duct. This is a normal flow pattern that represents helical flow in the MPA and can be differentiated by the ductal flow given its location and timing. Figure 27 (A) High parasternal view on 2D shows the bifurcation of the main pulmonary artery and a large PDA with large left to right shunt (B). (A) High parasternal view on 2D shows the bifurcation of the main pulmonary artery and a large PDA with large left to right shunt (B). © 2018 The authors Published by Bioscientifica Ltd © 2018 The authors Published by Bioscientifica Ltd This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Downloaded from Bioscientifica.com at 10/24/2024 04:45:27AM via Open Access. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. http://creativecommons.org/licenses/by-nc/4.0/ www.echorespract.com https://doi.org/10.1530/ERP-17-0062 Shunt lesions R14 5:1 Video 5 MPR with 3D of muscular VSDs. View Video 5 at http:// movie-usa.glencoesoftware.com/video/10.1530/ERP-17- 0062/video-5. A Deri and K English in a right aortic arch with aberrant left subclavian artery confirms the diagnosis of a vascular ring. 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https://openalex.org/W4311263970	https://zenodo.org/record/7429600/files/XEBERLER_70_3_2015-52-55.pdf	Russian	null	Activity of Brain Aconitase of Albino Rats after Prenatal Hypoxia	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	2,090	АМЕА-nın Xəbərləri (biologiya və tibb elmləri), cild 70, №3, səh. 52-55 (2015) АМЕА-nın Xəbərləri (biologiya və tibb elmləri), cild 70, №3, səh. 52-55 (2015) Активность Аконитазы Головного Мозга Белых Крыс После Пренатальной Гипоксии С.Н. Баба-заде, Т.М. Агаев С.Н. Баба-заде, Т.М. Агаев Институт физиологии им. А.И.Гараева НАНА, ул.Шариф-заде,2, Баку AZ 1100, Азер Исследованы изменения активности митохондриальной и цитоплазматической аконитазы в головном мозге белых крыс разного возраста, подвергнутых гипоксии в период органогенеза пренатального развития. Установлено, что пренатальная гипоксия в постнатальном развитии увеличивает активность аконитазы, причем, этот процесс носит обратимый характер. ВВЕДЕНИЕ Несмотря на то, что фермент изучается с 40-ых годов ХХ века, в литературе нет количе- ственных данных о содержании в тканях голов- ного мозга животных цитоплазматической и митохондриальных форм аконитазы. Аконитаза иначе называемая аконитатгид- ратаза, (КФ 4.2.1.3), является одним из фермен- тов цикла трикарбоновых кислот. Фермент об- ратимо превращает лимонную кислоту в изоли- монную кислоту и тем самым поддерживает, так называемое аконитазное равновесие состоящее из 91% цитрата, 6% изоцитрата и 3% цис- аконита. Аконитаза обнаружена в клетках жи- вотных, растений и микроорганизмов (Зайчи- кова,2010). Аконитаза относится к тем немно- гим белкам, которые в клетке выполняют не- сколько функций: в железосодержащей форме он катализирует одну из реакций цикла Кребса (обратимое превращение лимонной кислоты в изолимонную), а при окислительном стрессе в форме без иона железа связывается с регуля- торным элементом мРНК ферритина и репрес- сирует ее трансляцию, а также связывается с элементами нестабильности в мРНК рецептора трансферрина и защищает эту мРНК от дегра- дации. При увеличении в клетке количества двухвалентного железа фермент самопроиз- вольно регенерируется и образует цитозольную аконитазу (William, 2006). Гипоксия оказывает влияние на активность многих ферментов. Имеются данные, что гипок- сия, перенесенная плодом в период органогене- за сказывается в постнатальном периоде разви- тия (Журавин, 2009). Целью данной работы является изучение влияния пренатальной гипоксии в периоде ор- ганогенеза на активность аконитазы в различ- ных частях головного мозга белых крыс в пост- натальном развитии. Представленная работа может помочь в решении некоторых вопросов энергоснабжения головного мозга в стрессовых ситуациях. Исследованы изменения активности митохондриальной и цитоплазматической аконитазы в головном мозге белых крыс разного возраста, подвергнутых гипоксии в период органогенеза пренатального развития. Установлено, что пренатальная гипоксия в постнатальном развитии увеличивает активность аконитазы, причем, этот процесс носит обратимый характер. Ключевые слова: Гипоксия, головной мозг крыс, аконитаза МАТЕРИАЛЫ И МЕТОДЫ Эксперименты проводились на самках бес- породных белых крыс в количестве 20 голов. Самки в период органогенеза плода три дня в течение 10 мин подвергались гипоксии в спе- циальной камере газовой смесью, состоящей из 95% азота и 5% кислорода. Полученное от них потомство делилось на три группы и выращива- лось до достижения требуемого возраста: 17-ти дней, 1-го и 3-х месяцев. Эти возрасты считают- ся критическими в постнатальном развитии крыс. После окончания каждой серии опытов животных декапитировали, извлекали головной мозг и отделяли орбитальную., сенсомоторную., лимбическую кору, гипоталамус и мозжечок (Светухин,1968). Ткани гомогенизировали в 0,25 М растворе сахарозы в соотношении 1:9, центрифугировали при 20 000g в течение 20 мин для отделения митохондриальной фракции от цитозольной жидкости. Все эти процедуры про- Активные формы кислорода образуются в клетках в ходе нормальных метаболических ре- акций, однако, их содержание значительно по- вышается при гипоксии, которая приводит к окислительному стрессу. В настоящее время аконитазарассматривается в качестве чувстви- тельной и критической мишени действия сво- бодных радикалов в условиях окислительного стресса. При этом происходит резкое снижение активности фермента (Gardner, 1994; Mura- kamiand, Yoshino, 1997). Несмотря на то, что ферменты цикла трикарбоновых кислот локали- зованы в митохондриях, помимо митохондраль- ной, имеется и цитоплазматическая аконитаза. 52 Активность Аконитазы Головного Мозга (Анохина, 2010) в головном мозге появляется бе- лок HIF-1 (hepoxia inducible factоr), который способен регулировать транскрипцию более 60- ти белков, в том числе ферментов гликолиза (Меерсон, 1993). Имеются данные, что при этом активность гексокиназы (ГК) увеличивается на 23% и фосфофруктокиназы – на 50% (Sermnza, 2006). Гипоксия вынуждает организм животных оптимизировать энергетический обмен за счет появления в клетках митохондрий, обладающих меньшими размерами, со сниженным содержа- нием цитохромов, высокой активностью мито- хондриальных ферментов, имеющими более низ- кое сродство к своим субстратам, а также повы- шением эффективности окислительного фосфо- рилирования (Лукьянова, 2003). Активизация гликолиза приводит к увеличению количества ацетил-коэнзимаА, необходимый для синтеза лимонной кислоты – субстрата аконитазы. Уве- личение количества субстрата приводит к акти- вации как митохондриальной, так и цитоплазма- тической аконитазы. В мозге взрослых животных 65-70% изоцитрата окисляется НАД-зависимой цзоцитратдегидрогеназой, а у молодых, расту- щих животных большая часть цзоцитрата окис- ляется в цитозоле НАДФ-зависимой изоцитрат- дегидрогеназой и образующийся НАДФН ис- пользуется для биосинтеза липидов мозга (Аш- мариниСтукалов, 1996). Пренатальная гипоксия приводит к изменениям активности фермента в цитозоле изученных тканей животных всех воз- растов (Рис.2). В цитозоле тканей 17-ти дневных опытных животных наблюдается резкое (в 2-5 раз) увеличение аконитазной активности (p<0,001). водили при 0-4ºС. МАТЕРИАЛЫ И МЕТОДЫ В цитозольной и митохонд- риальных фракциях определяли активность аконитазы по методу (Guilbauli, 1976), белок определяли по методу Лоури (Кочетов, 1980). водили при 0-4ºС. В цитозольной и митохонд- риальных фракциях определяли активность аконитазы по методу (Guilbauli, 1976), белок определяли по методу Лоури (Кочетов, 1980). Опыты проводили в 6-кратной биологиче- ской и 2-кратной аналитической повторностях. Полученные данные обрабатывали с использо- ванием статистических критериев (Ллойд и Ле- дерман, 1990). Опыты проводили в 6-кратной биологиче- ской и 2-кратной аналитической повторностях. Полученные данные обрабатывали с использо- ванием статистических критериев (Ллойд и Ле- дерман, 1990). РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ Из результатов экспериментов видно (Рис.1), что в цитозоле исследованных тканей головного мозга контрольных крыс трех возрас- тов максимальная активность аконитазы наблю- дается у одномесячных животных, особенно в лимбической коре и мозжечке. В цитозоле тка- ней 17-ти дневных контрольных животных ак- тивность аконитазы намного ниже активности фермента остальных возрастных групп. Это наиболее четко видно на примере гипоталамуса, лимбической и сенсомоторной коры. Получен- ные данные хорошо согласуются с особенно- стями постнатального развития крыс. Так, из- вестно, что к17-му дню постнатального разви- тия у крысят открываются глаза, но головной мозг еще не завершил формирование и поэтому его функционирование не требует больших за- трат энергии. С 17 по 35 день развития в мозге происходит процесс миелинизации нейронов. На этот процесс требуется значительное коли- чество АТФ, а к 3-х месячному возрасту крысы вступают в половозрелый возраст. Рис. 2. Активность аконитазы в цитозольной фрак- ции гипоксированных крыс Эти результаты доказывают, что прена- тальная гипоксия в исследованных тканях го- Рис. 1. Активность аконитазы в цитозольной фрак- ции контрольных крыс Активность цикла Кребса неразрывно связа- на с процессом гликолиза. В ответ на гипоксию Рис. 2. Активность аконитазы в цитозольной фрак- ции гипоксированных крыс Эти результаты доказывают, что прена- тальная гипоксия в исследованных тканях го- Рис. 2. Активность аконитазы в цитозольной фрак- ции гипоксированных крыс Рис. 1. Активность аконитазы в цитозольной фрак- ции контрольных крыс Рис. 1. Активность аконитазы в цитозольной фрак- ции контрольных крыс Эти результаты доказывают, что прена- тальная гипоксия в исследованных тканях го- Активность цикла Кребса неразрывно связа- на с процессом гликолиза. В ответ на гипоксию Активность цикла Кребса неразрывно связа- на с процессом гликолиза. В ответ на гипоксию 53 Баба-заде и Агаев ловного мозга 17-ти дневных крыс усиливает биосинтетические процессы. Активность акони- тазы в цитозоле большинства тканей пренаталь- но гипоксированных одномесячных крыс уве- личивается. Лишь в мозжечке активность фер- мента в 2-2,5 раза ниже контроля (p<0,001) и в лимбической коре наблюдается недостоверное снижение активности фермента ( р> 0,05). ловного мозга 17-ти дневных крыс усиливает биосинтетические процессы. Активность акони- тазы в цитозоле большинства тканей пренаталь- но гипоксированных одномесячных крыс уве- личивается. Лишь в мозжечке активность фер- мента в 2-2,5 раза ниже контроля (p<0,001) и в лимбической коре наблюдается недостоверное снижение активности фермента ( р> 0,05). энергоснабжения, а у 3-х месячных крысах, за исключением мозжечка, в цитоплазме происхо- дит восстановление нормального функциониро- вания. энергоснабжения, а у 3-х месячных крысах, за исключением мозжечка, в цитоплазме происхо- дит восстановление нормального функциониро- вания. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ Интересно сравнить активность аконитазы в цитозольной и митохондриальной фракциях. Как уже отмечалось выше, эти ферменты в клетке используются для выполнения разных функций. В цитозоле 3-х месячных гипоксированных животных только в мозжечке наблюдается 3-х кратный рост активности аконитазы (p<0,001), а в остальных тканях имеют место незначитель- ные изменения. Результаты экспериментов показаны на (Рис.3). Как видно из рис.1 и 3, активности фер- мента в обеих фракциях 17-ти дневных и одно- месячных контрольных животных приблизи- тельно одинаковы. Кардинально меняется соот- ношение ферментов лишь в тканях 3х месяч- ных животных, где наблюдается значительное увеличение активности фермента в митохонд- риях по сравнению с цитозолем (p<0,0050). Эти результаты можно объяснить тем, что в голов- ном мозге 17-ти дневных и месячных крыс идет биосинтез липидов, образование необходимого для этого процесса НАДФН обеспечивается ци- топлазматической аконитазой. В тканях 3-х ме- сячных крыс процесс усиленного биосинтеза за- вершен и головной мозг переходит на путь по- лучения энергии в цикле Кребса, что приводит к активации ферментов цикла Кребса, в том числе и митохондриальной аконитазы. Рис. 3. Активность аконитазы в митохондриальной фракции контрольных крыс. Рис. 4. Активность аконитазы в митохондриальной фракции гипоксированных крыс На (Рис.4) показаны результаты действия пренатальной гипоксии на митохондрии. Мно- гократный рост активности фермента в мито- хондриях тканей 17-ти дневных животных по сравнению с контролем практически идентичен росту активности фермента в цитозольной фракции (р<0,01). Рис. 3. Активность аконитазы в митохондриальной фракции контрольных крыс. Рис. 3. Активность аконитазы в митохондриальной фракции контрольных крыс. Рис. 4. Активность аконитазы в митохондриальной ф В митохондриях одномесячных гипоксир- ованных крыс активность фермента выше, чем в других возрастных группах и в среднем в 2 раза превышает контрольные показатели, за исклю- чением мозжечка, в котором активность фер- мента не изменяется. Эти изменения вызваны теми же причинами, которые меняют актив- ность цитоплазматической акотиназы. Более сложная картина наблюдается в ми- тохондриях 3-х месячных животных. В мозжеч- ке происходит увеличение, в сенсо-моторной - снижение, а в лимбической и орбитальной коре и гипоталамусе активность аконитазы очень близка к контрольным значениям. Заметно, что изменения активности фермента в митохондри- альной фракции во многом идентичны измене- ниям активности в цитозоле. Разница только в том, что в митохондриях гипоксированных крыс активность фермента в сенсомоторной коре снижается в 5 раз, а в цитозоле она не меняется. СПИСОК ЛИТЕРАТУРЫ Ашмарин И.П., Стукалова П.В. 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(1980) Практикум по общей био- химии. М.: 331 с. Светухин В.М. (1968) Цитоархитектоника но- вой коры головного мозга в отряде грызунов. Архив анатомии, эмбриологии и гистологии, 42(№2): 31-45. Walden WE., Selezneva A.I., Dupuy J., Volbeda A., Fontecilla-Camps J.C., Theil E.C., VolzK. (2006) Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA. Journal of Science, 314(5807): 1903-1908. Ллойд Э., Ледерман У. (1990) Справочник по прикладной статистике. Финансы и статисти- ка. В двух томах. М.: 2:526. Рис. 4. Активность аконитазы в митохондриальной фракции гипоксированных крыс Рис. 4. Активность аконитазы в митохондриальной фракции гипоксированных крыс Рост активности аконитазы в цитозоле 17- ти дневных и месячных крыс можно объяснить сохранением в танях головного мозга крыс функционирования оптимизированной схемы На основании полученных данных можно сделать вывод, что пренатальная гипоксия пло- 54 Активность Аконитазы Головного Мозга Лукьянова Л.Д. (2003) Молекулярные меха- низмы тканевой гипоксии и адаптация орга- низма. Физиол. журн., 49(3): 17-35 да в период органогенеза в постнатальном онто- генезе увеличивает активность как митохондри- альной, так и цитоплазматической аконитазы, причем, этот процесс, имеет обратимый харак- тер. Меерсон Ф. З. (1993) Адаптационная медицина: механизмы и защитные эффекты адаптации. М.: Hypoxia Medical, 331с. Gardner P.R., Nguyen D.D., White C.W. (1994) Aconitase is a sensitive and critical target of oxy- gen poisoning in cultured mammalian cells and in rat lungs. Proc. Nat. Acad. Sci. USA, 91:12248- 12 252. AMEA A.İ.Qarayev adına Fiziologiya İnstitutu Prenatal inkişafın organogenez dövründə hipoksiyaya məruz qalmış müxtəlif yaşlı ağ siçovulların baş beynində mitoxondrial və sitoplazmatik akonitazanın fəallığının dəyişilməsi öyrənilib. Müəyyən edilib ki, prenatal hipoksiya posnatal inkişaf zamanı akonitazanın fəallığını artırır və bu prosses dönər xasiyyətlidir. Açar sözlər: Hipoksiya,siçanların baş beyni, akonitaza Institute of Physiology named after A.I. Garayev, ANAS Changes in activities of mitochondrial and cytoplasmic aconitase in the brains of albino rats of different ages subjected to hypoxia in the period of organogenesis of prenatal development were studied. It has been shown that prenatal hypoxia upregulates aconitase activity in postnatal development and this process, probably, has a reversible character. Key words: Hypoxia, albino rat’s brain, aconitase 55
https://openalex.org/W4317659290	https://www.biorxiv.org/content/biorxiv/early/2023/01/21/2023.01.20.524968.full.pdf	English	null	Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in<i>Aspergillus nidulans</i>	bioRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	17,579	Introduction peroxisome hitchhiking on early endosomes7,12. Both PxdA and DipA are found on early endosomes and loss of either compromises long distance motility of peroxisomes, result- ing in an accumulation of peroxisomes in the hyphal tip without affecting early endosome motility or distribu- tion7,12. Peroxisomes are the only organelle demonstrated to hitchhike on early endosomes in A. nidulans12, but the cel- lular basis for why peroxisomes hitchhike is unclear. peroxisome hitchhiking on early endosomes7,12. Both PxdA and DipA are found on early endosomes and loss of either compromises long distance motility of peroxisomes, result- ing in an accumulation of peroxisomes in the hyphal tip without affecting early endosome motility or distribu- tion7,12. Peroxisomes are the only organelle demonstrated to hitchhike on early endosomes in A. nidulans12, but the cel- lular basis for why peroxisomes hitchhike is unclear. Within a eukaryotic cell, organelles are precisely posi- tioned to regulate their cellular function. The intracellular trafficking of these cargos is primarily achieved by molecu- lar motors that bind different cargos and directly transport them along cytoskeletal filaments like microtubules or ac- tin1,2. Microtubules are polar structures that serve as the primary track for transporting cargo long distances in eu- karyotes. They possess a fast-growing plus end typically ori- ented towards the cell periphery and a minus end originat- ing at a microtubule organizing center. Kinesin motors transport cargo predominantly towards the plus end of mi- crotubules (anterograde)3. Conversely, cytoplasmic dynein motors (“dynein” here) transport cargo toward microtu- bule minus ends (retrograde)1. Using comparative genomics, we found that PxdA is conserved within the Pezizomycotina subphylum of asco- mycete filamentous fungi, suggesting that a function for pe- roxisome hitchhiking might also be conserved within this clade. We therefore investigated if hitchhiking was required for septal plugging, a peroxisome function unique to the Pezizomycotina. y Septal plugging is a critical process within Pezizomy- cotina fungi as they possess continuous hyphal compart- ments separated only by a septal wall containing a pore13. This interconnected hyphal network or “mycelium” has the advantage of quickly exchanging components throughout the organism14,15. However, if one portion of the organism is injured or lysed, significant cytoplasmic leakage can occur. In many species within the Pezizomycotina, peroxisome de- rivatives known as Woronin bodies plug septal pores upon mycelial injury, allowing the rest of the mycelium to remain intact16. Woronin bodies can also function to dynamically open and close pores to control hyphal heterogeneity14. Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Livia D. Songstera, Devahuti Bhuyanb, Jenna R. Christensenb, and Samara L. Reck-Petersonabc a Department of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA b Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA c Howard Hughes Medical Institute, Chevy Chase, MD, USA * Correspondence should be addressed to JRC (jrc039@health ucsd edu) or SLR-P (sreckpeterson@health ucsd edu) The proper functioning of organelles depends on their intracellular localization, mediated by motor protein- dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungus Aspergillus nidulans. However, the cellular function of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina, but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent wide- spread leakage. Here, we tested if peroxisome hitchhiking is important for Woronin body motility, distribution, and function in A. nidulans. We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of pe- roxisome hitchhiking by knocking out pxdA significantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Songster et al., 2023 (preprint) Introduction ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans d bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Figure 1 Phylogenetic analysis of the essential peroxisome hitchhiking proteins DipA bodies are thought to be transported to nascent septal sites, where they bud from peroxisomes and remain tethered at the septa18,19. However, the mechanism by which Woronin bodies are transported to septa is unknown. In this study, we investigated whether peroxisome hitchhiking is important for Woronin body motility, positioning, and function in A. nidulans. We find that Woro- nin body proteins associated with peroxi- somes move dynamically and bidirection- ally, and that this motility is dependent upon PxdA-mediated hitchhiking on early endosomes. We show that Woronin bodies separate from peroxisomes once localized to septa, but this fission process does not require PxdA. Finally, we show that the distribution of Woronin bodies in hyphal tips requires hitchhiking, but this is ulti- mately dispensable for the positioning of Woronin bodies at septa and their func- tion in septal closure after hyphal injury. Results Peroxisome hitchhiking proteins and Woronin body specialization are con- served within the Pezizomycotina fungi Peroxisome hitchhiking proteins and Woronin body specialization are con- served within the Pezizomycotina fungi Peroxisome hitchhiking has been directly observed in two species of fungi, A. nidulans and Ustilago maydis6,7. However, a cellular function for peroxisome hitchhiking has not been established. Two early endosome-associated proteins, a putative molecular tether (PxdA) and a phosphatase (DipA), have been shown to be essential for peroxisome hitchhiking in A. nidulans (Fig. 1A-B)7,12. To search for a potential cellular role for peroxisome hitchhiking, we used comparative genomics to identify which fungal clades likely exhibit PxdA- dependent peroxisome hitchhiking. We used BLASTp to search for DipA and PxdA protein homologs in 33 fungal species representing distinct clades across the fungal tree, with the underlying assumption that fungal species encoding both PxdA and DipA in their genomes would exhibit peroxisome hitchhiking. Putative PxdA homologs were only identified in the Pezizomycotina clade and were present in all 15 Pezizomycotina species we analyzed (Fig. 1C; Table S1). Peroxisome hitchhiking has also been observed outside of the Pezizomycotina, in the basidiomycete U. maydis6. We did not identify a homolog of PxdA in U. maydis, suggesting that peroxisome hitchhiking may function by a distinct mechanism in this species. However, it is possible that U. maydis possesses a PxdA Figure 1. Phyloge and PxdA. (A-B) Pr previously defined7 dicted in this study. species representin tions: Saccharo. = cotina; Usti. = Ustila = Chytridiomycota) cartoons are scaled different species. To search for a potential cellular role for peroxisome hitchhiking, we used comparative genomics to identify which fungal clades likely exhibit PxdA- dependent peroxisome hitchhiking. We used BLASTp to search for DipA and PxdA protein homologs in 33 fungal species representing distinct clades across the fungal tree, with the underlying assumption that fungal species encoding both PxdA and DipA in their genomes would exhibit peroxisome hitchhiking. Putative PxdA homologs were only identified in the Pezizomycotina clade and were present in all 15 Pezizomycotina species we analyzed (Fig. 1C; Table S1). Peroxisome hitchhiking has also been observed outside of the Pezizomycotina, in the basidiomycete U. maydis6. We did not identify a homolog of PxdA in U. maydis, suggesting that peroxisome hitchhiking may function by a distinct mechanism in this species. However, it is possible that U. maydis possesses a PxdA homolog that is sequence divergent and identified by our methods. Putative DipA homologs were identified Pezizomycotina species as well as in othe (Table S1). DipA functions in cellular pro peroxisome hitchhiking including septal Figure 1. Introduction Woronin body biogenesis is thought to initiate at the grow- ing hyphal tip, based on a spatial bias in mRNA expression of the essential Woronin body component Hex-1 in Neuro- spora crassa 17. After biogenesis at the hyphal tip, Woronin The canonical view of intracellular transport is that a cargo attaches directly to a motor protein for movement, typically via a protein adaptor1,4. However, a novel form of intracellular transport termed “hitchhiking” was recently discovered, whereby a primary cargo directly interacts with a motor and a secondary hitchhiking cargo is transported indirectly via association with the primary cargo (review5). Hitchhiking-like phenomena have been observed in diverse cell types and organisms such as filamentous fungi6,7, ani- mal neurons8,9, and plant cells10,11. In Aspergillus nidulans, peroxisomes move long dis- tances along microtubules by hitchhiking on motile early endosomes transported by kinesin-3 or dynein7. We previ- ously identified a putative molecular tether (peroxisome distribution mutant A; PxdA) and a phosphatase (DenA-in- teracting phosphatase A; DipA) that are essential for 1 Songster et al., 2023 (preprint) Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. Peroxisome hitchhiking proteins and Woronin body specialization are con- served within the Pezizomycotina fungi It is made available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: rint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans lly by hitchhiking on early endosomes in Aspergillus nidulans Figure 2. Characterization of Woronin body maturation from peroxi- somes in the cytoplasm versus at septa. (A) Diagram of Woronin body mat- uration from peroxisomes after import of HexA and SspA from the cytoplasm. (B-D) Example of SspA-mGFP5 and mCherry-PTS1 in the cytoplasm (B-C) and at tip apical septa (D) of adult wild type hyphae (sum-Z projections). White triangles indicate septa. CFW = calcofluor white, cell membrane dye; wb = Woronin body; p = peroxisome. Scale bars = 5 µm. (E) Average SspA-mGFP5 co-occurrence with mCherry-PTS1 while in the cytoplasm or associated with septa. Each dot represents one experiment with 5 to 20 regions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.87 ± 0.12; wild type septal = 6, 0.48 ± 0.08; pxdAΔ cytoplasmic = 10, 0.74 ± 0.19; pxdAΔ septal = 7, 0.47 ± 0.019). (F) Average intensity ratio of SspA-mGFP5 and mCherry-PTS1 in puncta at septa and in the cytoplasm (n puncta, mean ± SD: wild type cytoplasmic = 49, 1.28 ± 0.63; wild type septal = 56, 2.28 ± 0.84; pxdAΔ cytoplasmic = 48, 1.94 ± 0.82; pxdAΔ septal = 62, 3.11 ± 0.89). (G) Average mCherry-PTS1 co-occurrence with SspA-mGFP5 in the cytoplasm or associated with septa. Each dot represents one experiment with 5 to 20 re- gions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.67 ± 0.21; wild type septal = 6, 1.00 ± 0; pxdAΔ cytoplasmic = 10, 0.62 ± 0.19; pxdAΔ septal = 7, 1.00 ± 0). For E-G: Ordinary one-way ANOVA with post-hoc Tukey-Kramer test for multiple comparisons Error bars represent likely explaining why DipA is found more broadly in the fungal kingdom. Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans Each dot represents one experiment with 5 to 20 regions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.87 ± 0.12; wild type septal = 6, 0.48 ± 0.08; pxdAΔ cytoplasmic = 10, 0.74 ± 0.19; pxdAΔ septal = 7, 0.47 ± 0.019). (F) Average intensity ratio of SspA-mGFP5 and mCherry-PTS1 in puncta at septa and in the cytoplasm (n puncta, mean ± SD: wild type cytoplasmic = 49, 1.28 ± 0.63; wild type septal = 56, 2.28 ± 0.84; pxdAΔ cytoplasmic = 48, 1.94 ± 0.82; pxdAΔ septal = 62, 3.11 ± 0.89). (G) Average mCherry-PTS1 co-occurrence with SspA-mGFP5 in the cytoplasm or associated with septa. Each dot represents one experiment with 5 to 20 re- gions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.67 ± 0.21; wild type septal = 6, 1.00 ± 0; pxdAΔ cytoplasmic = 10, 0.62 ± 0.19; pxdAΔ septal = 7, 1.00 ± 0). For E-G: Ordinary one-way ANOVA with post-hoc Tukey-Kramer test for multiple comparisons. Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (*). p p p y p (B-D) Example of SspA-mGFP5 and mCherry-PTS1 in the cytoplasm (B-C) and at tip apical septa (D) of adult wild type hyphae (sum-Z projections). White triangles indicate septa. CFW = calcofluor white, cell membrane dye; wb = Woronin body; p = peroxisome. Scale bars = 5 µm. (E) Average SspA-mGFP5 co-occurrence with mCherry-PTS1 while in the cytoplasm or associated with septa. Each dot represents one experiment with 5 to 20 regions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.87 ± 0.12; wild type septal = 6, 0.48 ± 0.08; pxdAΔ cytoplasmic = 10, 0.74 ± 0.19; pxdAΔ septal = 7, 0.47 ± 0.019). (F) Average intensity ratio of SspA-mGFP5 and mCherry-PTS1 in puncta at septa and in the cytoplasm (n puncta, mean ± SD: wild type cytoplasmic = 49, 1.28 ± 0.63; wild type septal = 56, 2.28 ± 0.84; pxdAΔ cytoplasmic = 48, 1.94 ± 0.82; pxdAΔ septal = 62, 3.11 ± 0.89). (G) Average mCherry-PTS1 co-occurrence with SspA-mGFP5 in the cytoplasm or associated with septa. Peroxisome hitchhiking proteins and Woronin body specialization are con- served within the Pezizomycotina fungi f As putative homologs for both PxdA and DipA were found only in the Pezizomycotina, we hypothesized that a cellular function for peroxisome hitchhiking may also be specific to these fungi. The plugging of septal pores by Woronin bodies, which are derived from peroxisomes, also occurs exclusively within the Pezizomycotina ascomycetes21. Additionally, previous work has suggested that Woronin bodies might require active transport to reach nascent septal assembly sites prior to budding from peroxisomes19. We therefore sought to test if peroxisome hitchhiking affected Woronin body localization and function in septal pore plugging in A. nidulans. Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans To determine if Woronin body function requires hitch- hiking, we first needed to reliably distinguish peroxi- somes and Woronin bodies from one another in live cells. We achieved this by quantifying the colocalization and co-occurrence of fluorescently tagged proteins that were either an essential Woronin body component or a peroxisome marker. We chose SspA and HexA as repre- sentative Woronin body proteins for A. nidulans based on their involvement in Woronin body formation and function, which has been most extensively examined in N. crassa, another member of the Pezizomycotina (Fig. 1C; Table S1). Fi 2 Ch i ti f W i b d Figure 2. Characterization of Woronin body maturation from peroxi- somes in the cytoplasm versus at septa. (A) Diagram of Woronin body mat- uration from peroxisomes after import of HexA and SspA from the cytoplasm t ti f i In N. crassa, the proteins WSC (Woronin sorting com- plex; SspA in A. nidulans) and Hex-1 (HexA in A. nidu- lans) are essential for Woronin body formation and function, respectively. Previous work has shown that pe- roxisomes begin developing Woronin bodies with the import of Hex-1 via its C-terminal peroxisome targeting signal (PTS1)16. After import, Hex-1 self-assembles into a dense crystalline core, the main structural component of the Woronin body16,22. The Hex-1 complex then polar- izes to one side of the peroxisome due to the presence of the WSC protein in the peroxisome membrane23. This polarization is followed by organelle fission and separa- tion of the Woronin body from the peroxisome17,24. Figure 2. Characterization of Woronin body maturation from peroxi- Figure 2. Characterization of Woronin body maturation from peroxi somes in the cytoplasm versus at septa. (A) Diagram of Woronin body mat- uration from peroxisomes after import of HexA and SspA from the cytoplasm uration from peroxisomes after import of HexA and SspA from the cytoplasm. (B-D) Example of SspA-mGFP5 and mCherry-PTS1 in the cytoplasm (B-C) and at tip apical septa (D) of adult wild type hyphae (sum-Z projections). White triangles indicate septa. CFW = calcofluor white, cell membrane dye; wb = Woronin body; p = peroxisome. Scale bars = 5 µm. (E) Average SspA-mGFP5 co-occurrence with mCherry-PTS1 while in the cytoplasm or associated with septa. Peroxisome hitchhiking proteins and Woronin body specialization are con- served within the Pezizomycotina fungi Phylogenetic analysis of the essential peroxisome hitchhiki and PxdA. (A-B) Protein domain cartoons for A. nidulans DipA (A) and PxdA previously defined7 as CC1 and CC2-3 are indicated. Green bars represen dicted in this study. (C) Phylogenetic tree showing conservation of DipA an species representing major fungal clades, indicated by gray and pink boxe tions: Saccharo. = Saccharomycotina; Taphrino = Taphrinomycotina; Pucc cotina; Usti. = Ustilagomycotina; Agarico. = Agaricomycotina; Muc. = Mucoro = Chytridiomycota). Dictyostelium discoideum was included as an outgrou cartoons are scaled to one another to display amino acid length and doma different species. Figure 1. Phylogenetic analysis of the essential peroxisome hitchhiking proteins DipA g y g y p g p p and PxdA. (A-B) Protein domain cartoons for A. nidulans DipA (A) and PxdA (B). The domains previously defined7 as CC1 and CC2-3 are indicated. Green bars represent coiled coils pre- dicted in this study. (C) Phylogenetic tree showing conservation of DipA and PxdA across 33 species representing major fungal clades, indicated by gray and pink boxes (clade abbrevia- tions: Saccharo. = Saccharomycotina; Taphrino = Taphrinomycotina; Puccin. = Pucciniomy- cotina; Usti. = Ustilagomycotina; Agarico. = Agaricomycotina; Muc. = Mucoromycotina; Chytrid. = Chytridiomycota). Dictyostelium discoideum was included as an outgroup. Protein domain cartoons are scaled to one another to display amino acid length and domain organization in different species. homolog that is sequence divergent and therefore not identified by our methods. Putative DipA homologs were identified in all queried Pezizomycotina species as well as in other fungal clades (Table S1). DipA functions in cellular processes beyond peroxisome hitchhiking, including septal positioning20, Songster et al., 2023 (preprint) 2 Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans p y To better measure the relative amounts of peroxisome versus Woronin body proteins in the cytoplasm and at septa, we quantified the fluorescent intensity of SspA and HexA relative to PTS1. The ratio of SspA to HexA was con- sistent at both septa and in the cytoplasm (Fig. S1G), sug- gesting Woronin body composition does not vary much be- tween both locations. In contrast, septal puncta exhibited a significantly higher intensity ratio of SspA to PTS1 than cy- toplasmic puncta (Fig. 2F). We observed a similar pattern for the ratio of HexA to PTS1 (Fig. S1H). Together, this data supports the idea that most Woronin bodies have separated from peroxisomes by the time they associate with septa. ( g ) Next, we sought to test how frequently Woronin bodies and peroxisomes colocalize during long-distance runs (>3 µm travelled in one direction). The large majority of moving Woronin bodies were colocalized with a peroxisome, and vice versa (Fig. 3D-E). This finding was surprising as we previously observed that only two thirds of cytoplasmic peroxisomes had associated Woronin body signal (Fig. 2G). Furthermore, we found no obvious bias in whether the Woronin body was ‘leading’ or ‘trailing’ the peroxisome during motile runs, and instead observed the signals frequently overlapped during movement (Fig. 3F). This contrasts with our observations on stationary cytoplasmic peroxisomes and Woronin bodies, which frequently exhibited Woronin body signal that was polarized and offset to one side of the peroxisome signal (Fig. 2B). Together, this data suggests that Woronin body proteins move via association with a hitchhiking peroxisome, and not as a separate / distinct organelle. Finally, we wanted to estimate how many cytoplasmic peroxisomes were actively developing Woronin bodies. We observed that 67% of all cytoplasmic peroxisomes had an associated Woronin body (Fig. 2G). Overall, these data show that not all peroxisomes in the cytoplasm are actively devel- oping Woronin bodies in A. nidulans. However, for peroxi- somes that have begun developing Woronin bodies, the two organelles remain attached in the cytoplasm, and only sep- arate shortly before or immediately following recruitment to septa. Previous work has established that in most Pezizomy- cotina including A. nidulans, Woronin bodies are tethered at septa18. However, a small number of species including N. Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans peroxisome hitchhiking was responsible for Woronin body movement and transport to septa. These colocalization experiments in live cells estab- lished that SspA could serve as a sufficient marker for later stages of Woronin body maturation. We therefore used this as a tool to visually distinguish how Woronin body (SspA- mGFP5) and peroxisome (mCherry-PTS1) colocalization varies at different intracellular positions in wild type hy- phae (Fig. 2A). We began by scoring the fraction of colocal- ized Woronin body and peroxisome puncta in the cytoplasm (Fig. 2B) versus at septa (Fig. 2C-D). Cytoplasmic Woronin bodies were frequently associated with a peroxisome in wild type hyphae (Fig. 2E) and rarely found without an at- tached peroxisome (Fig. 2B). In comparison, at septa, about half of Woronin bodies had no associated peroxisome signal (Fig. 2D-E). For those septal Woronin bodies with corre- sponding peroxisome signal, the Woronin body signal was always closer to the septa (Fig. 2C; white arrow). This pre- ferred orientation is likely due to Woronin body tethering at septal sites by HexA27. We showed that Woronin bodies frequently colocalize with peroxisomes in the cytoplasm but are present alone at septa (Fig. 2E). This suggests that Woronin bodies could either (1) move in conjunction with hitchhiking peroxisomes and bud off once present at septa or (2) bud off peroxisomes in the cytoplasm and be transported to septa by a distinct mechanism. To test this, we first compared Woronin body and peroxisome movement in adult hyphae. In A. nidulans, only a small population of peroxisomes exhibit long-distance movements at a given time7,29. We found that Woronin bodies also exhibit infrequent but dynamic bidirectional movements, like peroxisomes (Movie S1). As expected7, 10% of total movements for both peroxisomes and Woronin body movements were processive over long-distances (>3 µm), indicative of microtubule-driven movement. During these long-distance movements, Woronin bodies moved at similar speeds (Fig. 3A) and over similar distances to peroxisomes (Fig. 3B). Both peroxisomes and Woronin bodies also exhibited bidirectional movements with no directional bias (Fig. 3C). Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans crassa use a divergent mechanism of Woronin body tether- ing, whereby Woronin bodies are delocalized from septa and instead are tethered to the actin cortex throughout a hyphal compartment28. Our observations in A. nidulans on the localization of Woronin body maturation and separation from peroxisomes after septal association is similar to what was previously reported for N. crassa28, despite these spe- cies exhibiting different Woronin body localization and tethering mechanisms. We next tested if Woronin body movement required the essential peroxisome hitchhiking protein PxdA. Compared to wild type hyphae, in which peroxisomes and Woronin bodies are consistently distributed across hyphae (Fig. 3G), pxdAΔ hyphae had an accumulation of both peroxisomes and Woronin bodies at the hyphal tip (Fig. 3H) and a drastic reduction in the number of Woronin bodies and peroxisomes moving in either direction (Fig. 3C; Movie S2). Furthermore, motile Woronin bodies colocalized with the putative hitchhiking tether PxdA (Fig. 3I-J). These data demonstrate that Woronin bodies require PxdA for long- distance, bidirectional movements in hyphal tips. Woronin bodies move bidirectionally using the peroxisome hitchhiking protein PxdA Characterization of Woronin body biogenesis and distri- bution in Aspergillus nidulans Each dot represents one experiment with 5 to 20 re- gions of interest scored (n fields of view, mean ± SD: wild type cytoplasmic = 9, 0.67 ± 0.21; wild type septal = 6, 1.00 ± 0; pxdAΔ cytoplasmic = 10, 0.62 ± 0.19; pxdAΔ septal = 7, 1.00 ± 0). For E-G: Ordinary one-way ANOVA with post-hoc Tukey-Kramer test for multiple comparisons. Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (**). y p The formation of Woronin bodies in A. nidulans is largely similar to that of N. crassa, as they also mature from peroxisomes following the import of HexA and SspA (Fig. 2A)25,26. To characterize the colocalization of Woronin bodies with peroxisomes in A. nidulans, we quantified the degree of overlap between endogenously tagged HexA, SspA, and the peroxisome marker PTS1 via fluorescence microscopy in germlings. We found almost all SspA puncta had co-occurring HexA signal. On the other hand, a large population of HexA puncta had no corresponding SspA signal (Fig. S1A-B). PTS1 co-oc- curred more frequently with HexA (Fig. S1C-D) than it did with SspA (Fig. S1E-F), suggesting that HexA is im- ported into peroxisomes prior to SspA. Consistent with this finding, PTS1 and HexA were often, but not always, closely overlapping (Fig. S1C), whereas PTS1 and SspA were often adjacent and only weakly overlapping with one an- other (Fig. S1E). Together, these data suggest that while the majority of peroxisome and Woronin body proteins exist an tri W co se sc w se m w px Av as gi 9, 0. po S 0. within the same membrane-bound compartment, distinct stages of Woronin body maturation can be visualized using fluorescent microscopy. 3 Songster et al., 2023 (preprint) Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. Essential Woronin body proteins are not required for perox- isome hitchhiking SspA mean ± SD: anterograde wild type = 0.94 0.95; anterograde pxdAΔ = 0.23 ± 0.53; retrograde wild type = -1.25 ± 1.1; retrograde pxdAΔ = -0.19 ± 0.50). (D) Kymograph depicting mCherry PTS1 and SspA-mGFP5 colocalization over time. This kymograph was generated by the blue dashed line shown in (G). The yellow dashed lin on the kymograph is an example of a line used for (F). X-scale bar = 1 µm, Y-scale bar = 10 s. (E) Percent of long-distance runs (>3 µm) tha are colocalized; each dot represents an individual experiment, where all runs from at least 10 hyphal tips were scored for colocalization. Th number of moving SspA puncta ranged from 11-18 for pxdAΔ and from 14-30 for wild type experiments. The number of moving PTS1 punct ranged from 6-13 for pxdAΔ and from 7-26 for wild type experiments (n experiments, mean ± SD: wild type SspA = 4, 0.84 ± 0.13; wild type PTS1 = 4, 0.91 ± 0.08; pxdAΔ SspA = 3, 0.71 ± 0.16; pxdAΔ PTS1 = 3, 0.95 ± 0.08). (F) Mean intensity of SspA-mGFP5 and mCherry-PTS during comigrating runs in wild type (n = 53 runs collected from 23 cells). The gray dashed line indicates the middle of the mCherry-PTS1 peak (G-H) SspA-mGFP5 and mCherry-PTS1 in a single plane of wild type (G) and pxdAΔ (H) hyphal tips. The blue dashed line was used to generat kymograph in (D). Scale bars = 10 µm. (I) SspA-mGFP5 and PxdA-mKate comigration towards a growing hyphal tip (not shown; to the left) White arrows indicate two comigration events. The dashed orange line in the last panel was used to generate the kymograph in (I). Scale ba = 2 µm. (J) Kymograph depicting SspA-mGFP5 and PxdA-mKate colocalization over time. X-scale bar = 1 µm, Y-scale bar = 3 s. White arrow i di h i i d h P dA K i h f l di S A GFP f ll i F B C d E E b As Woronin bodies and peroxisomes comigrated during long-distance runs, and most motile peroxisomes g , p p p g , Figure 3. Woronin bodies at the hyphal tip exhibit dynamic, bidirectional movement with peroxisomes, and this movement requires PxdA. Essential Woronin body proteins are not required for perox- isome hitchhiking (A) Histogram of speeds of long-distance runs (>3 µm travelled in one direction) for SspA-mGFP5 and mCherry-PTS1 puncta (n directed runs/total, mean ± SD: SspA-mGFP5 = 39/390, 1.71 ± 0.59 µm/s; mCherry-PTS1 = 34/330, 1.72 ± 0.79 µm/s). Solid lines indicate nonlinear Gaussian fit lines (SspA-mGFP5: R2 = 0.95; mCherry-PTS1: R2 = 0.76). (B) Mean distance of long-distance runs for SspA-mGFP5 and mCherry- PTS1 (n, mean ± SD: SspA-mGFP5 = 39 runs, 4.39 ± 1.2 µm; mCherry-PTS1 = 34 runs, 4.01 ± 0.96 µm). (C) Number of anterograde (towards the hyphal tip) and retrograde (towards the apical septum) movements of mCherry-PTS1 (top) and SspA-mGFP5 (bottom) in wild type and pxdAΔ adult hyphae (n: wild type = 32 cells and pxdAΔ = 64 cells. PTS1 mean ± SD puncta/30 s: anterograde wild type = 1.0 ± 1.2; anterograde pxdAΔ = 0.17 ± 0.42; retrograde wild type = -1.1 ± 0.94; retrograde pxdAΔ = -0.16 ± 0.37. SspA mean ± SD: anterograde wild type = 0.94 ± 0.95; anterograde pxdAΔ = 0.23 ± 0.53; retrograde wild type = -1.25 ± 1.1; retrograde pxdAΔ = -0.19 ± 0.50). (D) Kymograph depicting mCherry- PTS1 and SspA-mGFP5 colocalization over time. This kymograph was generated by the blue dashed line shown in (G). The yellow dashed line on the kymograph is an example of a line used for (F). X-scale bar = 1 µm, Y-scale bar = 10 s. (E) Percent of long-distance runs (>3 µm) that are colocalized; each dot represents an individual experiment, where all runs from at least 10 hyphal tips were scored for colocalization. The number of moving SspA puncta ranged from 11-18 for pxdAΔ and from 14-30 for wild type experiments. The number of moving PTS1 puncta ranged from 6-13 for pxdAΔ and from 7-26 for wild type experiments (n experiments, mean ± SD: wild type SspA = 4, 0.84 ± 0.13; wild type PTS1 = 4, 0.91 ± 0.08; pxdAΔ SspA = 3, 0.71 ± 0.16; pxdAΔ PTS1 = 3, 0.95 ± 0.08). (F) Mean intensity of SspA-mGFP5 and mCherry-PTS1 during comigrating runs in wild type (n = 53 runs collected from 23 cells). The gray dashed line indicates the middle of the mCherry-PTS1 peak. (G-H) SspA-mGFP5 and mCherry-PTS1 in a single plane of wild type (G) and pxdAΔ (H) hyphal tips. The blue dashed line was used to generate kymograph in (D). Scale bars = 10 µm. Essential Woronin body proteins are not required for perox- isome hitchhiking The number of moving PTS1 punct ranged from 6-13 for pxdAΔ and from 7-26 for wild type experiments (n experiments, mean ± SD: wild type SspA = 4, 0.84 ± 0.13; wild type PTS1 = 4, 0.91 ± 0.08; pxdAΔ SspA = 3, 0.71 ± 0.16; pxdAΔ PTS1 = 3, 0.95 ± 0.08). (F) Mean intensity of SspA-mGFP5 and mCherry-PTS during comigrating runs in wild type (n = 53 runs collected from 23 cells). The gray dashed line indicates the middle of the mCherry-PTS1 peak (G-H) SspA-mGFP5 and mCherry-PTS1 in a single plane of wild type (G) and pxdAΔ (H) hyphal tips. The blue dashed line was used to generat kymograph in (D). Scale bars = 10 µm. (I) SspA-mGFP5 and PxdA-mKate comigration towards a growing hyphal tip (not shown; to the left) White arrows indicate two comigration events. The dashed orange line in the last panel was used to generate the kymograph in (I). Scale ba = 2 µm (J) Kymograph depicting SspA mGFP5 and PxdA mKate colocalization over time X scale bar = 1 µm Y scale bar = 3 s White arrow some hitchhiking s Woronin bodies and peroxisomes comigrated during ong-distance runs, and most motile peroxisomes number of moving peroxisomes per cell (Fig. S2B). In hex hyphae, peroxisomes showed a slight accumulation hyphal tips (Fig. S2C), but to a lesser extent than Figure 3. Woronin bodies at the hyphal tip exhibit dynamic, bidirectional movement with peroxisomes, and this movement requires PxdA. (A) Histogram of speeds of long-distance runs (>3 µm travelled in one direction) for SspA-mGFP5 and mCherry-PTS1 puncta (n directe runs/total, mean ± SD: SspA-mGFP5 = 39/390, 1.71 ± 0.59 µm/s; mCherry-PTS1 = 34/330, 1.72 ± 0.79 µm/s). Solid lines indicate nonlinea Gaussian fit lines (SspA-mGFP5: R2 = 0.95; mCherry-PTS1: R2 = 0.76). (B) Mean distance of long-distance runs for SspA-mGFP5 and mCherry PTS1 (n, mean ± SD: SspA-mGFP5 = 39 runs, 4.39 ± 1.2 µm; mCherry-PTS1 = 34 runs, 4.01 ± 0.96 µm). (C) Number of anterograde (toward the hyphal tip) and retrograde (towards the apical septum) movements of mCherry-PTS1 (top) and SspA-mGFP5 (bottom) in wild type an pxdAΔ adult hyphae (n: wild type = 32 cells and pxdAΔ = 64 cells. PTS1 mean ± SD puncta/30 s: anterograde wild type = 1.0 ± 1.2; anterograde pxdAΔ = 0.17 ± 0.42; retrograde wild type = -1.1 ± 0.94; retrograde pxdAΔ = -0.16 ± 0.37. Woronin bodies move bidirectionally using the peroxisome hitchhiking protein PxdA While loss of PxdA abolished most long-distance movements, a small number of peroxisomes and Woronin bodies still moved processively in pxdAΔ hyphal tips (Fig. S1K-L). However, these Woronin bodies were still colocalized with peroxisomes during this movement (Fig. 3E). Additionally, the overall Woronin body and peroxisome co-occurrence (SspA/PTS1) in pxdAΔ hyphae was similar to what was observed in wild type (Fig. 2E-G, Woronin body biogenesis is thought to occur primarily in the cytoplasm near the hyphal tip17,18. Woronin bodies are then localized to their tethering site at or near septal pores, where they function to seal septal pores following injury19. However, the mechanism by which they are transported to septa is unknown. We therefore wanted to characterize Woronin body movement in hyphae and determine if 4 Songster et al., 2023 (preprint) Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidu S1F, and S1J). These findings demonstrate that the majority of Woronin body movement occurs via association with hitchhiking peroxisomes and that PxdA is not important for the assembly of Woronin bodies. S1F, and S1J). These findings demonstrate that the majority of Woronin body movement occurs via association with hitchhiking peroxisomes and that PxdA is not important for the assembly of Woronin bodies. colocalized with Woronin bodies while moving (Fig. 2E), we wondered if the presence of Woronin body proteins was required for peroxisome motility. We tested this using mutants lacking either HexA or SspA (Fig. S2A). Loss of HexA or SspA did not significantly affect the average number of moving peroxisomes per cell (Fig. S2B). In hexAΔ hyphae, peroxisomes showed a slight accumulation at hyphal tips (Fig. S2C), but to a lesser extent than the Essential Woronin body proteins are not required for perox- isome hitchhiking Essential Woronin body proteins are not required for perox- some hitchhiking As Woronin bodies and peroxisomes comigrated during ong-distance runs, and most motile peroxisomes HexA or SspA did not significantly affect the avera number of moving peroxisomes per cell (Fig. S2B). In hex hyphae, peroxisomes showed a slight accumulation hyphal tips (Fig. S2C), but to a lesser extent than Figure 3. Woronin bodies at the hyphal tip exhibit dynamic, bidirectional movement with peroxisomes, and this movement requires PxdA. (A) Histogram of speeds of long-distance runs (>3 µm travelled in one direction) for SspA-mGFP5 and mCherry-PTS1 puncta (n directe runs/total, mean ± SD: SspA-mGFP5 = 39/390, 1.71 ± 0.59 µm/s; mCherry-PTS1 = 34/330, 1.72 ± 0.79 µm/s). Solid lines indicate nonlinea Gaussian fit lines (SspA-mGFP5: R2 = 0.95; mCherry-PTS1: R2 = 0.76). (B) Mean distance of long-distance runs for SspA-mGFP5 and mCherry PTS1 (n, mean ± SD: SspA-mGFP5 = 39 runs, 4.39 ± 1.2 µm; mCherry-PTS1 = 34 runs, 4.01 ± 0.96 µm). (C) Number of anterograde (toward the hyphal tip) and retrograde (towards the apical septum) movements of mCherry-PTS1 (top) and SspA-mGFP5 (bottom) in wild type and pxdAΔ adult hyphae (n: wild type = 32 cells and pxdAΔ = 64 cells. PTS1 mean ± SD puncta/30 s: anterograde wild type = 1.0 ± 1.2; anterograde pxdAΔ = 0.17 ± 0.42; retrograde wild type = -1.1 ± 0.94; retrograde pxdAΔ = -0.16 ± 0.37. SspA mean ± SD: anterograde wild type = 0.94 0.95; anterograde pxdAΔ = 0.23 ± 0.53; retrograde wild type = -1.25 ± 1.1; retrograde pxdAΔ = -0.19 ± 0.50). (D) Kymograph depicting mCherry PTS1 and SspA-mGFP5 colocalization over time. This kymograph was generated by the blue dashed line shown in (G). The yellow dashed lin on the kymograph is an example of a line used for (F). X-scale bar = 1 µm, Y-scale bar = 10 s. (E) Percent of long-distance runs (>3 µm) tha are colocalized; each dot represents an individual experiment, where all runs from at least 10 hyphal tips were scored for colocalization. Th number of moving SspA puncta ranged from 11-18 for pxdAΔ and from 14-30 for wild type experiments. Woronin bodies localize at septa and plug damaged hyphae independently of hitchhiking Woronin bodies localize at septa and plug damaged hyphae independently of hitchhiking We next wanted to test whether peroxisome-Woronin body hitchhiking is required for Woronin bodies to localize to septa in undamaged hyphae. First, we quantified the cytoplasmic localization of Woronin bodies in strains lacking peroxisome movement (pxdAΔ and hookAΔ) as well as in a strain with non-functional Woronin bodies (hexAΔ). We included hookAΔ because HookA is the adaptor protein that links microtubule motors to early endosomes in A. nidulans 30, and loss of HookA therefore disrupts both early endosome and peroxisome distribution and motility7. As predicted, loss of peroxisome movement in both pxdAΔ and hookAΔ hyphae results in an accumulation of SspA-labeled Woronin bodies at the hyphal tip (Fig. 4A-B). There were occasionally very bright Woronin bodies at the apex of wild type hyphal tips, corresponding to a slight peak in intensity around 0.5 μm from the tip (Fig. 4B, black line) that was abolished in hexAΔ hyphae (Fig. 4B, red line). This peak in SspA intensity in wild type tips could correspond to higher levels of SspA import into peroxisomes in this region, or the Manual scoring of these localization patterns revealed that most tip apical septa had Woronin bodies on one or both sides in wild type hyphae (Fig. 4C-D). Furthermore, approximately one third of tip apical septa had Woronin bodies on both sides in wild type (Fig. 4D). As expected, most hexAΔ hyphae lacked Woronin bodies at apical septa. Both pxdAΔ and hookAΔ hyphae had an intermediate phenotype, with a slight reduction in the number of septa with Woronin bodies on both sides, but similar numbers of septa with Woronin bodies on one side compared to wild Songster et al 2023 (preprint) Figure 4. Woronin bodies require hitchhiking to localize at septa, but this localization is not required for septal plugging after hyphal damage. (A) SspA-mGFP5 localization at the hyphal tip in adult hyphae (max-Z projections). Scale bar = 10 µm. (B) Mean intensity of SspA- mGFP5 from the hyphal tip inwards (n hyphae: wild type = 140; hexAΔ = 92; pxdAΔ = 225; hookAΔ = 132). Gray bar indicates approximate position of a tip apical nucleus. (C) Association of Woronin bodies at tip apical septa when grown in glucose MM (max-Z projections). Hyphal tips would be to the right of each image. Scale bar = 2 µm. Essential Woronin body proteins are not required for perox- isome hitchhiking (I) SspA-mGFP5 and PxdA-mKate comigration towards a growing hyphal tip (not shown; to the left). White arrows indicate two comigration events. The dashed orange line in the last panel was used to generate the kymograph in (I). Scale bar = 2 µm. (J) Kymograph depicting SspA-mGFP5 and PxdA-mKate colocalization over time. X-scale bar = 1 µm, Y-scale bar = 3 s. White arrow indicates the time point at around 6 s where PxdA-mKate switches from leading SspA-mGFP5 to following. For B-C and E: Error bars represent SEM. Unpaired t-test; P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (**). 5 Songster et al., 2023 (preprint) Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidu presence of larger Woronin body/peroxisome organelles that have not yet undergone fission. accumulation observed in pxdAΔ hyphae (Fig. S2D). Despite nearly all moving peroxisomes possessing Woronin body proteins, these data show that the ability to form Woronin bodies is not required for peroxisome hitchhiking. We next examined Woronin body localization at tip apical septa in undamaged, wild type hyphae as well as pxdAΔ, hookAΔ, and hexAΔ mutants. Essential Woronin body proteins are not required for perox- isome hitchhiking We chose tip apical septa because these septa were most recently formed 31 and are still capable of dynamically opening and closing via septal plugging14. Transmission electron microscopy studies in A. nidulans have previously reported between four and nine Woronin bodies at septa18. In assessing Woronin body localization at septa via fluorescence microscopy, three classes of Woronin body localization were identified: (i) septa with Woronin body signal on either side likely representing more than one Woronin body, (ii) septa with Woronin body signal on only one side and (iii) no Woronin body signal (Fig. S3A-B). Woronin bodies localize at septa and plug damaged hyphae independently of hitchhiking (D) Fraction apical septa with Woronin bodies, quantified as being on one side (lighter bars; bottom) or both sides (darker bars; top) of septa. Each experiment scored 8-64 apical septa (n experiments, mean ± SD: one side wild type = 7, 0.55 ± 0.17; one side hexAΔ = 8, 0.16 ± 0.12; one side pxdAΔ = 6, 0.50 ± 0.15; one side hookAΔ = 4, 0.47 ± 0.13; both sides wild type = 7, 0.32 ± 0.12; both sides hexAΔ = 8, 0.012 ± 0.027; both sides pxdAΔ = 6, 0.18 ± 0.11; both sides hookAΔ = 4, 0.23 ± 0.092). (E) Hyphae expressing mCherry-SsoA as a cell membrane marker, when grown in 1% glucose agar versus 2% sorbose/0.1% glucose agar (sum-Z projections). Scale bar = 10 µm. The white circles indicate leaked cytoplasm due to hyphal tip bursting, and the red circle is an example of a background fluorescence measurements. (F) Leaked cytoplasm, measured as the integrated density – (background area) to correct for background intensity, and then normalized to the average wild type (n burst cells, mean ± SD: wild type = 73, 1.00 ± 0.64; hexAΔ = 63, 1.61 ± 0.81; pxdAΔ = 125, 1.12 ± 0.67; hookAΔ = 119, 1.25 ± 0.65). (G) Model of peroxisome (P) and Woronin body (WB) hitchhiking on early endosomes (EE). Dynein refers to dynein and dynactin complexes. For D and F: Error bars represent SEM. Ordinary one-way ANOVA with post-hoc Tukey-Kramer test; P > 0.05 (labeled ns for “not significant”), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (*). ocalization is not required for septal plugging after hypha bodies require hitchhiking to localize at septa, but this localization is not required for septal plugging after hypha Figure 4. Woronin bodies require hitchhiking to localize at septa, but this localization is not required for septal plugging after hyphal damage. (A) SspA-mGFP5 localization at the hyphal tip in adult hyphae (max-Z projections). Scale bar = 10 µm. (B) Mean intensity of SspA- mGFP5 from the hyphal tip inwards (n hyphae: wild type = 140; hexAΔ = 92; pxdAΔ = 225; hookAΔ = 132). Gray bar indicates approximate position of a tip apical nucleus. (C) Association of Woronin bodies at tip apical septa when grown in glucose MM (max-Z projections). Hyphal tips would be to the right of each image. Scale bar = 2 µm. Woronin bodies localize at septa and plug damaged hyphae independently of hitchhiking (D) Fraction apical septa with Woronin bodies, quantified as being on one side (lighter bars; bottom) or both sides (darker bars; top) of septa. Each experiment scored 8-64 apical septa (n experiments, mean ± SD: one side wild type = 7, 0.55 ± 0.17; one side hexAΔ = 8, 0.16 ± 0.12; one side pxdAΔ = 6, 0.50 ± 0.15; one side hookAΔ = 4, 0.47 ± 0.13; both sides wild type = 7, 0.32 ± 0.12; both sides hexAΔ = 8, 0.012 ± 0.027; both sides pxdAΔ = 6, 0.18 ± 0.11; both sides hookAΔ = 4, 0.23 ± 0.092). (E) Hyphae expressing mCherry-SsoA as a cell membrane marker, when grown in 1% glucose agar versus 2% sorbose/0.1% glucose agar (sum-Z projections). Scale bar = 10 µm. The white circles indicate leaked cytoplasm due to hyphal tip bursting, and the red circle is an example of a background fluorescence measurements. (F) Leaked cytoplasm, measured as the integrated density – (background area) to correct for background intensity, and then normalized to the average wild type (n burst cells, mean ± SD: wild type = 73, 1.00 ± 0.64; hexAΔ = 63, 1.61 ± 0.81; pxdAΔ = 125, 1.12 ± 0.67; hookAΔ = 119, 1.25 ± 0.65). (G) Model of peroxisome (P) and Woronin body (WB) hitchhiking on early endosomes (EE). Dynein refers to dynein and dynactin complexes. For D and F: Error bars represent SEM. Ordinary one-way ANOVA with post-hoc Tukey-Kramer test; P > 0.05 (labeled ns for “not significant”), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (*) Figure 4. Woronin bodies require hitchhiking to localize at septa, but this localization is not required for septal plugging after hyphal damage. (A) SspA-mGFP5 localization at the hyphal tip in adult hyphae (max-Z projections). Scale bar = 10 µm. (B) Mean intensity of SspA- mGFP5 from the hyphal tip inwards (n hyphae: wild type = 140; hexAΔ = 92; pxdAΔ = 225; hookAΔ = 132). Gray bar indicates approximate position of a tip apical nucleus. (C) Association of Woronin bodies at tip apical septa when grown in glucose MM (max-Z projections). Hyphal tips would be to the right of each image. Scale bar = 2 µm. (D) Fraction apical septa with Woronin bodies, quantified as being on one side (lighter bars; bottom) or both sides (darker bars; top) of septa. Concluding thoughts 13. Mouriño-Pérez, R. R. Septum development in filamentous ascomy- cetes. Fungal Biol Rev 27, 1–9 (2013). This study shows that Woronin bodies move dynamically and bidirectionally in the hyphal tip by hitchhiking on PxdA-labeled early endosomes in A. nidulans. Though Woronin bodies have been described to move in some fungi (Z. tritici33; A. fumigatus19; A. nidulans26), their motility has not been well characterized and the mechanism by which they move was previously unknown. We showed that Woronin bodies and peroxisomes often coexist as connected organelles in the cytoplasm, where they hitchhike together on early endosomes via PxdA with no directional bias (Fig. 4G). We have identified that virtually all peroxisomes colocalize with a Woronin body while hitchhiking, despite only two thirds of peroxisomes colocalizing with Woronin bodies while immotile in the cytoplasm. Despite this intriguing finding, we show that Woronin body proteins are not required for the identification of peroxisomes as hitchhiking cargo. 14. Bleichrodt, R.-J. et al. Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies. Mol Micro- biol 86, 1334–1344 (2012). 15. Tegelaar, M. & Wösten, H. A. B. Functional distinction of hyphal com- partments. Sci Rep 7, 6039 (2017). 16. Jedd, G. & Chua, N. H. A new self-assembled peroxisomal vesicle re- quired for efficient resealing of the plasma membrane. Nat Cell Biol 2, 226–231 (2000). 17. Tey, W. K., North, A. J., Reyes, J. L., Lu, Y. F. & Jedd, G. Polarized gene expression determines Woronin body formation at the leading Edge of the fungal colony. Mol Biol Cell 16, 2651–2659 (2005). 18. Momany, M., Richardson, E. A., Van Sickle, C. & Jedd, G. Mapping Woronin body position in Aspergillus nidulans. Mycologia 94, 260– 266 (2002). 19. Leonhardt, Y., Kakoschke, S. C., Wagener, J. & Ebel, F. Lah is a trans- membrane protein and requires Spa10 for stable positioning of Woronin bodies at the septal pore of Aspergillus fumigatus. Sci Rep 7, 44179 (2017). 20. Schinke, J. et al. The DenA/DEN1 interacting phosphatase DipA con- trols septa positioning and phosphorylation-dependent stability of cytoplasmatic DenA/DEN1 during fungal development. PLoS Genet 12, e1005949 (2016). It was previously hypothesized that Woronin body biogenesis begins in the hyphal tip, after which they are transported retrograde towards tethering sites at nascent septa17–19. We found that Woronin body movement in hyphal tips is bidirectional and requires the hitchhiking protein PxdA. References type. This finding suggests that PxdA-mediated hitchhiking could contribute to achieving Woronin bodies on both sides of nascent septa. However, fluorescence measurements show there is no significant difference in SspA signal at septa in wild type versus pxdAΔ or hookAΔ hyphae (Fig. S3C). Overall, our data suggest that Woronin body localization at septa does not depend on Woronin body hitchhiking. Though Woronin body hitchhiking may have a minor contribution to Woronin body recruitment at septa, it is not the primary mechanism by which these organelles localize at septa. Woronin bodies in pxdAΔ hyphae could be potentially recruited to tip-apical septa by other mechanisms, such as cytoplasmic movement via ‘poleward drift’32 or passive association with septal assembly sites that are marked as the hyphal tip grows31. 1. Reck-Peterson, S. L., Redwine, W. B., Vale, R. D. & Carter, A. P. The cy- toplasmic dynein transport machinery and its many cargoes. Nat Rev Mol Cell Biol 19, 382–398 (2018). 2. Titus, M. A. Myosin-driven intracellular transport. Cold Spring Harb Perspect Biol 10, a021972 (2018). 3. Hirokawa, N., Noda, Y., Tanaka, Y. & Niwa, S. Kinesin superfamily mo- tor proteins and intracellular transport. Nat Rev Mol Cell Biol 10, 682–696 (2009). 4. Olenick, M. A. & Holzbaur, E. L. F. Dynein activators and adaptors at a glance. J Cell Sci 132, jcs227132 (2019). 5. Christensen, J. R. & Reck-Peterson, S. L. Hitchhiking across kingdoms: Cotransport of cargos in fungal, animal, and plant cells. Annu Rev Cell Dev Biol 6, 155–178 (2022). 6. Guimaraes, S. C. et al. Peroxisomes, lipid droplets, and endoplasmic reticulum ‘hitchhike’ on motile early endosomes. J Cell Biol 211, 945– 954 (2015). 7. Salogiannis, J., Egan, M. J. & Reck-Peterson, S. L. Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA. Journal of Cell Biology 212, 289–296 (2016). Finally, we wanted to test if Woronin body hitchhiking was important for Woronin body-based sealing of septal pores following hyphal damage. Growth on sorbose media induced tip lysis in just under half of cells, regardless of genotype (Fig. S3D). Tip lysis and cytoplasmic puddling was clearly visible on sorbose media for all strains (Fig. 4E). The amount of leaked protein was higher in lysed hexAΔ than lysed wild type, pxdAΔ, or hookAΔ cells (Fig. 4F), despite leakage areas appearing similar in size (Fig. S3E). References This pattern of cytoplasmic leakage was recapitulated when dried mycelia were exposed to hypotonic shock via distilled water (Fig. S3F). Together, these data demonstrate that Woronin body hitchhiking is not required for proper septal plugging after hyphal damage. 8. Özkan, N. et al. ER – lysosome contacts at a pre-axonal region regulate axonal lysosome availability. Nat Commun 12, 4493 (2021). 9. Harbauer, A. B. et al. Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy. Neuron 110, 1516- 1531.e9 (2022). 10. Oikawa, K. et al. CHLOROPLAST UNUSUAL POSITIONING1 is essential for proper chloroplast positioning. Plant Cell 15, 2805–2815 (2003). 11. Luo, K.-R., Huang, N.-C., Chang, Y.-H. & Yu, T.-S. Arabidopsis cyclophil- ins direct plasmodesmata-targeting of mobile mRNA via organelle hitchhiking. Research Square (2022) doi:10.21203/rs.3.rs- 1088339/v1. 12. Salogiannis, J. et al. PxdA interacts with the DipA phosphatase to reg- ulate peroxisome hitchhiking on early endosomes. Molecular Biology of the Cell 32, 492–503 (2021). Woronin bodies localize at septa and plug damaged hyphae independently of hitchhiking Each experiment scored 8-64 apical septa (n experiments, mean ± SD: one side wild type = 7, 0.55 ± 0.17; one side hexAΔ = 8, 0.16 ± 0.12; one side pxdAΔ = 6, 0.50 ± 0.15; one side hookAΔ = 4, 0.47 ± 0.13; both sides wild type = 7, 0.32 ± 0.12; both sides hexAΔ = 8, 0.012 ± 0.027; both sides pxdAΔ = 6, 0.18 ± 0.11; both sides hookAΔ = 4, 0.23 ± 0.092). (E) Hyphae expressing mCherry-SsoA as a cell membrane marker, when grown in 1% glucose agar versus 2% sorbose/0.1% glucose agar (sum-Z projections). Scale bar = 10 µm. The white circles indicate leaked cytoplasm due to hyphal tip bursting, and the red circle is an example of a background fluorescence measurements. (F) Leaked cytoplasm, measured as the integrated density – (background area) to correct for background intensity, and then normalized to the average wild type (n burst cells, mean ± SD: wild type = 73, 1.00 ± 0.64; hexAΔ = 63, 1.61 ± 0.81; pxdAΔ = 125, 1.12 ± 0.67; hookAΔ = 119, 1.25 ± 0.65). (G) Model of peroxisome (P) and Woronin body (WB) hitchhiking on early endosomes (EE). Dynein refers to dynein and dynactin complexes. For D and F: Error bars represent SEM. Ordinary one-way ANOVA with post-hoc Tukey-Kramer test; P > 0.05 (labeled ns for “not significant”), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (*) 6 Songster et al., 2023 (preprint) ( ). Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Fungal growth conditions 36. Lee, S. B. & Taylor, J. W. 34 - Isolation of DNA from fungal mycelia and single spores. in PCR Protocols 282–287 (Academic Press, 1990). doi:10.1016/B978-0-12-372180-8.50038-X. A. nidulans strains were grown in either liquid or solid agar containing yeast-glucose (YG) complete media or minimal media (MM)34. YG and MM agar plates contained 10 g/L agar-agar/gum agar (USB 10654). YG com- plete media contained Bacto Yeast Extract (5 g/L), 2% final D+ glucose (20 g/L), trace elements solution (2 mL/L), uracil (56 mg/L), uridine (122.1 mg/L), and riboflavin (2.5 mg/L). The trace elements stock solution (500X) contained FeSO4·7H2O (1 g/L), ZnSO4·7H2O (8.8 g/L), CuSO4·5H2O (0.4 g/L), MnSO4·H2O (0.15 g/L), Na₂B₄O₇·10H2O (0.1 g/L), and (NH4)6Mo7O24·4H2O (0.05 g/L). Regular MM contained 1% final D+ glucose (10 g/L), MgSO4 (2 mL/L of 26% w/v), trace elements solution (2 mL/L), and stock salt solution (10 mL/L). Sorbose MM, to induce hyphal tip lysis41, contained 2% final L-sorbose (20 g/L), 0.1% final D+ glucose (1 g/L), trace elements solution (2 mL/L), and stock salt solution (10 mL/L). The stock salt solution (100x; pH 6-6.5) contained NaNO3 (60 g/L), KCl (5.2 g/L), and KH2PO4 (15.2 g/L). For strains with auxotrophic alleles, MM was supple- mented as follows: pyrG89 allele: 0.5 mM uracil and 0.5 mM uridine; pabaA1 allele: 1.46 µM p-aminobenzoic acid; riboB2 allele: 6.6 µM ribofla- vin; pyroA4 allele: 3.0 µM pyridoxine. 37. Rabe, B. A. & Cepko, C. A simple enhancement for Gibson isothermal assembly. bioRxiv (2020) doi:10.1101/2020.06.14.150979. 38. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to sev- eral hundred kilobases. Nat Methods 6, 343–345 (2009). 39. Tan, K., Roberts, A. J., Chonofsky, M., Egan, M. J. & Reck-Peterson, S. L. A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity. Molecular Biology of the Cell 25, 669–678 (2014). 40. Siemering, K. R., Golbik, R., Sever, R. & Haseloff, J. Mutations that sup- press the thermosensitivity of green fluorescent protein. Curr Biol 6, 1653–1663 (1996). 41. Soundararajan, S. et al. Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitro- gen starvation stress. Plant Cell 16, 1564–1574 (2004). 42. Nguyen, T. A. et al. Innovation and constraint leading to complex mul- ticellularity in the Ascomycota. Nat Commun 8, 14444 (2017). To grow adult hyphae for imaging, spores were inoculated into MM agar with supplements and incubated for 16-20hr at 37°C. Concluding thoughts Regardless, we ultimately determined that Woronin bodies do not require PxdA-mediated hitchhiking to reach nor associate with septa, thus disproving the hypothesis that they are actively transported retrograde towards septa. 21. Jedd, G. Fungal evo-devo: Organelles and multicellular complexity. Trends Cell Biol 21, 12–19 (2011). 22. Tenney, K. et al. hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores. Fungal Genet Biol 31, 205–217 (2000). 23. Liu, F. et al. Making two organelles from one: Woronin body biogen- esis by peroxisomal protein sorting. J Cell Biol 180, 325–339 (2008). 24. Escaño, C. S. et al. Disruption of the Aopex11-1 gene involved in pe- roxisome proliferation leads to impaired Woronin body formation in Aspergillus oryzae. Eukaryot Cell 8, 296–305 (2009). 25. Beck, J. & Ebel, F. Characterization of the major Woronin body protein HexA of the human pathogenic mold Aspergillus fumigatus. Int J Med Microbiol 303, 90–97 (2013). Songster et al., 2023 (preprint) 7 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Phylogenetic analysis of peroxisome hitchhiking proteins y g y p g p Pezizomycotina fungi included in this analysis were previously published to have Woronin bodies and/or have been referenced as representatives of different clades42,43. Species were only included if they had their whole genome sequenced and proteome annotated and/or drafted at the time of this analysis (30 August 2022). The phylogenetic tree for fungi was drawn manually based on the taxonomic rankings of each species on NCBI. 46. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocaliza- tion analysis in light microscopy. J Microsc 224, 213–232 (2006). 47. Fleiβner, A. & Glass, N. L. SO, a protein involved in hyphal fusion in Neurospora crassa, localizes to septal plugs. Eukaryot Cell 6, 84–94 (2007). Homologous genes for PxdA (AN1156) and DipA (AN10946) were identified using BLASTp available at NCBI (https://blast.ncbi.nlm.nih.gov/) (parameters: E = 1.0x10-5, word size of 6, BLOSUM62 matrix, gap costs existence 11 and extension 1, filtering low complexity regions, minimum percent coverage 20%). Reciprocal BLASTs were used to confirm all candidate homologs. The reference proteins data- base (refseq_proteins) was used to reduce redundancy of search results. For species where the refseq_proteins database was unavailable, the non- redundant protein sequences database (nr) was used instead. The E value, query coverage, and percent identity of the top hits for a reference strain of each species was recorded (Table S1). Author contributions 26. Dimou, S., Kourkoulou, A., Amillis, S., Percudani, R. & Diallinas, G. The peroxisomal SspA protein is redundant for purine utilization but es- sential for peroxisome localization in septal pores in Aspergillus nid- ulans. Fungal Genet Biol 132, 103259 (2019). LS, JRC, and SLR-P devised the experiments. LS and DB performed the ex- periments. JRC and SLR-P supervised the research. LS, JRC, and SLR-P wrote and edited the manuscript. 27. Leonhardt, Y., Beck, J. & Ebel, F. Functional characterization of the Woronin body protein WscA of the pathogenic mold Aspergillus fu- migatus. Int J Med Microbiol 306, 165–173 (2016). Strain construction A. nidulans strains used in this study are listed in Table S2. New strains were generated by transforming PCR-amplified targeting DNA into A. nid- ulans protoplasts34 or via genetic crosses35. The genotypes of novel strains were confirmed by PCR amplification from isolated genomic DNA36 and Sanger sequencing. 29. Egan, M. J., Tan, K. & Reck-Peterson, S. L. Lis1 is an initiation factor for dynein-driven organelle transport. J Cell Biol 197, 971–982 (2012). 30. Zhang, J., Qiu, R., Arst Jr, H. N., Peñalva, M. A. & Xiang, X. HookA is a novel dynein-early endosome linker critical for cargo movement in vivo. J Cell Biol 204, 1009–1026 (2014). Plasmid cloning l d d 31. Kaminskyj, S. G. W. Septum position is marked at the tip of Aspergil- lus nidulans hyphae. Fungal Genet Biol 31, 105–113 (2000). Plasmids and primers generated for this study are listed in Table S3 and Table S4, respectively. All DNA constructs were designed for homologous recombination at the endogenous locus with the endogenous promoter un- less otherwise stated. Novel plasmids were cloned using Gibson isothermal assembly37,38. Briefly, fragments were amplified using PCR (NEB Q5 high- fidelity DNA polymerase, M0491) from other plasmids or A. nidulans ge- nomic DNA. Then, fragments were assembled into the Blue Heron Biotech- nology pUC vector at the 5′EcoRI and 3′HindIII restriction sites. The clon- ing of mTagGFP2-rabA::AfpyrG, mCherry-FLAG-PTS1::AfpyroA, and 2xBFP- PTS1::AfriboB were described previously39. The cloning of pxdA- mKate2::AfpyroA and pxdAΔ::AfriboB were also previously published7,12. All fluorescently tagged proteins had a GA5 linker. The sequence for mGFP5 was optimized for expression in A. thalania plants40. 32. Lin, C. et al. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells. Nat Commun 7, 11814 (2016). 33. Steinberg, G., Schuster, M., Hacker, C., Kilaru, S. & Correia, A. ATP pre- vents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici. Cell Microbiol 19, e12764 (2017). 34. Szewczyk, E. et al. Fusion pcr and gene targeting in aspergillus nidu- lans. Nature Protocols 1, 3111–3120 (2006). 35. Todd, R. B., Davis, M. A. & Hynes, M. J. Genetic manipulation of Asper- gillus nidulans: Meiotic progeny for genetic analysis and strain con- struction. Nature Protocols 2, 811–821 (2007). Authors declare that they have no competing interests. 28. Ng, S. K., Liu, F., Lai, J., Low, W. & Jedd, G. A tether for Woronin body inheritance is associated with evolutionary variation in organelle po- sitioning. PLoS Genet 5, e1000521 (2009). oRxiv-word-template Competing interest statement p g Authors declare that they have no competing interests. Fungal growth conditions Individual colo- nies were cut from the agar and inverted onto 35 mm #1.5 glass-bottom imaging dishes (Cellvis D35C4201.5N). To grow germlings for imaging, spores were collected into 0.01% Tween-80 (Sigma-Aldrich P1754) and their density was measured using a hemocytometer. The spores were then inoculated into liquid MM with supplements at a density of 5x105 spores/mL in a four-chamber 35 mm dish and incubated for 16-20 hr at 30°C in an unsealed humid plastic container. 43. Prostak, S. M., Robinson, K. A., Titus, M. A. & Fritz-Laylin, L. K. The actin networks of chytrid fungi reveal evolutionary loss of cytoskele- tal complexity in the fungal kingdom. Curr Biol 31, 1192–1205 (2021). 44. R Core Team. R: A language and environment for statistical compu- ting. (2021). 45. Aaron, J. S., Taylor, A. B. & Chew, T.-L. Image co-localization – co-oc- currence versus correlation. J Cell Sci 131, jcs211847 (2018). Phylogenetic analysis of peroxisome hitchhiking proteins Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally b Mander’s coefficient of co-occurrence algorithms via the JACoP plugin in ImageJ45,46. To identify PxdA homologs, three query sequences were used: the PxdA coiled-coil region, previously defined as CC1-3 (aa 1466-2115)7; the PxdA uncharacterized N-terminus (aa 1-1465); and the full-length PxdA protein (aa 1-2236). The conserved stretch of amino acids in the PxdA CC3 domain are aa 2033-2058. The coiled-coil region of all PxdA homologs was individually predicted by querying each sequence in the Waggawagga coiled-coil prediction software (https://waggawagga.motorprotein.de/) with the following settings: Marcoil, Scorer 2.0 oligomerization, and win- dow length of 21. PxdA possesses a predicted coiled-coil region that is nec- essary and sufficient for peroxisome hitchhiking7. The coiled-coil predic- tion software Waggawagga estimated between three to six distinct coiled- coils in each PxdA homolog, with the total coiled-coil region varying from 167 to 307 aa long (Table S1). All PxdA homologs possessed an N-terminal extension of variable length with no discernable conserved domains, based on NCBI and PFAM databases. The fraction of SspA puncta with PTS1 and the fraction of SspA puncta with PTS1 was manually scored for four separate experiments containing at least ten germlings each. Germlings were imaged for 60 sec (300 ms in- terval) with a single z-plane using simultaneous dual-color settings. Puncta in the cytoplasm or at septa were manually segmented as an ROI and scored as colocalized if there was a punctum from both channels that over- lapped by >50% and if the mean gray value of the puncta of the opposite channel was at least 10% brighter than the background. Motile puncta were assessed by first generating kymographs, and for directed runs where a punctum traveled >2 µm, the co-occurrence of puncta in both channels was manually scored. To measure the fluorescence intensity ratio of different proteins, puncta from sum Z projections of germlings were manually circled as re- gions of interest (ROIs). The gray value of all puncta was measured in both the 488 and 561 channel and the intensity ratio for each ROI was calculated as the mean gray value of the 488 channels divided by the mean gray value of the 561 channels. For identifying DipA homologs, query sequences included both the full-length DipA protein (aa 1-704) and the DipA metallophosphatase do- main alone (aa 45-96). Spinning disk confocal microscopy Live cell imaging was performed at room temperature using a Nikon Ti2 microscope mounted with a Yokogawa W1 spinning-disk confocal scan head and two Prime95B cameras (Photometrics). All images were acquired in 16-bit format. The NIS Elements Advanced Research software (Nikon) was used to run the microscope using the 488 nm and 561 nm lasers of a six-line LUN-F-XL laser engine (405 nm, 445 nm, 488 nm, 515 nm, 561 nm, and 640 nm). The stage xy position was controlled by a ProScan linear mo- tor stage controller (Prior) and its z position was controlled by a piezo Nano-Z stage positioner (Mad City Labs). An Apo TIRF 100x 1.49 NA objec- tive (Nikon) was used for all experiments except for the imaging of apical septa for hyphae grown in glucose and sorbose agar, which required an S Fluor 40x 1.30 NA objective (Nikon) to capture a larger field of view. Hyphal tip line scans Whole adult hyphae were imaged live as a Z-stack (0.2 µm step size, 8-10 µm total depth). Max-Z intensity projections of fluorescence micrographs were obtained and the brightfield channel was used to trace from the hy- phal tip inwards using the segmented line tool (pixel width 20). The traces were superimposed on the fluorescence micrographs to project the aver- age fluorescence intensity along the line. The mean background intensity was measured for each cell and subtracted from that cell’s respective line scan prior to binning. Cytoplasmic leakage C l l k Cytoplasmic leakage was measured for burst hyphal tips in sorbose agar. First, complete Z stacks (0.2 µm step size, 4 µm total depth) were imaged, sum-Z projected, and blinded. The integrated density (mean intensity of puddle times puddle area) for each cytoplasmic puddle was measured in ImageJ and corrected by subtracting the product of the mean background intensity and puddle area. This background-corrected integrated density was then normalized to the mean observation for wild type to calculate the normalized amount of leaked cytoplasm. Organelle flux Total puncta movement was estimated from single-plane, triggered-acqui- sition movies (30 sec total length, 300 ms interval, 100 ms exposure per laser). The number of puncta crossing a perpendicular line approximately 10 μm from the hyphal tip was manually counted. Quantitative image analysis All image analysis was performed using ImageJ/FIJI (National Institutes of Health, Bethesda, MD). All microscopy images were blinded prior to anal- ysis using a custom script in R44. Statistical tests were performed using GraphPad Prism (Windows version 9.4.1). Code availability: All macros (ImageJ, Windows version 1.53q) and data processing scripts (R, Windows version 4.1.0; Rstudio, Windows version 2022.12.0 Build 353) are availa- ble on GitHub (https://github.com/LiviaSongster). Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans DipA homologs were defined to have both a metal- lophosphatase domain followed by a conserved domain of unknown func- tion (DUF2433). The amino acid boundaries of both the metallophospha- tase domains and the DUF2433 were identified for all DipA homologs using NCBI domain prediction and MUSCLE multiple protein sequence align- ments to the A. nidulans DipA reference sequence in SnapGene. Intensity profile during movement The intensity along a line (1 pixel width) was measured during a directed run for both the 488 and 561 channel. The line scans from multiple kymo- graphs were compiled in R. For each line scan, the maximum intensity in the 561 channel was set as x position 0 μm and then applied to the 488 channel. Woronin body localization at tip apical septa Cells expressing mCherry-SsoA and SspA-mGFP5 were grown overnight with supplements in either regular MM (1% glucose) or sorbose MM (2% sorbose/0.1% glucose). Adult hyphae were imaged using the 40x objective to collect complete volume Z-stacks (0.2 µm step size, 8-10 µm total depth). Images were max-Z projected and the apical septa were manually identi- fied as ROIs and cropped. Crops of single septa were blinded and Woronin body localization was manually scored as the fraction of apical septa with an associated Woronin body per experiment. The mean fluorescence inten- sity of SspA-mGFP5 from these max-Z projections was measured within a 0.5 µm-wide by 1 µm-long rectangle centered at an apical septum. The mean background intensity adjacent to each septum was measured and subtracted from the mean septal intensity. Background-corrected meas- urements were then normalized to the average septal intensity of wild type. Triggered acquisition of 488/561 was used when imaging Woronin body association at apical septa with the 40x objective and when imaging SspA-mGFP5 comigration with PxdA-mKate with the 100x objective. The firing of the 488 nm and 561 nm lasers was synchronized by the Prime95B camera trigger signal, which was integrated into a Nikon BB connected to a 6723 DAQ board housed in an external Pxi chassis (National Instru- ments). A quad bandpass filter (Chroma ZET405/488/561/640mv2) was placed in the emission path of the W1 scan head. Speed and distance travelled of migrating puncta Adult hyphae were imaged for 60 sec (300 ms interval) with a single z- plane using simultaneous dual-color settings. The line tool was used to trace puncta trajectories over time and movies were resliced along these lines to generate kymographs. The instantaneous speed and run lengths of individual moving puncta were calculated from the inverse of the slopes of manual kymograph traces. A punctum was scored as moving if it exhibited a directed run (no pauses) > 3 μm during its movement. Simultaneous dual-color imaging was used to assess colocalization of Woronin body and peroxisome proteins in germlings. Z-stacks were col- lected to capture the complete volume of germling cells in the field of view (0.2 µm step size, 8-10 µm total depth). The 488 nm and 561 nm lines were used to excite mGFP5- and mCherry-tagged fusion proteins, respectively. The emission was split with a Cairn TwinCam with a 580LP filter. The GFP/488 emission was reflected and passed through a 514/30 bandpass filter onto camera two. The mCherry/561 fluorescence was passed through a 617/73 and an additional 600/50 bandpass filter to camera one. The cameras were aligned manually in NIS Elements using 0.1 µm Tetra- Speck beads (Thermofisher T14792). Image acquisition settings, such as exposure time and laser power, were determined at the beginning of each imaging session to eliminate fluorescent bleed through between each channel. Acknowledgements LDS is funded by an NSF-GRFP and the UCSD Quantitative Integrative Biol- ogy T32 from the NIH (1T32GM127235). JRC is funded by a MOSAIC award (K99/R00) from the NIH (K99GM140269). SLRP is supported by HHMI and NIH (1R35GM141825). We thank John Salogiannis and Valentin Wernet for ideas and insight, Kaeling Tan for cloning RPB564, James Holcomb for clon- ing RPB2190, and Xin Xiang for the mGFP5 plasmid. This paper was typeset with the bioRxiv word template by @Chrelli: www.github.com/chrelli/bi- Songster et al., 2023 (preprint) 8 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Songster et al., 2023 (preprint) Colocalization and co-occurrence To prepare Z-stacks for colocalization analysis, whole germlings were cropped, max-Z projected, and saved as separate files. For both the 488 and 561 channel, a Gaussian Blur (sigma = 1) and background subtraction (roll- ing = 50) were applied prior to thresholding (Yen dark) to generate a bi- nary puncta mask. The threshold for the green channel was set at 15 to 65535, and for the red channel at 20 to 65535. All gray values outside of that channel’s mask were cleared to eliminate background. Colocalization was then quantified using Pearson’s coefficient of correlation and As a complimentary approach, mycelia were also subjected to hypo- tonic shock to induce global tip lysis, using an adapted protocol for A. nid- ulans47. Spores from strains expressing a cytoplasmic GFP were inoculated Songster et al., 2023 (preprint) 9 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans quid YG media and grown overnight at 37°C Mycelia was collected in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans into liquid YG media and grown overnight at 37°C. Mycelia was collected in Miracloth (EMD Millipore 475855) and dried with paper towels. Approxi- mately 300 ± 10 mg dried mycelia were submerged in 1 mL sterile ddH2O and vortexed to induce hypotonic damage at hyphal tips. Samples were spun at 15,000 x g for 1 min in a tabletop centrifuge to pellet cell debris and the supernatant was diluted 1:10 in sterile ddH2O for a 96-well micro- plate Bradford microassay (Bio-Rad 5000201) to estimate the amount of leaked protein. The absorbance at 595 nm was measured using a Cytation 5 plate reader (BioTek) and Gen5 data analysis software (Windows version 3.05). Colocalization and co-occurrence For B, D, H, and J: unpaired t-test. Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.00 (), P ≤ 0.0001 (**). . CC BY 4.0 International license perpetuity. It is made available under a Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans peroxisome colocalization and co-occurrence in whole germlings. (A, C, E, I) Example max-Z projections of Figure S1. Woronin body and peroxisome colocalization and co-occurrence in whole germlings. (A, C, E, I g y p g g ( , , , ) p p j colocalization between GFP and mCherry-tagged markers for Woronin bodies and peroxisomes in wild type (A, C, E) or pxdAΔ (I) germlings. White arrows indicate the septum closest to the spore head. Scale bars = 5 µm. (B, D, F) Average Mander’s coefficient for each pair of markers in wild type germlings. B) SspA-mGFP5 and mCherry-HexA puncta in wild type (n = 19 germlings; mean ± SD: SspA w/ HexA = 0.92 ± 0.13; HexA w/ SspA = 0.44 ± 0.29). D) mGFP5-HexA and mCherry-PTS1 puncta in wild type (n = 31 germlings; mean ± SD: HexA w/ PTS1 = 0.60 ± 0.13; PTS1 w/ HexA = 0.69 ± 0.16) F) SspA-mGFP5 and mCherry-PTS1 puncta in wild type (n = 27 germlings; mean ± SD: SspA w/ PTS1 = 0.84 ± 0.085; PTS1 w/ SspA = 0.55 ± 0.16). (G) Average intensity ratio of SspA-mGFP5 and mCherry-HexA in puncta at septa and in the cytoplasm (n puncta, mean ± SD: septal = 24, 0.44 ± 0.45; cytoplasmic = 33, 0.29 ± 0.19). (H) Average intensity ratio of mGFP5-HexA and mCherry-PTS1 in puncta at septa and in the cytoplasm (n, mean ± SD: septal = 36, 2.59 ± 0.89; cytoplasmic = 47, 1.1 ± 1.1). (J) SspA-mGFP5 and mCherry-PTS1 puncta in pxdAΔ germlings (n = 43 germlings; mean ± SD: SspA w/ PTS1 = 0.73 ± 0.21; PTS1 w/ SspA = 0.45 ± 0.18). Colocalization and co-occurrence Songster et al., 2023 (preprint) 10 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Figure S1. Woronin body and peroxisome colocalization and co-occurrence in whole germlings. (A, C, E, I) Example max-Z projections colocalization between GFP and mCherry-tagged markers for Woronin bodies and peroxisomes in wild type (A, C, E) or pxdAΔ (I) germlings. Whi arrows indicate the septum closest to the spore head. Scale bars = 5 µm. (B, D, F) Average Mander’s coefficient for each pair of markers in wild typ germlings. B) SspA-mGFP5 and mCherry-HexA puncta in wild type (n = 19 germlings; mean ± SD: SspA w/ HexA = 0.92 ± 0.13; HexA w/ SspA 0.44 ± 0.29). D) mGFP5-HexA and mCherry-PTS1 puncta in wild type (n = 31 germlings; mean ± SD: HexA w/ PTS1 = 0.60 ± 0.13; PTS1 w/ Hex = 0.69 ± 0.16) F) SspA-mGFP5 and mCherry-PTS1 puncta in wild type (n = 27 germlings; mean ± SD: SspA w/ PTS1 = 0.84 ± 0.085; PTS1 w/ Ssp = 0.55 ± 0.16). (G) Average intensity ratio of SspA-mGFP5 and mCherry-HexA in puncta at septa and in the cytoplasm (n puncta, mean ± SD: sept = 24, 0.44 ± 0.45; cytoplasmic = 33, 0.29 ± 0.19). (H) Average intensity ratio of mGFP5-HexA and mCherry-PTS1 in puncta at septa and in th cytoplasm (n, mean ± SD: septal = 36, 2.59 ± 0.89; cytoplasmic = 47, 1.1 ± 1.1). (J) SspA-mGFP5 and mCherry-PTS1 puncta in pxdAΔ germlings = 43 germlings; mean ± SD: SspA w/ PTS1 = 0.73 ± 0.21; PTS1 w/ SspA = 0.45 ± 0.18). (K-L) Histograms of the relative frequency of all movemen (retrograde and anterograde) for SspA-mGFP5 (K) and mCherry-PTS1 (L) in adult hyphae (n hyphal tips: wild type = 32; pxdAΔ = 64). Colocalization and co-occurrence (K-L) Histograms of the relative frequency of all movements (retrograde and anterograde) for SspA-mGFP5 (K) and mCherry-PTS1 (L) in adult hyphae (n hyphal tips: wild type = 32; pxdAΔ = 64). For B, D, F- H, and J: unpaired t-test. Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (**). Songster et al., 2023 (preprint) 11 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Figure S2. Peroxisome distribution and movement in cells lacking complete Woronin bodies. (A) Diagram of Woronin bodies lacking SspA or HexA. (B) Cumulative frequency plot of the number of moving mCherry-PTS1 puncta per 30 s in adult hyphal tips (n, mean ± SD puncta/30 s: wild type = 53 hyphae, 1.09 ± 1.5; pxdAΔ = 48 hyphae, 0.33 ± 0.56; hexAΔ = 132 hyphae, 1.02 ± 1.4; sspAΔ = 110 hyphae, 1.60 ± 1.86). The results of a Kruskal-Wallis with post-hoc Dunn’s multiple comparisons test versus wild type are shown; P > 0.05 labeled ns for “not significant”; P ≤ 0.05 (). (C) Examples of mCherry-PTS1 in adult hyphal tips (max-Z projections). Scale bar = 10 µm. (D) Mean intensity of mCherry-PTS1 from the hyphal tip inwards (n: wild type = 33 hyphae; pxdAΔ = 30 hyphae; hexAΔ = 53 hyphae; sspAΔ = 54 hyphae). Error bars represent SEM. Figure S2. Peroxisome distribution and movement in cells lacking complete Woronin bodies. (A) Dia Figure S2. Peroxisome distribution and movement in cells lacking complete Woronin bodies. (A) Diagram of Woronin bodies lacking SspA or HexA. (B) Cumulative frequency plot of the number of moving mCherry-PTS1 puncta per 30 s in adult hyphal tips (n, mean ± SD puncta/30 s: wild type = 53 hyphae, 1.09 ± 1.5; pxdAΔ = 48 hyphae, 0.33 ± 0.56; hexAΔ = 132 hyphae, 1.02 ± 1.4; sspAΔ = 110 hyphae, 1.60 ± 1.86). Songster et al., 2023 (preprint) Colocalization and co-occurrence ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Figure S3. Woronin bodies at tip apical septa and cytoplasmic leakage assays. (A) Examples of Woronin body localization at septa. SspA- mGFP5 puncta were scored on max-Z projections as either present on both sides, one side, or none. Scale bar = 2 µm. (B) Line scans showing fluorescence intensity of Woronin bodies (SspA) or cell membrane at septa (SsoA) for each score category from A. Arrows indicate peak intensities for each fluorescent protein. (C) Normalized mean intensity of SspA-mGFP5 on either side (±0.5 µm) of apical septa for cells grown in glucose MM agar (n septa, mean ± SD AU: wild type = 43, 1.00 ± 1.24; hexAΔ = 45, 0.12 ± 0.6; pxdAΔ = 68, 1.93 ± 2.08; hookAΔ = 50, 1.80 ± 1.98). (D) Fraction of burst or damaged hyphal tips when grown in glucose or sorbose agar. Each circle represents the fraction burst hyphae from all tips imaged in a single experiment with 8-50 hyphae each (Glucose n experiments, mean ± SD: wild type = 4, 0.02 ± 0.03; hexAΔ = 4, 0 ± 0; pxdAΔ = 4, 0.01 ± 0.02; hookAΔ = 4, 0 ± 0. Sorbose n, mean ± SD: wild type = 4, 0.38 ± 0.07; hexAΔ = 4, 0.39 ± 0.17; pxdAΔ = 4, 0.46 ± 0.04; hookAΔ = 4, 0.49 ± 0.08. (E) Leakage area of cytoplasmic puddles for hyphae grown in sorbose (n burst hyphae, mean ± SD: wild type = 73, 43.7 ± 22.7 µm2; hexAΔ = 63, 62.7 ± 26.8 µm2; pxdAΔ = 125, 55.6 ± 28.9 µm2; hookAΔ = 119, 62.5 ± 32.5 µm2). (F) Quantification of cytoplasmic leakage after hypotonic shock. The amount of leaked protein in the supernatant was quantified via Bradford assay (n samples, mean ± SD: water = 5, -0.45 ± 1.3 µg/mL; wild type = 12, 62 ± 18 µg/mL; hexAΔ = 10, 189 ± 39 µg/mL; sspAΔ = 11, 66 ± 35 µg/mL; pxdAΔ = 10, 58 ± 28 µg/mL; hookAΔ = 10, 52 ± 28 µg/mL). For C: Kruskal- Wallis test with Dunn’s multiple comparisons for nonparametric data. Colocalization and co-occurrence For D-F: Ordinary one-way ANOVA with post-hoc Tukey-Kramer test. For C- F: Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (*). Figure S3. Woronin bodies at tip apical septa and cytoplasmic leakage assays. (A) Examples of Woroni Figure S3. Woronin bodies at tip apical septa and cytoplasmic leakage assays. (A) Examples of Woronin body localization at septa. SspA- mGFP5 puncta were scored on max-Z projections as either present on both sides, one side, or none. Scale bar = 2 µm. (B) Line scans showing fluorescence intensity of Woronin bodies (SspA) or cell membrane at septa (SsoA) for each score category from A. Arrows indicate peak intensities for each fluorescent protein. (C) Normalized mean intensity of SspA-mGFP5 on either side (±0.5 µm) of apical septa for cells grown in glucose MM agar (n septa, mean ± SD AU: wild type = 43, 1.00 ± 1.24; hexAΔ = 45, 0.12 ± 0.6; pxdAΔ = 68, 1.93 ± 2.08; hookAΔ = 50, 1.80 ± 1.98). (D) Fraction of burst or damaged hyphal tips when grown in glucose or sorbose agar. Each circle represents the fraction burst hyphae from all tips imaged in a single experiment with 8-50 hyphae each (Glucose n experiments, mean ± SD: wild type = 4, 0.02 ± 0.03; hexAΔ = 4, 0 ± 0; pxdAΔ = 4, 0.01 ± 0.02; hookAΔ = 4, 0 ± 0. Sorbose n, mean ± SD: wild type = 4, 0.38 ± 0.07; hexAΔ = 4, 0.39 ± 0.17; pxdAΔ = 4, 0.46 ± 0.04; hookAΔ = 4, 0.49 ± 0.08. (E) Leakage area of cytoplasmic puddles for hyphae grown in sorbose (n burst hyphae, mean ± SD: wild type = 73, 43.7 ± 22.7 µm2; hexAΔ = 63, 62.7 ± 26.8 µm2; pxdAΔ = 125, 55.6 ± 28.9 µm2; hookAΔ = 119, 62.5 ± 32.5 µm2). (F) Quantification of cytoplasmic leakage after hypotonic shock. The amount of leaked protein in the supernatant was quantified via Bradford assay (n samples, mean ± SD: water = 5, -0.45 ± 1.3 µg/mL; wild type = 12, 62 ± 18 µg/mL; hexAΔ = 10, 189 ± 39 µg/mL; sspAΔ = 11, 66 ± 35 µg/mL; pxdAΔ = 10, 58 ± 28 µg/mL; hookAΔ = 10, 52 ± 28 µg/mL). Songster et al., 2023 (preprint) Colocalization and co-occurrence The results of a Kruskal-Wallis with post-hoc Dunn’s multiple comparisons test versus wild type are shown; P > 0.05 labeled ns for “not significant”; P ≤ 0.05 (). (C) Examples of mCherry-PTS1 in adult hyphal tips (max-Z projections). Scale bar = 10 µm. (D) Mean intensity of mCherry-PTS1 from the hyphal tip inwards (n: wild type = 33 hyphae; pxdAΔ = 30 hyphae; hexAΔ = 53 hyphae; sspAΔ = 54 hyphae). Error bars represent SEM. Figure S2. Peroxisome distribution and movement in cells lacking complete Woronin bodies. (A) Diagram of Woronin bodies lacking SspA or HexA. (B) Cumulative frequency plot of the number of moving mCherry-PTS1 puncta per 30 s in adult hyphal tips (n, mean ± SD puncta/30 s: wild type = 53 hyphae, 1.09 ± 1.5; pxdAΔ = 48 hyphae, 0.33 ± 0.56; hexAΔ = 132 hyphae, 1.02 ± 1.4; sspAΔ = 110 hyphae, 1.60 ± 1.86). The results of a Kruskal-Wallis with post-hoc Dunn’s multiple comparisons test versus wild type are shown; P > 0.05 labeled ns for “not significant”; P ≤ 0.05 (). (C) Examples of mCherry-PTS1 in adult hyphal tips (max-Z projections). Scale bar = 10 µm. (D) Mean intensity of mCherry-PTS1 from the hyphal tip inwards (n: wild type = 33 hyphae; pxdAΔ = 30 hyphae; hexAΔ = 53 hyphae; sspAΔ = 54 hyphae). Error bars represent SEM. 12 Songster et al., 2023 (preprint) 12 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. Songster et al., 2023 (preprint) Colocalization and co-occurrence For C: Kruskal- Wallis test with Dunn’s multiple comparisons for nonparametric data. For D-F: Ordinary one-way ANOVA with post-hoc Tukey-Kramer test. For C- F: Error bars represent SEM. P > 0.05 (labeled ns for “not significant,” or not shown), P ≤ 0.05 (), P ≤ 0.01 (), P ≤ 0.001 (), P ≤ 0.0001 (***). Songster et al., 2023 (preprint) 13 Songster et al., 2023 (preprint) . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Movie S1 (separate file). Woronin body (SspA-mGFP5) and peroxisome (mCherry-PTS1) comigration in a wild type hyphal tip. A single Z plane image was taken every 300 ms via simultaneous dual-color spinning disk confocal microscopy (Nikon). The white arrow indicates a comigrating, bidirectional run. Scale bar = 10 µm. Movie S2 (separate file). Woronin bodies (SspA-mGFP5) and peroxisomes (mCherry-PTS1) in a pxdAΔ hyphal tip. A taken every 300 ms via simultaneous dual-color spinning disk confocal microscopy (Nikon). Scale bar = 10 µm. Table S1 (separate file). PxdA (sheet 1) and DipA (sheet 2) BLASTp results for all 33 fungal species tested. The amino acid sequences used for each BLASTP search are shown in row 1 on both sheets. 14 Songster et al., 2023 (preprint) 14 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. Table S3. Plasmids generated for this study. Songster et al., 2023 (preprint) Colocalization and co-occurrence ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Strain Alias Genotype Source RPA1256 RPA1257 SspA-GFP, mCherry- HexA [mCherry-HexA::Afpyro], pyroA4, [SspA-mGFP5::AfpyrG], pyrG89, nkuA::Bar This study RPA1252 RPA1254 SspA-GFP, mCherry- PTS1 yA::[gpdA(p)::mCherry-FLAG-PTS1::Afpyro], pyroA4, [SspA-mGFP5::AfpyrG], pyrG89, nkuA::bar This study RPA1247 RPA1248 GFP-HexA, mCherry- PTS1 yA::[gpdA(p)::mCherry-FLAG-PTS1::Afpyro], pyroA4, [mGFP5-HexA::AfpyrG], pyrG89, nkuA::bar This study RPA1249 RPA1250 SspA-GFP, mCherry- PTS1, pxdAΔ yA::[gpdA(p)::mCherry-FLAG-PTS1::Afpyro], pyroA4, [SspA-mGFP5::AfpyrG], pyrG89, [pxdAΔ::Afribo], riboB2, pabaA1, nkuA::argB+ This study RPA1273 SspA-GFP, BFP- PTS1, PxdA-mKate wA::[gpdA(p)::2xtagBFP-PTS1::AfriboB], riboB2, [PxdA-mKate::Afpyro], pyroA4, [SspA-mGFP5::AfpyrG], pyrG89, nkuA:: argB+ This study RPA1326 SspA-GFP, mCherry- SsoA yA1, [SspA-mGFP5::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, nkuA::argB+ This study RPA1327 SspA-GFP, mCherry- SsoA [SspA-mGFP5::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, nkuA::argB+ This study RPA1320 SspA-GFP, mCherry- SsoA, pxdAΔ yA1, [SspA-mGFP5::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, [pxdAΔ::AfriboB], riboB2, pabaA1, nkuA::argB+ This study RPA1532 SspA-GFP, mCherry- SsoA, hookAΔ yA1, [SspA-mGFP5::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, [hookAΔ::AfriboB], riboB2, pabaA1, nkuA::argB+ This study RPA1321 RPA1322 SspA-GFP, mCherry- SsoA, hexAΔ yA1, [SspA -mGFP5::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, [hexAΔ::AfriboB], riboB2, pabaA1, nkuA::argB+ This study RPA1328 RPA1329 GFP, mCherry-SsoA yA1, wA::[mTagGFP2::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, nkuA::argB+ This study RPA1315 RPA1316 GFP, mCherry-SsoA, pxdAΔ yA1, wA::[mTagGFP2::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, [pxdAΔ::AfriboB], ri- boB2, nkuA::argB+ This study RPA1312 RPA1323 GFP, mCherry-SsoA, hookAΔ yA1, wA::[mTagGFP2::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, [hookAΔ::AfriboB], riboB2, nkuA::argB+ This study RPA1313 RPA1314 GFP, mCherry-SsoA, hexAΔ yA1, wA::[mTagGFP2::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, [hexAΔ::AfriboB], ri- boB2, nkuA::argB+ This study RPA1317 RPA1318 GFP, mCherry-SsoA, sspAΔ yA1, wA::[mTagGFP2::AfpyrG], pyrG89, [mCherry-SsoA::Afpyro], pyroA4, pabaA1, [sspAΔ::AfriboB], ri- boB2, nkuA::argB+ This study RPA528 GFP-RabA, mCherry- PTS1 yA::[gpdA(p)-mCherry-FLAG-PTS1::Afpyro], pyroA4, [mTagGFP2-RabA::AfpyrG], pyrG89, riboB2, pabaA1, nkuA::argB+ Tan et al., 201439 RPA861 GFP-RabA, mCherry- PTS1, pxdAΔ yA::[gpdA(p)-mCherry-FLAG-PTS1::Afpyro], pyroA4, [mTagGFP2-RabA::AfpyrG], pyrG89, [pxdAΔ::Afri- boB], riboB2, pabaA1, nkuA::argB+ Salogiannis et al., 201639 RPA1330 RPA1344 GFP-RabA, mCherry- PTS1, hexAΔ yA::[gpdA(p)-mCherry-FLAG-PTS1::Afpyro], pyroA4, [mTagGFP2-RabA::AfpyrG], pyrG89, [hexAΔ::Afri- boB], riboB2, pabaA1, nkuA::argB+ This study RPA1345 RPA1346 GFP-RabA, mCherry- PTS1, sspAΔ yA::[gpdA(p)-mCherry-FLAG-PTS1::Afpyro], pyroA4, [mTagGFP2-RabA::AfpyrG], pyrG89, [sspAΔ::Afri- boB], riboB2, pabaA1, nkuA::argB+ This study 15 Songster et al., 2023 (preprint) 15 Songster et al., 2023 (preprint) . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans . CC-BY 4.0 International license perpetuity. Colocalization and co-occurrence It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Plasmid ID Plasmid Name Source RPB2188 hookAΔ::AfriboB This study RPB2007 hexAΔ::AfriboB This study RPB2184 sspAΔ::AfriboB This study RPB2042 wA::mTagGFP2::AfpyrG This study RPB2049 SspA-mGFP5::AfpyrG This study RPB2048 mGFP5-HexA::AfpyrG This study RPB2190 mCherry-SsoA::AfpyroA This study RPB564 mCherry-HexA::AfpyroA This study Songster et al., 2023 (preprint) 16 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. Songster et al., 2023 (preprint) Colocalization and co-occurrence ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Primer Sequence (5'-3') Purpose Plasmid LS65 AGCTTGGCGTAATCATGGTCATAGCTG F amplify pBH without overhangs all LS66 CACTGGCCGTCGTTTTACAACGT R amplify pBH without overhangs all JC192 AAAACGACGGCCAGTCATGCTTGCTTCCTCTTGCG F amplify upstream hookA RPB2188 LS119 cgattacatgaaggtccacctgtcatttgtcagCTTGAGCGGGCGCAGGG R amplify upstream hookA RPB2188 LS120 CGCCCCTGCGCCCGCTCAAGctgacaaatgacaggtggaccttcatgtaa F amplify AfriboB RPB2188 LS121 GGGTCATTTGTCAAGATTGAGCATGatgagtgtgacgagcatacaccgtg R amplify AfriboB RPB2188 LS122 cggtgtatgctcgtcacactcatCATGCTCAATCTTGACAAATGACCCCT F amplify downstream hookA RPB2188 JC96 GAAACAGCTATGACCATGATTACGCCAAGCAATAACTGTTGAAGGA- GATCCTGACAGTGG R amplify downstream hookA RPB2188 JC202 CATGCTTGCTTCCTCTTGCGCTCGC F targeting primer RPB2188 JC118 AATAACTGTTGAAGGAGATCCTGAC R targeting primer RPB2188 JC938 GTCACGACGTTGTAAAACGACGGCCAGTGTAGCACTTCGCACGTCGAACG F amplify upstream hexA RPB2007 JC939 cgagccagactcctgaacggcctcttCTTGCGAGCCTTGGGGTGATCTAC R amplify upstream hexA RPB2007 JC940 GTAGTAGATCACCCCAAGGCTCGCAAGaagaggccgttcaggagtctgg F amplify AfriboB RPB2007 JC941 AGGTCGTTTTAGAGAACCCCTGTAGACAGCatgagtgtgacgagcatacaccgtg R amplify AfriboB RPB2007 JC942 gtcgacacggtgtatgctcgtcacactcatGCTGTCTACAGGGGTTCTCTAAAACGACC F amplify downstream hexA RPB2007 JC915 GAAACAGCTATGACCATGATTACGCCAAGCTCACCTGAACGATCCAAC- GGTTTCGACG R amplify downstream hexA RPB2007 JC950 TAGCACTTCGCACGTCGAACGG F targeting primer RPB2007 JC953 CACCTGAACGATCCAACGGTTTCGACG R targeting primer RPB2007 LS11 CGACGTTGTAAAACGACGGCCAGTGtggaaagacgccatatctgggtctg F amplify upstream sspA RPB2184 LS26 AGCCAGACTCCTGAACGGCCTCTTaggaaaagcaatgaacaggaaacgcg R amplify upstream sspA RPB2184 LS27 ttcgcgtttcctgttcattgcttttcctAAGAGGCCGTTCAGGAGTCTGG F amplify AfriboB RPB2184 LS83 ACGGTGTATGCTCGTCACACTCATgaccaaaatggtccgtgctatcccat R amplify AfriboB RPB2184 LS29 TATGCTCGTCACACTCATaggatgtaatttcctcccggacaaatgatgac F amplify downstream sspA RPB2184 LS20 AGGAAACAGCTATGACCATGATTACGCCAAGCTgcggaggcggctcgacg R amplify downstream sspA RPB2184 LS137 tggaaagacgccatatctgggtctgg F targeting primer RPB2184 LS38 gcggaggcggctcgac R targeting primer RPB2184 JC192 AAAACGACGGCCAGTCATGCTTGCTTCCTCTTGCG F amplify upstream hookA RPB2582 LS628 actgtattattatattcagttatataagcgccggCTTGAGCGGGCGCAGG R amplify upstream hookA RPB2582 LS630 ccggcgcttatataactgaatataat F amplify AfpabA RPB2582 LS631 ttggaatgtttgcagcagc R amplify AfpabA RPB2582 LS629 acttgctgctgcaaacattccaaCATGCTCAATCTTGACAAATGACCCCT F amplify downstream hookA RPB2582 JC96 GAAACAGCTATGACCATGATTACGCCAAGCAATAACTGTTGAAGGA- GATCCTGACAGTGG R amplify downstream hookA RPB2582 JC202 CATGCTTGCTTCCTCTTGCGCTCGC F targeting primer RPB2582 JC118 AATAACTGTTGAAGGAGATCCTGAC R targeting primer RPB2582 JC165 AGTCACGACGTTGTAAAACGACGGCCAGTGCTCTGGAACAGTCTCGCCG F amplify upstream wA RPB2042 JC4 TGATTCTATAGGAAGATCCAGGCACCGGTCGATCAGGAGAAGGAGAG- TCAAGTCCGCTGA R amplify upstream wA RPB2042 JC3 TCAGCGGACTTGACTCTCCTTCTCCTGATCGACCGGTGCCTGGATCTTCC- TATAGAATCA F amplify gpdA promoter RPB2042 JC745 CAATTCTTCACCTCCTGACATTGTGATGTCTGCTCAAGCGGGG R amplify gpdA promoter RPB2042 JC743 CGCTTGAGCAGACATCACAATGTCAGGAGGTGAAGAATTGTTCG- CAGGTATTGTTCCTGT F amplify mTagGFP2::AfpyrG::downstream wA RPB2042 wA DS R GAAACAGCTATGACCATGATTACGCCAAGCTACTCG- GAAAGGGTGGTGAATGCGGCGTGC R amplify mTagGFP2::AfpyrG::downstream wA RPB2042 LS45 CTCTGGAACAGTCTCGCCGTCTTG F targeting primer RPB2042 LS46 ACTCGGAAAGGGTGGTGAATGCG R targeting primer RPB2042 LS21 TCACGACGTTGTAAAACGACGGCCAGTGtttccttgagctcaccgcgttc F amplify sspA CDS RPB2049 LS22 TGGCACCGGCTCCAGCGCCTGCACCAGCTCCccggtagtcgggtccaggg R amplify sspA CDS RPB2049 LS23 gagtacgagaagggccctggacccgactaccggGGAGCTGGTGCAGGCGC F amplify mGFP5 RPB2049 LS24 ggaaattacatcctTTATTTGTATAGTTCATCCATGCCATGTGTAATCCC R amplify mGFP5 RPB2049 LS25 GGATGAACTATACAAATAAaggatgtaatttcctcccggacaaatgatga F amplify sspA 3' UTR RPB2049 LS16 GCGAAGAGGGTGAAGAGCATTGtaaagaaataaagatgcaccgagcggcg R amplify sspA 3' UTR RPB2049 LS17 gccgctcggtgcatctttatttctttaCAATGCTCTTCACCCTCTTCGCG F amplify AfpyrG RPB2049 LS18 atgggatagcacggaccattttggtcCTGTCTGAGAGGAGGCACTGATGC R amplify AfpyrG RPB2049 LS19 GCATCAGTGCCTCCTCTCAGACAGgaccaaaatggtccgtgctatcccat F amplify downstream sspA RPB2049 LS20 AGGAAACAGCTATGACCATGATTACGCCAAGCTgcggaggcggctcgacg R amplify downstream sspA RPB2049 LS37 tttccttgagctcaccgcgttct F targeting primer RPB2049 LS38 gcggaggcggctcgac R targeting primer RPB2049 LS30 CGACGTTGTAAAACGACGGCCAGTGggaagatactcgaacctctgcccct F amplify upstream hexA RPB2048 LS31 ACTCCAGTGAAAAGTTCTTCTCCTTTCATcttgcgagccttggggtgatc R amplify upstream hexA RPB2048 LS32 ccccaaggctcgcaagATGAAAGGAGAAGAACTTTTCACTGGAGTTGTCC F amplify mGFP5 RPB2048 LS33 taacccatTCCGGAGCCACTTCCTTTGTATAGTTCATCCATGCCATGTGT R amplify mGFP5 RPB2048 LS34 ATGAACTATACAAAGGAAGTGGCTCCGGAatgggttactacgacgacgac F amplify hexA CDS + 3' UTR RPB2048 JC956 GGTATTTCAGACCCGCGAAGAGGGTGAAGAGCATT- Gacaggccctacatgcagccttatc R amplify hexA CDS + 3' UTR RPB2048 JC958 ttttacttttttgataaggctgcatgtagggcctgtCAATGCTCTTCACCCTCTTCGCGG F amplify AfpyrG RPB2048 JC959 tttcgacccatacggtgccgagcagcgcgacctgcCTGTCTGAGAGGAGGCACTGATGCG R amplify AfpyrG RPB2048 JC960 GATAACAGCTTGGCATCACGCATCAGTGCCTCCTCTCAGA- CAGgcaggtcgcgctgctcg F amplify downstream hexA RPB2048 JC915 GAAACAGCTATGACCATGATTACGCCAAGCTcacctgaacgatccaacggtttcgacg R amplify downstream hexA RPB2048 LS39 ggaagatactcgaacctctgcccct F targeting primer RPB2048 LS40 cacctgaacgatccaacggtttcga R targeting primer RPB2048 LS77 AGTCACGACGTTGTAAAACGACGGCCAGTGcaccatgttgggggtc F amplify upstream ssoA RPB2190 LS107 tcctcgcccttgctcaccatCGTGAAGATATAATGGAATCAATGAATCAA R amplify upstream ssoA RPB2190 LS103 TGATTCATTGATTCCATTATATCTTCACGatggtgagcaagggcgaggag F amplify mCherry RPB2190 LS104 CACCGGCTCCAGCGCCTGCACCAGCTCCcttgtacagctcgtccatgccg R amplify mCherry RPB2190 LS108 gccactccaccggcggcatggacgagctgtacaagGGAGCTGGTGCAGGC F amplify ssoA CDS + 3' UTR RPB2190 LS124 AGCATCTGATGTCCgtaacaaaaagatattaagattattaggtgatgact R amplify ssoA CDS + 3' UTR RPB2190 LS125 tcttaatatctttttgttacGGACATCAGATGCTGGATTACTAAGGTAAT F amplify AfpyroA RPB2190 Songster et al., 2023 (preprint) 17 . Colocalization and co-occurrence CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524968 doi: bioRxiv preprint Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans onin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans Primer Sequence (5'-3') Purpose Plasmid LS126 gattagattatatatacccaGCGAGTGTCTACATAATGAAGGACAAATGC R amplify AfpyroA RPB2190 LS123 TATGTAGACACTCGCtgggtatatataatctaatccacgttaacagctcc F amplify downstream ssoA RPB2190 JC925 AAACAGCTATGACCATGATTACGCCAAGCTaagcgcggcgagagataagg R amplify downstream ssoA RPB2190 JC954 caccatgttgggggtcttggagtatc F targeting primer RPB2190 JC955 aagcgcggcgagagataagg R targeting primer RPB2190 K147 CGAACAGCGAGTTCGCGCG F amplify upstream hexA RPB564 K148 GAGGGAGAAGTCAACCTCGTGGACATCAGATGCTGGATTACTAAGGTAAT R amplify upstream hexA RPB564 S669 GGACATCAGATGCTGGATTAC F amplify AfpyroA RPB564 S670 CTTCATTATGTAGACACTCGC R amplify AfpyroA RPB564 K149 CATTTGTCCTTCATTATGTAGACACTCGCTAGCACTTCGCACGTCGAACG F amplify hexA promoter RPB564 K150 CACCCCAAGGCTCGCAAGATGGTGAGCAAGGGCGAGGAGGATAACA R amplify hexA promoter RPB564 K071 GTGAGCAAGGGCGAGGAGG F amplify mCherry RPB564 K152 GGCATGGACGAGCTGTACAAGGGAGCTGGTGCAGGCGCTG- GAGCCGGTGCC R amplify mCherry RPB564 K154 GGTGCAGGCGCTGGAGCCGGTGCCGGTTACTACGACGACGACGGTAACT F amplify hexA CDS + downstream RPB564 K155 GACACGAAACGAACGCCTCG R amplify hexA CDS + downstream RPB564 K157 CCGTCAACGAAGGGTATGGAAC F targeting primer RPB564 K156 CAGACAAGCGCGCGAGCTCC R targeting primer RPB564 Table S4. Primers used to make plasmids for this study. Songster et al., 2023 (preprint) 18
https://openalex.org/W3037206681	https://europepmc.org/articles/pmc7349252?pdf=render	English	null	Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool	Genes	2,020	cc-by	10,221	Keywords: anisakiasis; Anisakis spp.; proteomics; MALDI–TOF MS; LC–ESI–MS/MS Keywords: anisakiasis; Anisakis spp.; proteomics; MALDI–TOF MS; LC–ESI–MS/MS Keywords: anisakiasis; Anisakis spp.; proteomics; MALDI–TOF MS; LC–ESI–MS/MS genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes Article Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool Valeria Marzano 1 , Stefania Pane 2 , Gianluca Foglietta 2, Stefano Levi Mortera 1, Pamela Vernocchi 1, Andrea Onetti Muda 3 and Lorenza Putignani 4,* Valeria Marzano 1 , Stefania Pane 2 , Gianluca Foglietta 2, Stefano Levi Mortera 1, Pamela Vernocchi 1, Andrea Onetti Muda 3 and Lorenza Putignani 4,* 1 Unit of Human Microbiome, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy; valeria.marzano@opbg.net (V.M.); stefano.levimortera@opbg.net (S.L.M.); pamela.vernocchi@opbg.net (P.V.) 1 Unit of Human Microbiome, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy; valeria.marzano@opbg.net (V.M.); stefano.levimortera@opbg.net (S.L.M.); pamela.vernocchi@opbg.net (P.V.) 1 Unit of Human Microbiome, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy; valeria.marzano@opbg.net (V.M.); stefano.levimortera@opbg.net (S.L.M.); pamela.vernocchi@opbg.net (P.V.) p p g 2 Unit of Parasitology, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Ita stefania.pane@opbg.net (S.P.); gianluca.foglietta@opbg.net (G.F.) p p g g g p g 3 Department of Laboratories, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy; andrea.onettimuda@opbg.net 4 Units of Parasitology and Human Microbiome, Bambino Gesù Children’s Hospital IRCCS, Piazza Sant’Onofrio 4, 00165 Rome, Italy y * Correspondence: lorenza.putignani@opbg.net; Tel.: +39-0668594127 Received: 30 May 2020; Accepted: 22 June 2020; Published: 24 June 2020 Abstract: Anisakiasis is nowadays a well-known infection, mainly caused by the accidental ingestion of Anisakis larvae, following the consumption of raw or undercooked ﬁshes and cephalopods. Due to the similarity of symptoms with those of common gastrointestinal disorders, this infection is often underestimated, and the need for new speciﬁc diagnostic tools is becoming crucial. Given the remarkable impact that MALDI–TOF MS biotyping had in the last decade in clinical routine practice for the recognition of bacterial and fungi strains, a similar scenario could be foreseen for the identiﬁcation of parasites, such as nematodes. In this work, a MALDI–TOF MS proﬁling of Anisakis proteome was pursued with a view to constructing a ﬁrst spectral library for the diagnosis of Anisakis infections. At the same time, a shotgun proteomics approach by LC–ESI–MS/MS was performed on the two main fractions obtained from protein extraction, to evaluate the protein species enriched by the protocol. A set of MALDI–TOF MS signals associated with proteins originating in the ribosomal fraction of the nematode extract was selected as a potential diagnostic tool for the identiﬁcation of Anisakis spp. 1. Introduction Among food biological hazards, parasites are particularly dangerous for human health. Globalization has markedly increased the change in eating habits, including the widespread consumption of raw, marinated, or smoked ﬁsh. Moreover, a quota of food allergies of unknown origin among the general population may be due to sensitization to Anisakis spp. (roundworms), representing a public health issue, whose clinical manifestations are characterized by digestive disorders, asthma, dermatitis, and even anaphylaxis. Nematodes of the Anisakidae family are ﬁsh parasites that can be found all over the world. The larvae live in the gut, visceral peritoneum, and ﬂesh of many marine ﬁsh and cephalopod species and can colonize through diﬀerent trophic bridges, ensuring and widening the parasite life cycle. Genes 2020, 11, 693; doi:10.3390/genes11060693 www.mdpi.com/journal/genes www.mdpi.com/journal/genes Genes 2020, 11, 693 2 of 18 Humans can become accidental hosts of the Anisakis parasite by eating parasitized raw or undercooked ﬁsh containing larvae in stage 3 [1,2]. Within hours of being ingested, Anisakis larvae penetrate the mucosal layers of the gastrointestinal tract, causing direct tissue damage that may lead to the zoonotic disease known as anisakiasis. This acute gastrointestinal form of Anisakis infection is usually transient, with the worm dying within a few weeks. It is manifested by clinical symptoms ranging from nausea to vomiting, diarrhea, mild to severe abdominal pain, and intestinal obstruction [3], mimicking other common gastrointestinal disturbances, such as acute appendicitis, gastric ulcer, or tumors, thus making the diagnosis of anisakiasis extremely diﬃcult. Diagnosis is generally obtained through anamnestic data, endoscopy, radiography, serum-speciﬁc anti-Anisakis IgE determination, or surgery if the worm has embedded. Visualization and morphological identiﬁcation of the larva(e), removed by endoscopy or surgery or expelled by patients’ cough is the conclusive assessment. To date, no unambiguous diagnostic criteria and laboratory algorithm have been established; the lack of highly skilled microscopists in biomedical laboratories and the high number of false positives due to cross-reactivities with numerous panallergens have underlined the need to improve the diagnostic approaches. Indeed, because of technical diﬃculties in their diagnosis, these infections are likely to be under-diagnosed. Nowadays, mass spectrometry (MS) biotyping is a rapid, easy, and validated method for accurate microbial phenotypic identiﬁcation (ID). It takes advantage of the matrix-assisted laser desorption/ionization time-of-ﬂight MS (MALDI–TOF MS) technology. 1. Introduction In particular, three platforms, i.e., MALDI Biotyper (Bruker Daltonics GmbH, Bremen, Germany), VITEK MS (bioMerieux, Marcy-l’Étoile, France), and Andromas MS (Paris, France) are currently approved for routine use and adopted by many clinical microbiological laboratories [4–7], facilitating the clinical identiﬁcation of many pathogenic microorganisms, including bacteria, yeasts, and ﬁlamentous fungi. However, an increase of applications of this technology has been observed in the last 10 years, with encouraging results also in studies regarding parasites [8,9]. A recent application of MALDI–TOF MS for Diroﬁlaria and Ascaris protein-based proﬁling showed a promising scenario towards clinical applications involving nematodes biotyping [10]. The aim of this study is to extend the MALDI–TOF MS Biotyper approach to Anisakis spp. ID and to deﬁne a preliminary assessment of its potential in anisakiasis diagnosis. In parallel, a shotgun identiﬁcation proﬁling of Anisakis proteins by liquid chromatography–electrospray ionization–tandem MS (LC–ESI–MS/MS) was performed on the two main fractions of the extraction protocol, with the aim of detecting a relationship with the speciﬁcally detected MALDI–TOF MS Biotyper signals. 2. Materials and Methods Infection by several live parasite larvae isolated from salmon portions, bought in a local supermarket in Rome in late 2019, was assessed by macroscopic observation. Additionally, microscopic analysis was performed using an Axiovert 25 microscope (Zeiss, Jena, Germany). Specimens were identiﬁed as Anisakis spp. larvae. 2.1. Protein Extraction Five specimens were washed several times in 0.9% NaCl solution, and each larva was stored in a sterile 0.9% NaCl solution in a ratio of 1:3 (parasite weight/volume of the isotonic solution) at −80 ◦C. Mechanical homogenization of the frozen material was carried out with a steel pestle, followed by an ultrasonic treatment of the biomass at 100% power (BactoSonic, BANDELIN electronic GmbH & Co KG, Berlin, Germany) in ﬁve cycles of 30 s. Two-hundred µL of Lysis buﬀer from the MALDI Sepsityper Kit (Bruker Daltonics GmbH, Bremen, Germany) was added to the resulting sample and mixed by vortexing for 10 s. After centrifugation, the pellet was suspended with 1 mL of Washing Buﬀer (MALDI Sepsityper Kit) and, after another centrifugation, the supernatant (“sample A”) was used for LC–ESI–MS/MS analysis. Genes 2020, 11, 693 3 of 18 Last, the obtained pellet was resuspended in 1.2 mL of a solution water/ethanol 1:3, and 2/3 of it was centrifuged, obtaining a second pellet (“B”), which was resuspended in 35% formic acid (FA), 15% water, and 50% acetonitrile (ACN) for MALDI–TOF MS analysis (“sample BM”, M as the abbreviation for MALDI–TOF MS). Genes 2020, 11, x FOR PEER REVIEW 3 of 18 15% water, and 50% acetonitrile (ACN) for MALDI–TOF MS analysis (“sample BM”, M as the abbreviation for MALDI–TOF MS). In parallel, 1/3 of the solution was centrifuged for 2 min at 14,000 rpm, and the resulting pellet (“B”) was sonicated (VibraCell Ultrasonic Liquid Processor, Sonics & Materials Inc, Newtown, CT, USA) 7 times, 60% amplitude, in Sample Buﬀer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 40 mM trizma, and 50 mM dithiothreitol (DTT)) and incubated for 1 h at 37 ◦C. After centrifugation at 14,000 rpm for 15 min, the supernatant (“sample BLC “, LC as the abbreviation for LC–ESI–MS/MS) was collected in order to perform the LC–ESI–MS/MS analysis (Figure 1). In parallel, 1/3 of the solution was centrifuged for 2 min at 14,000 rpm, and the resulting pellet (“B”) was sonicated (VibraCell Ultrasonic Liquid Processor, Sonics & Materials Inc, Newtown, CT, USA) 7 times, 60% amplitude, in Sample Buffer (7 M urea, 2 M thiourea, 4% 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 40 mM trizma, and 50 mM dithiothreitol (DTT)) and incubated for 1 h at 37 °C. 2.1. Protein Extraction After centrifugation at 14,000 rpm for 15 min, the supernatant (“sample BLC “, LC as the abbreviation for LC–ESI–MS/MS) was collected in order to perform the LC–ESI–MS/MS analysis (Figure 1). Figure 1. Sketch of the two proteomic experimental approaches exploited for Anisakis spp. identification. Figure 1. Sketch of the two proteomic experimental approaches exploited for Anisakis spp. identiﬁcation. 2 Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF MS) Figure 1. Sketch of the two proteomic experimental approaches exploited for Anisakis spp. Figure 1. Sketch of the two proteomic experimental approaches exploited for Anisakis spp. identiﬁcation. ntification. trix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF MS) identification. .2. Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF MS) 2.2. Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF MS) One microliter of sample BM was placed on an MSP 96 polished steel target (Bruker Daltonics, GmbH, Bremen, Germany), air-dried, and overlaid with 1 µL of the matrix, consisting in a solution of 5 mg/mL of α-cyano-4-hydroxycinnamic acid (Bruker Daltonics GmbH, Bremen, Germany) in 50% ACN, 47.5% water, 2.5% trifluoroacetic acid (TFA). Each sample was spotted onto eight target spots of the MALDI target plate, and spectral measurements were performed with a Microflex LT mass spectrometer (Biotyper, Bruker Daltonics GmbH, Bremen, Germany), equipped with the FlexControl software package, (version 3.4, Bruker Daltonics GmbH, Bremen, Germany), operating in the positive linear mode (laser frequency 20 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 18.4 kV; lens One microliter of sample BM was placed on an MSP 96 polished steel target (Bruker Daltonics, GmbH, Bremen, Germany), air-dried, and overlaid with 1 µL of the matrix, consisting in a solution of 5 mg/mL of α-cyano-4-hydroxycinnamic acid (Bruker Daltonics GmbH, Bremen, Germany) in 50% ACN, 47.5% water, 2.5% triﬂuoroacetic acid (TFA). Each sample was spotted onto eight target spots of the MALDI target plate, and spectral measurements were performed with a Microﬂex LT mass spectrometer (Biotyper, Bruker Daltonics GmbH, Bremen, Germany), equipped with the FlexControl software package, (version 3.4, Bruker Daltonics GmbH, Bremen, Germany), operating in the positive linear mode (laser frequency 20 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 18.4 kV; lens voltage, 6 kV; mass range, 2000 to 20,000 m/z). 2.4. Liquid Chromatography–Electrospray Ionization–Tandem MS (LC–ESI–MS/MS) LC–ESI–MS/MS experiments were performed on an UltiMate3000 RSLCnano System directly coupled to an Orbitrap Fusion Tribrid mass spectrometer, operating in positive ionization mode, equipped with a nanoESI source (EASY-Spray NG) (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The digested proteins (1.25 µg) were ﬁrst trapped and desalted onto a µ-precolumn cartridge C18 PepMap100 (5 µm particle size, 100 Å pore size, 300 µm i.d. × 5 mm length, Thermo Fisher Scientiﬁc, Waltham, MA, USA) for 3 min at 10 µL/min, with an aqueous solution of 2% ACN and 0.1% TFA, and then separated by reverse-phase chromatography performed on an EASY-Spray PepMap RSLC C18 column (2 µm particle size, 100 Å pore size, 75 µm i.d. × 50 cm length, Thermo Fisher Scientiﬁc, Waltham, MA, USA) at a ﬂow rate of 250 nL/min and a temperature of 35 ◦C, by a one-step linear gradient from 95% eluent A (0.1% FA in water) to 25% eluent B (99.9% ACN, 0.1% FA) in 113 min and total LC run of 160 min. Precursor (MS1) survey scans were recorded in the Orbitrap, at resolving powers of 120 K (at m/z 200). Data-dependent MS/MS (MS2) analysis was performed in top speed mode with a 3 s cycle time, during which most abundant multiple-charged (2+–7+) precursor ions detected within the range of 375–1500 m/z were selected for activation in order of abundance and detected in ion trap at rapid scan rate. Quadrupole isolation with a 1.6 m/z isolation window was used, and dynamic exclusion was enabled for 60 s after a single scan. Automatic gain control targets were 4.0 × 105 for MS1 and 2.0 × 103 for MS2, with 50 and 300 ms maximum injection times, respectively. For MS2, the signal intensity threshold was 5.0 ×103, and the option “Injection Ions for All Available Parallelizable Time” was set. High-energy collisional dissociation (HCD) was performed using 30% normalized collision energy. Lock mass was set as an internal calibration using polydimethylcyclosiloxane (445.12003 m/z). 2.1. Protein Extraction Three independent mass spectra with 240 shots (from 4 of 18 Genes 2020, 11, 693 diﬀerent positions of the target spot) for each spectrum were acquired from each spot, to obtain 24 spectra replicas, externally calibrated by using the Bacterial Test Standard (Bruker Daltonics GmbH, Bremen, Germany). Subsequently, spectra datasets were imported into ClinPro Tools software (version 3.0, Bruker Daltonics, GmbH, Bremen, Germany) for data mining after peak picking on the calculated total average spectrum, setting the signal-to-noise (S/N) threshold at 3, baseline subtraction (Top Hat), and peak intensity calculation. 2.3. In-Solution Protein Digestion The protein extracts (sample A and BLC) were subjected to reduction, alkylation, and trypsin digestion according to the ﬁlter-aided sample preparation (FASP) protocol [11]. Brieﬂy, the protein extracts were loaded on the Microcon-10kDa Centrifugal Filter Unit with an Ultracel-10 membrane (Merck, Burlington, MA, USA) in the presence of 8 M urea and 100 mM Tris-HCl, pH 8.5; disulﬁde bonds were reduced for 15 min at 37 ◦C with 8 mM DTT, then the samples were incubated with 50 mM iodoacetamide for 15 min and subsequently with DTT and digested with 1 µg of sequencing-grade trypsin (Promega, Milan, Italy) at 37 ◦C in 50 mM ammonium bicarbonate buﬀer pH 8.0, overnight (16 h). Peptides were eluted from the Microcon, speedvac-dried, and resuspended in a water solution with 2% ACN and 0.1% FA. Total peptide content was determined by NanoDrop 2000 (Thermo Fisher Scientiﬁc, Waltham, MA, USA) analysis, with a standard curve of MassPrep Escherichia coli digestion (Waters, Milford, MA, USA). 2.5. Database Searching and Protein Identiﬁcation Protein IDs were obtained with the embedded search engine (Sequest HT) of the Proteome Discoverer software (PD, version 2.4, Thermo Fisher Scientiﬁc, Waltham, MA, USA) after searching a custom-made database containing the complete UniProtKB/Swiss-Prot sequence entries catalogue (561,568 proteins, release: 2019_11) to which Anisakis UniProtKB/TrEMBL (25,874 proteins, release: 2019_11) and “Salmon” UniProtKB/TrEMBL (233,298 proteins, release: 2020_01) sequence entries were appended. The search parameters included trypsin as the proteolytic enzyme with a maximum of 2 missed cleavages per peptide allowed and oxidation of methionine as a variable modiﬁcation, 5 of 18 Genes 2020, 11, 693 whereas carbamidomethylation of cysteine was set as static modiﬁcation. Precursor and fragment mass tolerance were set to 10 ppm and 0.6 Da, respectively. False discovery rate (FDR) was calculated by the Percolator algorithm, and a cut-oﬀof 0.01 was used for the identiﬁcations (i.e., the expected fraction of incorrect protein match in the entire data set was set to less than 1%, calculated on a decoy database). At least two peptides were considered for protein ID. 3.1. Experimental Pipeline After mechanical homogenization and sonication, the samples obtained from ﬁve larvae were treated according with the MALDI Sepsityper Kit protocol (Bruker Daltonics, GmbH, Bremen, Germany). To exploit the advantages of diﬀerent proteomic strategies, a combined approach based on two mass spectrometry platforms was undertaken in the current study (Figure 1). 2.6. Functional Analysis The mapping of orthologous genes from protein lists to Caenorhabditis elegans was carried out by the g:Orth tool of the g:Proﬁler web server based on data collected into the Ensembl database [12,13] and by STRING (version 11.0, database 11_0) [14,15]. Bioinformatic analyses were performed by the g:GOSt function of the g:Proﬁler software in order to perform statistical enrichment analysis to ﬁnd over-representation of information from Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways. The most signiﬁcant categories associated with the uploaded datasets were identiﬁed by calculating the related signiﬁcance (p-value) when comparing the protein list to the whole C. elegans proteome. The p-value measures the likelihood that the association between the genes/proteins in the datasets and each GO and KEGG terms is not due to random chance alone, identifying a signiﬁcant over-representation of molecules in association with a given process. We applied an experiment-wide p-value threshold of 0.05, limiting the FDR (i.e., the expected fraction of false positives among signiﬁcant terms) to less than 5%; g:GOSt uses multiple testing correction and applies the tailor-made algorithm g:SCS for reducing signiﬁcance scores. Protein–protein interaction networks analysis was performed using the STRING application. The highest conﬁdence of 0.9 was chosen as the minimum required interaction score threshold, such that only interactions above this score were included in the predicted networks. Networks were clustered by the Markov clustering (MCL) algorithm with inﬂation parameter set as 3 (indirectly related to the precision of the clustering, i.e., the higher the inﬂation, the more abundant the clusters). 3.2. Protein Proﬁling by MALDI–TOF MS Analysis Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. Pseudogel view of the ﬁve larvae (samples BM) from MALDI–TOF MS proﬁling. The mass-to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). The phenotypic variability of the larvae, inspected by the clustering analysis performed on t plete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed o with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). The phenotypic variability of the larvae, inspected by the clustering analysis performed on th mplete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed on de with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). Figure 4 Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all Figure 4. Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all MALDI–TOF MS spectral replicas from Anisakis larvae. Fi 4 S d d (b i d hi hi l l i ) b i d b l i ll Figure 4. Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all MALDI–TOF MS spectral replicas from Anisakis larvae. Figure 4. Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all MALDI–TOF MS spectral replicas from Anisakis larvae. g p g y p g y y g MALDI–TOF MS spectral replicas from Anisakis larvae. 3.2. Protein Proﬁling by MALDI–TOF MS Analysis Pseudogel view of the ﬁve larvae (samples BM) from MALDI–TOF MS proﬁling. The mass-to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). The phenotypic variability of the larvae, inspected by the clustering analysis performed on the mplete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed one de with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. Pseudogel view of the ﬁve larvae (samples BM) from MALDI–TOF MS proﬁling. The mass-to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). The phenotypic variability of the larvae, inspected by the clustering analysis performed on lete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed o with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). Figure 3. 3.2. Protein Proﬁling by MALDI–TOF MS Analysis The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). The phenotypic variability of the larvae, inspected by the clustering analysis performed on th plete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed on e with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). Figure 3. Pseudogel view of the ﬁve larvae (samples BM) from MALDI–TOF MS proﬁling. The mass-to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). The phenotypic variability of the larvae, inspected by the clustering analysis performed on th plete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed on with larva 5; larva 2 and 3 were more similar and identified a different cluster (Figure 4). Figure 4. Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all MALDI–TOF MS spectral replicas from Anisakis larvae. Figure 4. Spectra dendrogram (by unsupervised hierarchical clustering) obtained by analyzing all MALDI–TOF MS spectral replicas from Anisakis larvae. The mass spectra dataset of each larva were grouped in a diﬀerent class, and a cut-oﬀ≤8 on erence between the maximum and the minimum average peak intensities of all classes (DAve Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. The mass- to-charge values (m/z, Da) are reported on the x-axis, while the gray scale bar, reported on the right y-axis, shows the relationship between the color intensity and the peak intensity, expressed by arbitrary units (arb. u.). The left y-axis evidences the spectra numbers (Sp.#). Figure 3. 3.2. Protein Proﬁling by MALDI–TOF MS Analysis After protein extraction, spotting, and MS analysis, high-intensity peaks in the range of 2000–12,000 Da (m/z) were highlighted from the ﬁve larvae (sample BM), with the highest density in the region comprised between 2500 and 8000 Da, with clusters of signals in ranges corresponding to 2700–2900, 5400–5700, and 7100–7500 Da (Figure 2; Figure 3). The phenotypic variability of the larvae, inspected by the clustering analysis performed on the complete dataset, was underlined: mass spectra of larva 1 and 4 clustered together and formed one clade with larva 5; larva 2 and 3 were more similar and identiﬁed a diﬀerent cluster (Figure 4). 6 of 18 6 f 18 Genes 2020, 11, 693 Genes 2020, 11, x FOR PEER REVIEW 6 of 18 Figure 2. Representative protein profiling of Anisakis spp. by Biotyper MALDI–TOF MS. The average spectrum for each sample is depicted, as well as the average spectrum obtained from the entire spectra dataset. The m/z values are expressed in Da, and the signal intensities are reported in a scale of arbitrary units (arb. u.). Figure 2. Representative protein proﬁling of Anisakis spp. by Biotyper MALDI–TOF MS. The average spectrum for each sample is depicted, as well as the average spectrum obtained from the entire spectra dataset. The m/z values are expressed in Da, and the signal intensities are reported in a scale of arbitrary units (arb. u.). Figure 2. Representative protein profiling of Anisakis spp. by Biotyper MALDI–TOF MS. The average spectrum for each sample is depicted, as well as the average spectrum obtained from the entire spectra dataset. The m/z values are expressed in Da, and the signal intensities are reported in a scale of arbitrary units (arb. u.). Figure 2. Representative protein proﬁling of Anisakis spp. by Biotyper MALDI–TOF MS. The average spectrum for each sample is depicted, as well as the average spectrum obtained from the entire spectra dataset. The m/z values are expressed in Da, and the signal intensities are reported in a scale of arbitrary units (arb. u.). 7 of 18 7 of 18 Genes 2020, 11, 693 G 2020 11 FOR 2020, 11, x FOR PEER REVIEW 7 of Figure 3. Pseudogel view of the five larvae (samples BM) from MALDI–TOF MS profiling. 3.2. Protein Proﬁling by MALDI–TOF MS Analysis # Peak m/z DAve Ave CV (%) 1 2 3 4 5 1 2 3 4 5 1 2597.26 0.97 2.63 2.57 2.55 3.52 3.12 19.32 16.09 15.34 15.86 18.74 2 2654.85 3.6 4.41 7.84 7.78 4.24 5.08 17.66 15.12 12.49 10.57 14.7 3 2704.25 1.26 2.91 3.7 3.51 4.15 2.89 10.86 11.12 15.85 16.27 14.07 4 2724.12 3.66 4.6 7.84 7.4 4.18 4.47 18.86 12.1 12.99 13.48 17.98 5 2733.01 0.98 3.52 4.5 4.26 3.68 3.56 16.22 14.21 9.88 17.98 13.54 6 2741.79 1.25 3.63 4.47 4.47 3.94 3.22 15.67 13.38 11.7 13.73 15.86 7 2758.46 1.13 4.21 3.22 3.98 4.35 3.35 14.8 12.06 8.77 11.37 18.26 8 2781.06 1.35 4.0 4.5 5.11 4.0 3.76 9.79 11.77 9.01 11.98 19.07 9 2807.87 0.82 3.39 3.71 3.61 3.75 2.92 16.69 12.95 11.4 17.88 16.87 10 2827.16 0.6 3.25 3.72 3.55 3.29 3.12 14.97 14.59 14.48 17.82 19.86 11 2834.32 0.71 3.85 4.56 3.99 4.15 4.13 10.02 14.2 14.82 12.6 16.51 12 2840.23 1.8 3.91 2.82 3.98 4.54 4.63 14.68 7.22 11.06 10.73 19.48 13 4969.15 1.4 4.23 4.33 4.24 3.73 2.92 12.47 14.59 12.16 10.67 12.37 14 5335.65 3.25 2.98 4.36 5.38 2.81 6.05 14.79 12.28 12 11.62 15.81 15 5370.57 1.75 2.73 3.57 4.47 3.14 3.91 16.47 16.83 16.68 14.81 12 16 5656.33 7.86 8.48 13.73 12.95 5.87 5.9 10.17 11.46 9.92 14.32 19.42 17 5694.2 1.87 5.67 5.31 5.46 7.19 7.08 7.24 7.8 10.33 18.31 12.79 18 5712.76 6.26 5.97 11.04 9.91 4.78 5.74 8.99 9.07 9.14 13.13 13.56 19 7166.45 5.19 7.98 2.79 3.43 2.98 5.14 14.63 14.19 14.1 14.43 15.45 3 3 P t i P ﬁli b T d M S t t 3.3. Protein Proﬁling by Tandem Mass Spectrometry After enzymatic digestion and peptide puriﬁcation, we identiﬁed the total protein content of two extraction steps of the adopted protocol by LC–ESI–MS/MS on a high-resolution platform. From sample A of larvae 1, 2, 3, 4, and 5 we identiﬁed an overall number of 2179 diﬀerent proteins, of which 561 were identiﬁed as shared; in sample BLC, we identiﬁed a total of 3091 diverse proteins, of which 210 were common to the ﬁve larvae (Supplementary material S1). 3.2. Protein Proﬁling by MALDI–TOF MS Analysis The mass spectra dataset of each larva were grouped in a different class, and a cut-off ≤8 on the difference between the maximum and the minimum average peak intensities of all classes (DAve), a The mass spectra dataset of each larva were grouped in a diﬀerent class, and a cut-oﬀ≤8 on the diﬀerence between the maximum and the minimum average peak intensities of all classes (DAve), a minimum peak intensity average of 2.5 (Ave), and 20% of the coeﬃcient of variation (CV) for each class were applied. Regardless of the variability addressed by dendrogram analysis, 19 signals (among a total of 179) for all samples were furthermore identiﬁed, representing a collection of ﬁngerprinting classiﬁers of Anisakis spp. (Table 1). 8 of 18 Genes 2020, 11, 693 Table 1. Selected reference signals for the identiﬁcation of Anisakis spp. DAve, diﬀerence between the maximum and the minimum average peak intensities, Ave, peak intensity average, CV, coeﬃcient of variation. 3.2. Protein Proﬁling by MALDI–TOF MS Analysis We focused our functional analysis only on identiﬁed proteins belonging to Anisakis species and to organisms selected according to phylogenetic similarity (Rhabditida order [16], 1732 and 1543 total diﬀerent proteins in sample A and B, respectively) (Figure 5, Supplementary Material S2). Lists of identiﬁed proteins belonging to the Rhabditida order were ﬁltered by a three-step process (Figure 6): 1. only proteins identiﬁed in at least four out of ﬁve larvae (Supplementary material S3) were considered; 2. proteins with diﬀerent UniProtKB accession code but the same name were collapsed into single hit; 3. proteins deﬁned as unknown were deleted (Supplementary material S4). 9 of 18 9 f 18 Genes 2020, 11, 693 G 11 FO enes 2020, 11, x FOR PEER REVIEW 9 of 18 Figure 5. Overview of proteins identified by LC–ESI–MS/MS analysis. Venn diagrams show the number of Rhabditida proteins and their distribution in the five analyzed larvae, highlighting shared and exclusive proteins (numbers in colored areas) in samples A (a) and BLC (b). The organism distribution (as percentage of identified proteins) at the Rhabditida order taxonomy level in sample A and B is depicted with histograms (c). Figure 5. Overview of proteins identiﬁed by LC–ESI–MS/MS analysis. Venn diagrams show the number of Rhabditida proteins and their distribution in the ﬁve analyzed larvae, highlighting shared and exclusive proteins (numbers in colored areas) in samples A (a) and BLC (b). The organism distribution (as percentage of identiﬁed proteins) at the Rhabditida order taxonomy level in sample A and B is depicted with histograms (c). Figure 5. Overview of proteins identified by LC–ESI–MS/MS analysis. Venn diagrams show the number of Rhabditida proteins and their distribution in the five analyzed larvae, highlighting shared and exclusive proteins (numbers in colored areas) in samples A (a) and BLC (b). The organism distribution (as percentage of identified proteins) at the Rhabditida order taxonomy level in sample A and B is depicted with histograms (c). Figure 5. Overview of proteins identiﬁed by LC–ESI–MS/MS analysis. Venn diagrams show the number of Rhabditida proteins and their distribution in the ﬁve analyzed larvae, highlighting shared and exclusive proteins (numbers in colored areas) in samples A (a) and BLC (b). The organism distribution (as percentage of identiﬁed proteins) at the Rhabditida order taxonomy level in sample A and B is depicted with histograms (c). 3.2. Protein Proﬁling by MALDI–TOF MS Analysis 10 of 18 10 of 18 Genes 2020, 11, 693 Genes 2020 11 x FO Figure 6. Filters applied to protein lists prior to performing functional bioinformatic analyses. KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure 6. Filters applied to protein lists prior to performing functional bioinformatic analyses. KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure 6. Filters applied to protein lists prior to performing functional bioinformatic analyses. KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure 6. Filters applied to protein lists prior to performing functional bioinformatic analyses. KEGG, Kyoto Encyclopedia of Genes and Genomes. Functional Analysis Functional Analysis y The filtered protein lists were examined for their known GO terms, retrieved by the ProteinCenter application of PD software, and grouped in the respective categories as percentage with respect to the total terms of each sample. The most represented biological process, cellular component, and molecular function were, as expected, similar between sample A and BLC and were linked to metabolic process membrane and catalytic activity (Figure 7) The ﬁltered protein lists were examined for their known GO terms, retrieved by the ProteinCenter application of PD software, and grouped in the respective categories as percentage with respect to the total terms of each sample. The most represented biological process, cellular component, and molecular function were, as expected, similar between sample A and BLC and were linked to metabolic process, membrane, and catalytic activity (Figure 7). linked to metabolic process, membrane, and catalytic activity (Figure 7). In order to highlight an over-representation of information from GO terms and biological pathways, as well as protein–protein interaction networks, related to our dataset, we converted the two protein lists into orthologous genes of C. elegans (Figure 6 and Supplementary Material S5), as this model organism is the most investigated one for biomedical research and available in enrichment analysis web applications, contrary to Anisakis spp. Orthologous genes are likely conserved through evolution from a common ancestor, may carry out similar function, and are therefore relevant in functional analysis. 11 of 18 11 of 18 Genes 2020, 11, 693 Genes 2020, 11, x FO Figure 7 Pie chart of Gene Ontology (GO) distribution terms associated with the identified proteins Figure 7. Pie chart of Gene Ontology (GO) distribution terms associated with the identiﬁed proteins. Figure 7. Pie chart of Gene Ontology (GO) distribution terms associated with the identiﬁed proteins. Figure 7. Pie chart of Gene Ontology (GO) distribution terms associated with the identiﬁed proteins. Figure 7. Pie chart of Gene Ontology (GO) distribution terms associated with the identified proteins. In order to highlight an over-representation of information from GO terms and biological pathways, as well as protein–protein interaction networks, related to our dataset, we converted the two protein lists into orthologous genes of C. Functional Analysis Functional Analysis The x-axis represents functional terms that are grouped and color-coded by data sources (e.g., Molecular Function, MF, Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly significant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the specific term retrieved from the dataset, correspond to Figure 8. Manhattan plot of GO terms and biological pathways (KEGG) enrichment analysis, results of a multiquery with the two input gene lists (A and BLC, obtained from protein). The x-axis represents functional terms that are grouped and color-coded by data sources (e.g., Molecular Function, MF, Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly signiﬁcant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the speciﬁc term retrieved from the dataset, correspond to larger circles). functional terms that are grouped and color-coded by data sources (e.g., Molecular Function, MF, Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly significant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the specific term retrieved from the dataset, correspond to Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly signiﬁcant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the speciﬁc term retrieved from the dataset, correspond to larger circles). larger circles). Table 2. Functional Analysis Functional Analysis GO terms and biological pathways (KEGG) enrichment analysis. Term Group Circle Number (Figure 8) Term Name Term id Adjusted p-value Sample A Sample BLC MF 1 structural constituent of ribosome GO:0003735 4.07 × 10−26 1.10 × 10-04 2 structural molecule activity GO:0005198 1.42× 10−18 6.53 × 10-07 BP 3 translation GO:0006412 5.30× 10−21 1.10 × 10-04 4 peptide biosynthetic process GO:0043043 1.11× 10−20 1.38 × 10-04 5 amide biosynthetic process GO:0043604 2.53× 10−20 2.32 × 10−05 20 generation of precursor metabolites and energy GO:0006091 1.23× 10−16 6.24 × 10−18 CC 6 cytoplasm GO:0005737 2.19× 10−82 6.58 × 10−40 7 intracellular GO:0005622 7.43× 10−55 1.82 × 10−22 8 cytosolic ribosome GO:0022626 7.86× 10−38 4.04 × 10−10 9 cytosol GO:0005829 3.35× 10−36 1.37 × 10−21 10 ribosome GO:0005840 4.02× 10−28 8.32 × 10−06 11 ribosomal subunit GO:0044391 1.05× 10−27 5.91 × 10−07 12 intracellular organelle GO:0043229 7.65× 10−23 1.76 × 10−05 13 organelle GO:0043226 1.01× 10−21 8.60 × 10−05 14 cytosolic large ribosomal subunit GO:0022625 1.98× 10−20 9.33 × 10−05 15 protein-containing complex GO:0032991 6.21× 10−20 5.83 × 10−08 16 mitochondrion GO:0005739 3.42× 10−19 6.19 × 10−13 17 ribonucleoprotein complex GO:1990904 6.95× 10−18 5.88 × 10−03 18 cytosolic small ribosomal subunit GO:0022627 4.34× 10−17 1.23 × 10−04 KEGG 19 carbon metabolism KEGG:01200 9.55× 10−20 1.69 × 10−27 Table 2. GO terms and biological pathways (KEGG) enrichment analysis. Functional Analysis Functional Analysis elegans (Figure 6 and Supplementary material S5), as h d l h d f b d l h d l bl h Our protein extraction pipeline followed by tandem mass spectrometry analysis led us to a great enrichment of proteins from ribosome and related to carbon metabolism pathway (Figure 8 and Table 2) as well as many other GO terms and biological pathways, such as glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, metabolic pathways (Supplementary Material S6). 12 of 18 Genes 2020, 11, 693 nes 2020, 11, 693 12 o Genes 2020, 11, x FOR PEER REVIEW 12 of 18 Figure 8. Manhattan plot of GO terms and biological pathways (KEGG) enrichment analysis, results of a multiquery with the two input gene lists (A and BLC, obtained from protein). The x-axis represents functional terms that are grouped and color-coded by data sources (e.g., Molecular Function, MF, Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly significant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the specific term retrieved from the dataset, correspond to Figure 8. Manhattan plot of GO terms and biological pathways (KEGG) enrichment analysis, results of a multiquery with the two input gene lists (A and BLC, obtained from protein). The x-axis represents functional terms that are grouped and color-coded by data sources (e.g., Molecular Function, MF, Biological Process, BP, Cellular Component, CC from GO, and KEGG pathways, are red, green, orange, and violet, respectively). The y-axis shows the adjusted enrichment p-values in negative log10 scale). Term circles with p-values less than 1.0 × 10−16 (highly signiﬁcant) in at least one sample are highlighted and numbered. The circle sizes are in accordance with the corresponding term sizes (i.e., larger terms, which means more hits of the speciﬁc term retrieved from the dataset, correspond to larger circles). Figure 8. Manhattan plot of GO terms and biological pathways (KEGG) enrichment analysis, results of a multiquery with the two input gene lists (A and BLC, obtained from protein). Functional Analysis Functional Analysis Term Group Circle Number (Figure 8) Term Name Term id Adjusted p-Value Sample A Sample BLC MF 1 structural constituent of ribosome GO:0003735 4.07 × 10−26 1.10 × 10−04 2 structural molecule activity GO:0005198 1.42 × 10−18 6.53 × 10−07 BP 3 translation GO:0006412 5.30 × 10−21 1.10 × 10−04 4 peptide biosynthetic process GO:0043043 1.11 × 10−20 1.38 × 10−04 5 amide biosynthetic process GO:0043604 2.53 × 10−20 2.32 × 10−05 20 generation of precursor metabolites and energy GO:0006091 1.23 × 10−16 6.24 × 10−18 CC 6 cytoplasm GO:0005737 2.19 × 10−82 6.58 × 10−40 7 intracellular GO:0005622 7.43 × 10−55 1.82 × 10−22 8 cytosolic ribosome GO:0022626 7.86 × 10−38 4.04 × 10−10 9 cytosol GO:0005829 3.35 × 10−36 1.37 × 10−21 10 ribosome GO:0005840 4.02 × 10−28 8.32 × 10−06 11 ribosomal subunit GO:0044391 1.05 × 10−27 5.91 × 10−07 12 intracellular organelle GO:0043229 7.65 × 10−23 1.76 × 10−05 13 organelle GO:0043226 1.01 × 10−21 8.60 × 10−05 14 cytosolic large ribosomal subunit GO:0022625 1.98 × 10−20 9.33 × 10−05 15 protein-containing complex GO:0032991 6.21 × 10−20 5.83 × 10−08 16 mitochondrion GO:0005739 3.42 × 10−19 6.19 × 10−13 17 ribonucleoprotein complex GO:1990904 6.95 × 10−18 5.88 × 10−03 18 cytosolic small ribosomal subunit GO:0022627 4.34 × 10−17 1.23 × 10−04 KEGG 19 carbon metabolism KEGG:01200 9.55 × 10−20 1.69 × 10−27 circles). Table 2. GO terms and biological pathways (KEGG) enrichment analysis. Genes 2020, 11, 693 Genes 2020, 11, x FOR 13 of 18 13 of 18 Consequently, network analysis evidenced a main cluster of ribosomal proteins in sample A (Figure 9) and sample BLC (Figure 10). In fact, a group of proteins at least partially biologically connected present more interactions than a random set of proteins of similar size drawn from the genome; it indicated an enrichment of the ribosome-associated structural constituents and translation. (Figure 9) and sample BLC (Figure 10). In fact, a group of proteins at least partially biologically connected present more interactions than a random set of proteins of similar size drawn from the genome; it indicated an enrichment of the ribosome-associated structural constituents and translation. Figure 9. Network analysis of sample A. The network nodes are proteins, and the edges represent the predicted functional associations. The highlighted red cluster is related to ribosomal proteins. Figure 9. Network analysis of sample A. 4. Discussion 4. Discussion Clinical laboratories take advantage of the MALDI–TOF MS technology to identify pathogenic microorganisms, including bacteria, yeasts, and filamentous fungi, thanks to the ease of use, speed of analysis, cost-effectiveness, and accuracy of IDs. MALDI–TOF MS analysis allows the detection of different types of biomolecules in a range of concentrations close to sub-femtomoles; in clinical diagnosis, peptides and proteins are fingerprinting classifiers [17]. In particular, one of the main advantages of this particular approach, i.e., rapidity, is grounded on the availability of mass spectra collections unambiguously identifying an organism by matching these spectral libraries with signals Clinical laboratories take advantage of the MALDI–TOF MS technology to identify pathogenic microorganisms, including bacteria, yeasts, and ﬁlamentous fungi, thanks to the ease of use, speed of analysis, cost-eﬀectiveness, and accuracy of IDs. MALDI–TOF MS analysis allows the detection of diﬀerent types of biomolecules in a range of concentrations close to sub-femtomoles; in clinical diagnosis, peptides and proteins are ﬁngerprinting classiﬁers [17]. In particular, one of the main advantages of this particular approach, i.e., rapidity, is grounded on the availability of mass spectra collections unambiguously identifying an organism by matching these spectral libraries with signals obtained from the samples. collections unambiguously identifying an organism by matching these spectral libraries with signals obtained from the samples. Moreover, the applications comply with the European In Vitro Diagnostic Devices Directive (98/79/EC), which means that MALDI–TOF MS is a CE Marking for all in vitro diagnostic (IVD) devices, may be legally commercialized in the EU as a diagnostic tool, and the associated processes are similar to those of medical devices. In fact, the Bruker Biotyper and the VITEK MS system received first CE-IVD status in 2009 and 2011, respectively, and FDA clearance (for both) in 2013 [17]. Functional Analysis Functional Analysis The network nodes are proteins, and the edges represent the predicted functional associations. The highlighted red cluster is related to ribosomal proteins. Figure 9. Network analysis of sample A. The network nodes are proteins, and the edges represent the predicted functional associations. The highlighted red cluster is related to ribosomal proteins. Figure 9. Network analysis of sample A. The network nodes are proteins, and the edges represent the predicted functional associations. The highlighted red cluster is related to ribosomal proteins. 14 of 18 14 of 18 Genes 2020, 11, 693 Genes 2020 11 x FOR Figure 10. Network analysis of sample BLC. The highlighted blue cluster is related to ribosomal Figure 10. Network analysis of sample BLC. The highlighted blue cluster is related to ribosomal proteins. Figure 10. Network analysis of sample BLC. The highlighted blue cluster is related to ribosomal Figure 10. Network analysis of sample BLC. The highlighted blue cluster is related to ribosomal proteins. 4. Discussion y The aim of our study was to assess the proof of concept that MBT Sepsityper Kit-based protein extraction from Anisakis larvae and subsequent MALDI–TOF MS Biotyper-based collection of spectra may provide diagnostic signals of biomedical interest related to this Anisakis life stage. The proposed method will work for the identiﬁcation of already extracted nematode larvae from patients to improve the current diagnostic approaches. Although a variability in the MS peaks was ascertained, a few detected signals were statistically signiﬁcant in representing all the larvae samples. This evidence is promising for the development of new diagnostic tools for Anisakis. We are aware that a deﬁnite assessment of the herein hypothesized diagnostic pipeline will require a collection of more Anisakis larvae samples, also with diﬀerent geographical origin, as well as control samples from diﬀerent nematodes in order to obtain a precise taxonomic typing. Hopefully, in the near future, eﬀorts towards this development will be made in a consortium of research and clinical laboratories. An interesting outcome of our study was the conﬁrmation that the adopted protein extraction protocol and the MALDI–TOF MS analysis resulted in ﬁngerprinting spectra attributable to peptides and proteins belonging to the plethora of ribosome molecular species, as already known [21]. The molecular weight of ribosomal proteins is within/around the mass range of the linear MALDI–TOF MS acquisition set-up, and our shotgun proﬁling experiments, by tandem MS on an higher resolution platform, highlighted the enrichment of ribosome proteins in two experimental steps. g g p p p Moreover, among the 448 proteins identiﬁed in four out of ﬁve larvae (Supplementary material S4), we identiﬁed also proteins related to infection molecular mechanisms, which have not yet been fully understood [22]. Ingested Anisakis has to survive in the highly acidic human stomach, penetrate the gastrointestinal wall, through degradation of the mucosa and submucosa, and migrate to the ﬁnal location, causing tissue damage and inﬂammation. At the end, anisakiasis arises from both the tissue damage and the interplay between the host immune system and substances secreted by or contained within the larvae [2]. 4. Discussion The technique and the expertise have been growing over time; the implementation of associated software and reference databases, as well as sample preparation kits commercialized by the vendor have allowed researchers and laboratory specialists to both increase the type of microorganisms that can be identified and potentially treat/process “direct samples” (e.g., blood cultures), facilitating the management of infected patients As an example Bruker has developed the MBT Sepsityper IVD Kit Moreover, the applications comply with the European In Vitro Diagnostic Devices Directive (98/79/EC), which means that MALDI–TOF MS is a CE Marking for all in vitro diagnostic (IVD) devices, may be legally commercialized in the EU as a diagnostic tool, and the associated processes are similar to those of medical devices. In fact, the Bruker Biotyper and the VITEK MS system received ﬁrst CE-IVD status in 2009 and 2011, respectively, and FDA clearance (for both) in 2013 [17]. The technique and the expertise have been growing over time; the implementation of associated software and reference databases, as well as sample preparation kits commercialized by the vendor have allowed researchers and laboratory specialists to both increase the type of microorganisms that can be identiﬁed and potentially treat/process “direct samples” (e.g., blood cultures), facilitating the management of infected patients. As an example, Bruker has developed the MBT Sepsityper IVD Kit for the identiﬁcation of microorganisms from blood cultures using a MALDI–TOF MS platform [18–20]. Interestingly, a recent Genes 2020, 11, 693 15 of 18 article reported the use of the MBT Sepsityper Kit in order to proﬁle four nematodes (Diroﬁlaria repens, Diroﬁlaria immitis, Ascaris suum, and Ascaris lumbricoides) [10], which intrigued and prompted us to test this method on Anisakis spp. In fact, our future goal, that has driven the preliminary study here presented, is to develop a pipeline for a proteomic proﬁling of Anisakis spp. for diagnostic purposes; the existence of an IVD kit will facilitate this future application of our method. Although widely used as a diagnostic tool for accurate identiﬁcation of bacteria, very few studies have tried to translate the advantages of the MALDI–TOF MS technique to clinical parasitology [8,9], and therefore, as far as we know, there are no related commercially MALDI–TOF MS ID spectral library databases. 4. Discussion We identiﬁed some harmful antigens (proteinase inhibitor, somatic paramyosin, tropomyosin, and heat shock proteins), including Ani s 2 (UniProtKB accession number: L7V1I9), Ani s 4 (Q14QT4), Ani s 5 (A1IKL2), and Ani s 8 allergens (two isoforms: 1 and 10, A7M6S9 and A1IKL2, respectively), which are present in the Allergome database [23] and registered as Anisakis allergenic proteins by WHO/IUIS [24]. Ani s 2 is a cytoskeletal paramyosin protein, showing highly conserved sequences with respect to paramyosins of diﬀerent origin (other nematodes, insects, shellﬁsh), which account for the cross-reactivity in IgE binding. The excretory–secretory (ES) Ani s 4 allergen is a cysteine-type endopeptidase with inhibitor activity, present both in excretory glands and below the cuticle. Ani s 5 and Ani s 8 are other heat-stable ES allergens and members of the nematode SXP/RAL-2 protein family. In particular, Ani s 5 is assumed to be secreted in the human gastrointestinal tract from the third-stage larvae of Anisakis simplex; its putative magnesium ion transporter functional feature may be inferred by its magnesium ion binding capability and structural similarity to Calmodulin [25–27]. ES molecules are responsible for a multitude of functions during infection, such as penetration of host tissues and evasion of host immune responses, and at the same time are known to elicit immune responses. Therefore, ES proteins are hypothesized to be the major contributors in clinical manifestation of the disease in humans [28]. The identiﬁed Synthase trehalose-6-phosphate (A0A0M3JTQ2) may be important for the infection mechanism because it can be associated with a plethora of physiological and biochemical adaptive Genes 2020, 11, 693 16 of 18 16 of 18 mechanisms that parasitic nematodes put in place under adverse environmental conditions, such as the unfavorable pH that Anisakis ﬁnds in the human gastrointestinal milieu, in order to survive [29]. Proteolytic enzymes, such as peptidases, are responsible for Anisakis pathogenicity because of their role in biological pathways linked to fundamental host–pathogen interactions. Among the several peptidases present in our dataset, we identiﬁed Metalloendopeptidase (A0A3G5BC99) belonging to the Astacin peptidase family M12A (Pfam code: Pf01400, secreted or membrane-anchored proteases that requires zinc for catalysis) and Carboxypeptidase (A0A158PN74), part of the Peptidase_S10 family (Pf00450). Interestingly, both protein families were recently identiﬁed amongst the upregulated transcripts in the pharynx of A. simplex and Anisakis pegreﬃi [22]. Moreover, the Metalloendopeptidase mRNA expression level was found to be higher in A. 4. Discussion simplex third-stage larvae compared to the fourth-stage larvae, enforcing the role of this enzyme as a signiﬁcant player in host tissues invasion [30]. 5. Conclusions A set of MALDI-TOF MS signals was identiﬁed as potential consensus “biomarkers” peak list, characterized by speciﬁc averaged m/z and intensity, for the identiﬁcation of Anisakis spp. from nematode larvae present in patients tissue to improve the current diagnostic approaches. In fact, due to the similarity of symptoms with those of common gastrointestinal disorders and lack of highly skilled microscopists in biomedical laboratories, Anisakis infection is often underestimated and alternative diagnostic tools are enviable. Additionally, the shotgun bottom-up analysis of Anisakis proteins, obtained by the extraction method based on the MBT Sepsityper Kit and performed by LC–ESI–MS/MS on a high-resolution platform, underlined the presence, in the nematode extract, of both an enrichment of ribosome proteins and speciﬁc proteins potentially associated with molecular mechanisms that accompany infection. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/6/693/s1, Supplementary material S1: List of identiﬁed proteins by LC–ESI–MS/MS; Supplementary material S2: List of identiﬁed proteins by LC–ESI–MS/MS and belonging to Rhabditida taxonomy order; Supplementary material S3: List of Rhabditida proteins identiﬁed in at least four out of ﬁve larvae of samples A and BLC, Supplementary material S4: List of Rhabditida proteins identiﬁed in at least four out of ﬁve larvae of samples A and BLC and ﬁltered by collapsing proteins with the same name into one single hit and deleting unknown proteins; Supplementary material S5: Results of gene orthology conversion; Supplementary material S6: Functional analysis enrichment. Author Contributions: Conceptualization, L.P.; Funding acquisition, L.P.; Investigation, V.M., S.P. and G.F.; Supervision, L.P.; Visualization, V.M. and G.F.; Writing—original draft, V.M. and S.P.; Writing—review & editing, V.M., S.P., G.F., S.L.M., P.V., A.O.M. and L.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fondazione Bambino Gesù, grant number 201903_SBG, and Ricerca Corrente of the Minister of Italian Health, grant number 201905_genetica, to L.P. Funding: This research was funded by Fondazione Bambino Gesù, grant number 201903_SBG, and Ric Corrente of the Minister of Italian Health, grant number 201905_genetica, to L.P. Acknowledgments: We would like to thank A. Buchicchio (Bruker Italia S.r.l. Daltonics Division) for helpful ad Acknowledgments: We would like to thank A. Buchicchio (Bruker Italia S.r.l. Daltonics Division) for helpful advice. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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Biochem. Parasitol. 2018, 226, 24–33. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4200505600	https://www.frontiersin.org/articles/10.3389/fevo.2021.723558/pdf	English	null	Data_Sheet_1.zip	null	2,021	cc-by	14,698	Citation: Cardoso D, Moonlight PW, Ramos G, Oatley G, Dudley C, Gagnon E, Queiroz LP, Pennington RT and Särkinen TE (2021) Deﬁning Biologically Meaningful Biomes Through Floristic, Functional, and Phylogenetic Data. Front. Ecol. Evol. 9:723558. doi: 10.3389/fevo.2021.723558 ORIGINAL RESEARCH published: 24 December 2021 doi: 10.3389/fevo.2021.723558 Keywords: biome delimitation, functional diversity, macroecology, phylogenetic diversity, SDTF, species distribution modeling, transition zones, bioregionalization Specialty section: Specialty section: This article was submitted to Biogeography and Macroecology, a section of the journal Frontiers in Ecology and Evolution Received: 10 June 2021 Accepted: 22 November 2021 Published: 24 December 2021 Reviewed by: Reviewed by: Thaís Guedes, State University of Maranhão, Brazil Adeline Fayolle, University of Liège, Belgium Reviewed by: Thaís Guedes, State University of Maranhão, Brazil Adeline Fayolle, University of Liège, Belgium 1 National Institute of Science and Technology in Interdisciplinary and Transdisciplinary Studies in Ecology and Evolution (INCT IN-TREE), Instituto de Biologia, Universidade Federal da Bahia, Salvador, Brazil, 2 Tropical Diversity Section, Royal Botanic Garden Edinburgh, Edinburgh, United Kingdom, 3 Department of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom, 4 Department of Geography, University of Exeter, Exeter, United Kingdom, 5 Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Feira de Santana, Brazil Correspondence: Domingos Cardoso cardosobot@gmail.com †ORCID: †ORCID: While we have largely improved our understanding on what biomes are and their utility in global change ecology, conservation planning, and evolutionary biology is clear, there is no consensus on how biomes should be delimited or mapped. Existing methods emphasize different aspects of biomes, with different strengths and limitations. We introduce a novel approach to biome delimitation and mapping, based upon combining individual regionalizations derived from ﬂoristic, functional, and phylogenetic data linked to environmentally trained species distribution models. We deﬁne “core Biomes” as areas where independent regionalizations agree and “transition zones” as those whose biome identity is not corroborated by all analyses. We apply this approach to delimiting the neglected Caatinga seasonally dry tropical forest biome in northeast Brazil. We delimit the “core Caatinga” as a smaller and more climatically limited area than previous deﬁnitions, and argue it represents a ﬂoristically, functionally, and phylogenetically coherent unit within the driest parts of northeast Brazil. “Caatinga transition zones” represent a large and biologically important area, highlighting that ecological and evolutionary processes work across environmental gradients and that biomes are not categorical variables. We discuss the differences among individual regionalizations in an ecological and evolutionary context and the potential limitations and utility of individual and combined biome delimitations. Our integrated ecological and evolutionary deﬁnition of the Caatinga and associated transition zones are argued to best describe and map biologically meaningful biomes. Domingos Cardoso orcid.org/0000-0001-7072-2656 Peter W. Moonlight orcid.org/0000-0003-4342-2089 Gustavo Ramos orcid.org/0000-0002-4539-394X Graeme Oatley orcid.org/0000-0003-0896-9994 Edeline Gagnon orcid.org/0000-0003-3212-9688 Luciano Paganucci de Queiroz orcid.org/0000-0001-7436-0939 R. Toby Pennington orcid.org/0000-0002-8196-288X Tiina E. Särkinen orcid.org/0000-0002-6956-3093 ‡These authors have contributed equally to this work and share ﬁrst authorship Edited by: Danilo M. Neves, Federal University of Minas Gerais, Brazil Edited by: Danilo M. Neves, Federal University of Minas Gerais, Brazil Domingos Cardoso1†‡, Peter W. Moonlight2†‡, Gustavo Ramos2,3†, Graeme Oatley4†, Christopher Dudley2, Edeline Gagnon2†, Luciano Paganucci de Queiroz5†, R. Toby Pennington2,4† and Tiina E. Särkinen2† Domingos Cardoso1*†‡, Peter W. Moonlight2†‡, Gustavo Ramos2,3†, Graeme Oatley4†, Christopher Dudley2, Edeline Gagnon2†, Luciano Paganucci de Queiroz5†, R. Toby Pennington2,4† and Tiina E. Särkinen2† INTRODUCTION Biomes are a key concept in ecology and biogeography (Higgins et al., 2016; Mucina, 2019) and have been largely used in global change ecology (Prentice et al., 2007; Williams et al., 2007; Lehmann et al., 2014; Moncrieﬀet al., 2016), conservation planning (Hoekstra et al., 2005), and evolutionary biology (Donoghue and Edwards, 2014; Landis et al., 2021a). Although biome deﬁnitions have diﬀered (Mucina, 2019), the scientiﬁc community has generally settled on an December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 1 Biologically Meaningful Biomes Cardoso et al. agreed biome deﬁnition: “a biotic community ﬁnding its expression at large geographic scales, shaped by climatic factors, and perhaps better characterized by physiognomy and functional aspects, rather than by species or life-form composition” (Mucina, 2019). Despite this accord over the deﬁnition of a biome, there remains no universally recognized method of delimiting and mapping biomes. Diﬀerent approaches focus upon diﬀerent elements of biomes – their physiognomic, ﬂoristic, environmental, or functional characteristics – which in turn produce diﬀerent biome maps. Although such single-criterion- based biome schemes are helpful for understanding plant communities from an operational point of view, and at the local to the global scale (Conradi et al., 2020), they cannot deﬁne the nature of biomes through time and fail to capture the distribution, structure, and functioning of biomes in an evolutionary continuum. leading to distinct biome assemblies (see also Donoghue and Edwards, 2014). Biome conservatism and environmental ﬁltering are of course not universal. For example, depending on the “evolutionary accessibility” of a new ecological setting or a lineage’s biome aﬃnity and location relative to the spatial distribution of other biomes at any given time, there is a varying spectrum of possible biogeographical scenarios, including those at which lineages can transcend ecological barriers more easily than geographical barriers (Edwards and Donoghue, 2013; Landis et al., 2021a). Evidence from global tropical grasslands shows that many lineages were able to colonize the biome over the past 10 million years from other biomes (Simon et al., 2009; Maurin et al., 2014), indicating that perhaps some biomes are more open to outsiders (i.e., non-native or pre-adapted lineages; Edwards and Donoghue, 2013; Donoghue and Edwards, 2014). INTRODUCTION Such evolutionary biome switching reﬂects that some environmental gradients (i.e., biome borders) are more easily crossed than others, perhaps due to the ease at which adaptations to these gradients can be acquired (Pennington et al., 2006; Simon and Pennington, 2012). The intermediate disturbance hypothesis (Connell, 1978) posits that if a disturbance is not too extreme, many plant lineages may already have or can evolve traits required to survive it, but the more extreme the disturbance (e.g., extreme drought and extreme cold or extreme heat), an increasingly small number of species will have these traits because they are hard to evolve. Quantitative evidence across ﬂoras is now needed to understand the relative roles of niche conservatism and the species-environment interactions (environmental ﬁltering) across ecological gradients, particularly in the Neotropics where much biome complexity is found (Pennington et al., 2009; Hughes et al., 2013; Dexter et al., 2018; Silva-de-Miranda et al., 2018). Most recently, a global-scale conceptual view of biomes has been proposed which considers biomes as the conﬂuence of ecology, evolution, and biogeography (Pennington et al., 2009, 2018; Oliveira-Filho et al., 2013a,b; Moncrieﬀet al., 2016; Pennington and Lavin, 2016; Mucina, 2019, 2020; Nürk et al., 2020; Ringelberg et al., 2020). While there is a consensus in ecology and biogeography that biomes should be deﬁned based on physiognomy and functional aspects (Mucina, 2019; Pennington et al., 2018), an evolutionary dimension emphasizes the processes that have led to current biome distributions. This concept deﬁnes biomes as “evolutionary theaters” within which lineages interact and evolve through time, and as meta-communities regulated by community assembly at large spatial scale (Pennington et al., 2009; Oliveira-Filho et al., 2013b; Pennington and Lavin, 2016). The concept has emerged partly in response to increasing evidence for the prevalence of phylogenetic niche conservatism (Crisp et al., 2009; Pennington et al., 2009; Oliveira-Filho et al., 2013a,b; Kerkhoﬀet al., 2014; Gagnon et al., 2019; Ringelberg et al., 2020; Segovia et al., 2020). This tendency of plant lineages to inherit their overall ancestral environmental niches is based upon evidence that many plant lineages have dispersed across large distances over evolutionary timescales yet occupy similar ecological conditions. The general lack of dispersal limitation and diﬃculty of accruing novel environmental adaptations had led to the popularity of the phrase that for plants, it is “easier to move than evolve” (Donoghue, 2008). Frontiers in Ecology and Evolution \| www.frontiersin.org INTRODUCTION If phylogenetic niche conservatism and environmental ﬁltering have shaped the macroevolutionary patterns of ﬂoristic and functional diversity that make up the evolutionary theaters or biomes, then exploring variation in taxonomic, functional trait, and community phylogenetic data may help delimiting more biologically meaningful biomes that are globally comparable. While biomes deﬁned exclusively by individual, distinct operational criteria will result in diﬀerent biome maps ﬁt for diﬀerent purposes such as comparative ecology and global change research (Conradi et al., 2020), comparisons of biomes delimited under diﬀerent or combinations of criteria remain rare, particularly across geologically and ecologically complex biogeographic regions like the Caatinga domain of northeast Brazil (NE Brazil, Figure 1) (Queiroz et al., 2017; Fernandes et al., 2020). That plant phylogenies are often more structured ecologically than geographically suggests that ecological gradients are evolutionarily important (Crisp et al., 2009; Oliveira-Filho et al., 2013a). The concept of phylogenetic niche conservatism is strongly linked to environmental ﬁltering, i.e., the process whereby environmental gradients act as strong ﬁlters for species distributions (Cavender-Bares et al., 2009; Hardy et al., 2012; Blonder et al., 2015). For example, a previous study across plant lineages has shown that environmental ﬁltering has played an important role in shaping the ﬂora of the Galapagos Islands (Carvajal-Endara et al., 2017). In the presence of environmental ﬁltering, lineages cannot successfully establish unless they have traits that leave them pre-adapted to pass environmental ﬁlters, The Caatinga region is often treated in biodiversity, evolutionary, conservation, and biogeographical studies of plants and animals as a single, homogeneous unit, generally termed a “biome” (Instituto Brasileiro de Geograﬁa e Estatística [IBGE], 2012; The Brazil Flora Group [BFG], 2015; Garda et al., 2017; Mesquita et al., 2017; Araujo and Silva, 2017; Antonelli et al., 2018; Manhães et al., 2018; Nascimento et al., 2018; Silva and Souza, 2018; Medeiros et al., 2019; Prieto-Torres et al., 2019; Souza-e-Silva et al., 2019; Correia et al., 2020; December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 2 Biologically Meaningful Biomes Cardoso et al. FIGURE 1 \| The main biogeographical regions of northeast Brazil (including the state of Minas Gerais) where three biomes predominate: the geographically disjunct Amazonian and Atlantic rainforests [(A), green; (D), blue], the succulent-rich Caatinga seasonally dry tropical forests [(B), red], and the grass-rich, ﬁre-prone savannas of the Cerrado [(C), yellow]. INTRODUCTION For geographic reference, the whole north and northeastern borders of the inset are the Atlantic Ocean. Photos by PWM (A) and DC (B–D). FIGURE 1 \| The main biogeographical regions of northeast Brazil (including the state of Minas Gerais) where three biomes predominate: the geographically disjunct Amazonian and Atlantic rainforests [(A), green; (D), blue], the succulent-rich Caatinga seasonally dry tropical forests [(B), red], and the grass-rich, ﬁre-prone savannas of the Cerrado [(C), yellow]. For geographic reference, the whole north and northeastern borders of the inset are the Atlantic Ocean. Photos by PWM (A) and DC (B–D). Dória and Dobrovolski, 2021). However, this deﬁnition of the Caatinga as a “biome” is in conﬂict with the more generally accepted deﬁnition of a biome at a global scale; it is in fact a biogeographic region. Whilst the Caatinga region may have a broadly similar, seasonally dry climate, it includes interdigitating, distinct biomes (as deﬁned and recognized at global scale) such as rainforest and ﬁre-prone savannas within a predominant matrix of Caatinga seasonally dry tropical forests (SDTF biome). The use of maps with a geographic delimitation of the Caatinga as a “biome” may impact upon downstream analyses aimed at disentangling the ecological and historical drivers that have shaped the evolutionary trajectories of all Caatinga species diversity (Queiroz, 2006; Cardoso and Queiroz, 2010; Queiroz et al., 2017; Guedes et al., 2014). It may also impact assessing priority areas for conservation in the severely impacted SDTF biome across Brazil, which it is not just conﬁned to the Caatinga, but is found across the Cerrado and Pantanal regions (DRYFLOR, 2016; the Cerrado and Pantanal are also termed “biomes” by IBGE). Dória and Dobrovolski, 2021). However, this deﬁnition of the Caatinga as a “biome” is in conﬂict with the more generally accepted deﬁnition of a biome at a global scale; it is in fact a biogeographic region. Whilst the Caatinga region may have a broadly similar, seasonally dry climate, it includes interdigitating, distinct biomes (as deﬁned and recognized at global scale) such as rainforest and ﬁre-prone savannas within a predominant matrix of Caatinga seasonally dry tropical forests (SDTF biome). Frontiers in Ecology and Evolution \| www.frontiersin.org INTRODUCTION The use of maps with a geographic delimitation of the Caatinga as a “biome” may impact upon downstream analyses aimed at disentangling the ecological and historical drivers that have shaped the evolutionary trajectories of all Caatinga species diversity (Queiroz, 2006; Cardoso and Queiroz, 2010; Queiroz et al., 2017; Guedes et al., 2014). It may also impact assessing priority areas for conservation in the severely impacted SDTF biome across Brazil, which it is not just conﬁned to the Caatinga, but is found across the Cerrado and Pantanal regions (DRYFLOR, 2016; the Cerrado and Pantanal are also termed “biomes” by IBGE). from nearby and interdigitating ecologically and evolutionarily distinct rainforest and savanna biomes and occupies a unique environmental space. We use the three distinct data sources to measure the degree to which the Caatinga SDTF biome diﬀers ﬂoristically, phylogenetically, and in functional trait composition, as ﬁrst step to understand the ease at which these diﬀerences can and have been traversed adaptively by plant lineages through time (Wiens et al., 2010; Crisp and Cook, 2012; Donoghue and Edwards, 2014). We use standard clustering algorithms, which in a geographic context have been termed regionalization approaches (Kreft and Jetz, 2010; Linder et al., 2012; Vilhena and Antonelli, 2015; Daru et al., 2017), to delimit clusters. Several authors have used biogeographic regionalization methods on a relatively similar (Linder et al., 2012; Fayolle et al., 2018; Aleman et al., 2020) or even larger scales (e.g., Kreft and Jetz, 2010; Holt et al., 2013; Vilhena and Antonelli, 2015; Ficetola et al., 2017) and argued that the results are comparable to biomes (Vilhena and Antonelli, 2015; Aleman et al., 2020). However, while extremely useful, previous regionalizations have been based on only a single type of data (e.g., ﬂoristic data; Linder et al., 2012) so cannot encompass all aspects of biomes. Our approach diﬀers because we use three independent regionalizations, each based upon a diﬀerent type of data (ﬂoristic, functional, and phylogenetic). By both combining and comparing the results of these regionalizations, Here we explore how taxonomic, functional, and community phylogenetic data can be used to delimit biomes in NE Brazil and explore how biomes deﬁned in these diﬀerent ways are shaped by climatic variables. INTRODUCTION We aim to demonstrate that a Caatinga SDTF biome, lying at the extremely dry end of the tropical seasonality and rainfall gradient of NE Brazil, greatly diﬀers December 2021 \| Volume 9 \| Article 723558 3 Biologically Meaningful Biomes Cardoso et al. homogenous in ﬂoristic, functional, and phylogenetic space, so it was important to include areas of all biomes that surround the Caatinga (i.e., areas known to diﬀer in these respects). According to the Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012) classiﬁcation, this includes the Cerrado (Brazilian savanna), Mata Atlantica (Brazilian Atlantic rainforest), and Amazonia (Amazon rainforest). Our study area has an area of 2.144 million km2 and includes a buﬀer of at least 200 km around the IBGE deﬁnition of the Caatinga in all directions (Figure 1). we argue that our results approach for the ﬁrst time a data-driven and repeatable biome map that considers all facets of agreed biome deﬁnitions. MATERIALS AND METHODS Our approach diﬀers from classical and modern approaches that fall under the umbrella of “biogeographical regionalization” or “bioregionalization” (e.g., Kreft and Jetz, 2010; Linder et al., 2012; Holt et al., 2013; Vilhena and Antonelli, 2015; Daru et al., 2016, 2017; Edler et al., 2017; Ficetola et al., 2017). Most such analyses are essentially focused on understanding the signature of historical (i.e., geographic barriers) processes in explaining the spatial distribution of speciﬁc “groups” of organisms across geographically conﬁned areas, rather than delimiting biomes that are applicable across the tree of life. Those that do include broader ranges of taxa still rely only on ﬂoristic (e.g., Linder et al., 2012) or phylogenetic (e.g., Daru et al., 2017) data to classify the “bioregions,” rather than attempting to integrate ﬂoristic, functional, and phylogenetic data. The resulting areas are more akin to “biogeographic regions” (sensu Wallace, 1876; Holt et al., 2013) than “biomes.” In our study, biomes were delimited by comparing and combining independent regionalization analyses based upon ﬂoristic, functional, and phylogenetic data. We therefore consider the impact of geography and the evolutionary and functional distinctiveness of areas, as well as how the environment [here incorporated indirectly by the use of Species Distribution Models (SDMs)] deﬁnes where lineages are conﬁned. We consider the results of our analyses biologically meaningful biomes. Species Distribution Modeling Species Distribution Modeling Climatic predictors were derived from remotely sensed temperature (MODIS; Wan and Dozier, 1996; Wan, 2014), rainfall (CHIRPS; Funk et al., 2014), and cloud cover data (MODCF; Wilson and Jetz, 2016). These data are calibrated with data from ground weather stations and outperform those derived from ground data alone (e.g., WorldClim) for SDMs (Deblauwe et al., 2016). All data were downloaded at a 0.05◦resolution (c. 5.5 km2 at the equator). Edaphic data were derived from the SoilGrids 250 m database (accessed February 2017)4 interpolated to a 0.05◦resolution. Edaphic factors are believed to be important in determining species distributions in NE Brazil (Queiroz et al., 2017) and have been shown to increase SDM performance in the tropical Americas (Figueiredo et al., 2017; Moulatlet et al., 2017; Species Distribution Data The ongoing Flora do Brasil 2020 project (2021)1 provides a robust taxonomic framework to work in the region with currently 16,351 species of ﬂowering plants recorded for NE Brazil (Flora do Brasil 2020, 2021). A relatively large amount of species occurrence data is available for the region (Supplementary Figure 1) thanks to two dynamic network of local herbaria across Brazil available through CRIA speciesLink2 and the Reﬂora specimen database3. In this study, we attempted to produce an SDM for every angiosperm species with recorded distributions in NE Brazil (Reﬂora, 2021) using data from CRIA speciesLink and the Reﬂora specimen database. While many NE Brazilian species have distributions outside Brazil, we only included Brazilian specimen records due to diﬃculties matching taxonomic backbones across countries. We used the latest version of Reﬂora (2021) to harmonize the taxonomy of the two specimen databases and update synonymy. Data were cleaned in six stages as described in Appendix S2 of Moonlight et al. (2020), which were designed to remove misidentiﬁed specimens and those with coordinate errors. We addressed environmental bias in occurrence data using spatial ﬁltering by retaining a single specimen record within each 10 km radius for every species following Kramer- Schadt et al. (2013). We retained and attempted to model all species with ≥5 records collected in diﬀerent grid cells at a 0.05◦resolution, resulting in 296,439 unique occurrence records for 9,134 species. Floristic data were based on thresholded, statistically signiﬁcant SDMs for 3,457 ﬂowering plant species, which were also included in a molecular phylogeny. SDMs were used to estimate species lists for all grid cells across the study area. Functional and phylogenetic data for each grid cell were based on seven functional traits and a community phylogeny generated for all the species present for the study area, respectively. Regionalization analyses were carried out using hierarchical clustering based upon ﬂoristic, functional, and phylogenetic distance among all cells. All three distance matrices (ﬂoristic, functional, and phylogenetic) were summed and used to delimit “total evidence” biomes. Study Area Four and ﬁve axes were selected, which each explained >80% of variation (see Appendix S4 of Moonlight et al., 2020). This process reduces the number of explanatory variables, thus minimizing collinearity (Dormann et al., 2013) and model overﬁtting (Peterson et al., 2007) while maximizing the explanatory data available for modeling. SDMs for 9,134 were run using MaxEnt v.3.3.3 in the R package “dismo” (Hijmans et al., 2017). MaxEnt was chosen because it has been shown to outperform other SDM methodologies, particularly when species have few distribution points. MaxEnt was used with the default settings, with 5-fold cross-validation, and all feature classes allowed. Background data (also known as pseudo-absence data) were sampled from a unique area for each species, consisting of NE Brazil plus circles of 250 km around each species’ known occurrence points. This was a compromise between predicting species into areas not covered by background points, providing a large number of climatically unsuitable points (Anderson and Raza, 2010), and including a biologically realistic extent for each species’ model. We controlled for bias in sampling eﬀort (Stolar and Nielsen, 2015) by selecting 10,000 background points for each species using an Epanechnikov kernel (Wiegand and Moloney, 2013) calculated from all angiosperm presence data for Brazil. g We evaluated model performance using the Continuous Boyce Index (CBI; Hirzel et al., 2006). This evaluation index that relies upon presence and pseudo-absence data is based upon the Boyce Index (Boyce et al., 2002), calculated with code available at https: //rdrr.io/github/adamlilith/enmSdm/man/contBoyce.html. CBI has been shown to be less stochastic to variation at low numbers of presence points than alternative indices (Hirzel et al., 2006). A CBI of ≥0 indicates that a model is better than chance and from the SDMs of 9,134 species we retained all models with a mean CBI of ≥0.25 over the ﬁve replicates (6,823 species, 75%, see Supplementary Table 3). Retained replicates were summed and converted into binary presence-absence maps using the 10th percentile logistic threshold. This was chosen because it was the strictest of the commonly used threshold values, so it limits the well-documented over-estimated of summed SDMs. To maximize the compatibility of analyses, we retained models only for species included in the community phylogeny (see section “Phylogenetic Delimitation” below) for downstream analyses (3,457 species). Predicted species lists were estimated for every 0.05◦grid cell in NE Brazil, which were aggregated to a 0.25◦resolution due to computation constraints. 5https://www.ncbi.nlm.nih.gov/genbank/ Study Area We applied our biome delimitation approaches to NE Brazil because of our long-term experience working on the taxonomy, distribution, ecology, and evolution of ﬂowering plants in the region (e.g., Rocha et al., 2004; Queiroz, 2006; Queiroz et al., 2010, 2017; Särkinen et al., 2011; Santos et al., 2012; Cardoso et al., 2014; Fernandes et al., 2020; Moonlight et al., 2020). We deﬁned our study area as NE Brazil (including the state of Minas Gerais) in order to include all areas deﬁned as the Caatinga by Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012) and alternative biome classiﬁcations (e.g., Queiroz et al., 2017; Silva-de-Miranda et al., 2018; Moonlight et al., 2020). We have a particular interest in identifying “core” areas that are relatively 1http://ﬂoradobrasil.jbrj.gov.br/ 2http://www.splink.org.br/ 3http://ﬂoradobrasil.jbrj.gov.br/reﬂora/herbarioVirtual/ 4https://soilgrids.org/ December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 4 Biologically Meaningful Biomes Cardoso et al. grid cells and not nestedness (Baselga, 2010). The ﬂoristic distance matrix was used in unbiased cluster analysis, where the row order of the distance matrix was randomized 100 times using the “recluster” package in R (Dapporto et al., 2013, 2015). RogueNaRok (Aberer et al., 2012; Available at: https://rnr.h-its.org/) was used to identify rogue grid cells responsible for reducing resolution in the resulting 50% majority rule consensus dendrogram. A total of 138 rogue grid cells were identiﬁed and removed. Clusters were mapped based upon a process of reciprocal illumination following Moonlight et al. (2020) and analogous to the approaches taken by similar analyses (Silva-de-Miranda et al., 2018; Moonlight et al., 2020). Clusters were labeled based on comparing the mapped distribution of sub-clusters to our biological knowledge of the vegetation patterns in NE Brazil. Our priorities were to delimit clusters that could be matched with conﬁdence to four biomes recognized by the Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012) classiﬁcation, and to maximize similarity with the mapped clusters from the phylogenetic and functional biome classiﬁcations (see below). We acknowledge that this approach is not fully repeatable, but argue that it is the best currently available considering that alternative approaches (e.g., k-means clustering, Amaral et al., 2017) also rely upon prior knowledge to deﬁne an expected number of biomes. We term this approach “ﬂoristic regionalization.” Rapini et al., 2021). Climatic and edaphic predictors (35 and 55, respectively) were converted into two independent principal component analysis (PCA) axes. Study Area Presence in a species list was assigned based upon predicted presence in any constituent cell at the original 0.05◦resolution. Phylogenetic Delimitation To delimit clusters based upon phylogenetic distances between plant communities (phylogenetic regionalization), we produced a novel community phylogeny for the ﬂora of NE Brazil based on DNA sequences mostly downloaded from GenBank (Benson et al., 2017). We attempted to download sequence data for all 16,351 species recorded in NE Brazil by Flora do Brasil 2020 (2021) for the following regions: matK, atpB, ndhF, rbcL, and trnL. Regions were chosen based upon: (i) wide use across angiosperms; (ii) ease of alignment across angiosperms; (iii) adequate level of sequence variation across orders, families, and genera. To augment our species sampling in the community phylogeny, we have newly generated 445 matK and 444 rbcL sequences from herbarium and ﬁeld-collected leaf tissues preserved through silica gel desiccation of 546 species. DNA extraction, PCR ampliﬁcation, and robotic sequencing largely followed standard protocols of DNA barcoding for community phylogenetics (e.g., Kress et al., 2009). The newly generated DNA sequences are publicly available in GenBank (see Supplementary Table 1 for voucher and accession number details)5. The R package “rentrex” (Winters, 2017) was used to query GenBank, specifying a sequence length of 500–5,000 base pairs. For species in NE Brazil for which no sequence data were available in GenBank, we repeated the steps above to locate sequences for congeners from outside of Brazil (for genera with one or two species within NE Brazil) because any one or two species for these genera would provide the same phylogenetic distance in our analyses as those present in NE Brazil. Alignments Combined Cluster Delimitation Combined analyses of ﬂoristic, phylogenetic, and functional distance matrices were run to delimit “total evidence” clusters based on all three approaches. All possible combinations of the three approaches were also run to see subsets of results (i.e., ﬂoristic + phylogenetic, ﬂoristic + functional, and phylogenetic + functional). Distance matrices were scaled from 0 to 1 to give equal weight to each matrix before being summed, so the distance matrix values could range from 0 to 2 in analyses with two approaches, or 0 to 3 in analyses with three approaches. Clusters were estimated, mapped, and named following the hierarchical clustering method described under section “Floristic Delimitation.” The results of the “total evidence” clusters are named herein as biomes because they are the result of three, independent lines of evidence. No rogue grid cells were removed. Phylogenetic distances between all grid cells in NE Brazil were calculated by estimating the phylogenetic β-diversity among estimated species lists. The phylogenetic Simpson’s index was used following Chave et al. (2007) because it is comparable with β-sim (see above). Phylogenetic regionalization was carried out following the hierarchical clustering method described above under section “Floristic Delimitation.” No rogue grid cells were identiﬁed and removed. Comparison of Regionalizations: Clusters as Biomes Comparisons of regionalizations based on individual approaches (functional, phylogenetic, and ﬂoristic) were carried out in both geographic and environmental space to highlight areas of “core” biomes (areas where all regionalizations analyses agreed on biome identity) and transition zones (areas where regionalizations diﬀered between analyses). “Core” biome areas were visualized by highlighting areas of agreement between the three individual regionalizations (e.g., “core Caatinga”). Transitional biome areas were visualized by highlighting areas of disagreement where one or two approaches showed disagreement regarding biome distribution (e.g., “transitional Caatinga”). A raster ﬁle depicting the core Caatinga and associated transition zones is available as Supplementary Figure 5. Cluster Delimitation Floristic Delimitation To delimit clusters based upon ﬂoristic data (ﬂoristic regionalization), a distance matrix was computed based on β-diversity (Simpson’s dissimilarity: β-sim) in the R package “betapart” (Baselga et al., 2018). This approach was chosen because it measures ﬂoristic turnover (i.e., dissimilarity) between December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 5 Biologically Meaningful Biomes Cardoso et al. were done in Maﬀt v.7.450 for each DNA region with default settings with a maximum of six iterations performed per region. Data cleaning was done by identifying poorly aligned sequences based on visual assessment, using Vsearch v2.14.2 to identify highly variable sequences with <40% sequence similarity, and by identifying misplaced species based on neighbor-joining trees run for each DNA region with FastTree v2.1.10 (Price et al., 2010). Replacements for any species removed during the cleaning were searched in GenBank if alternative sequences were available. The ﬁnal cleaned community phylogeny contained 10,279 sequences from GenBank and 662 newly generated sequences for a total of 6,296 species (Supplementary Table 2). All DNA regions were combined and analyzed using RaxML-HPC2 on XSEDE on the CIPRES Science Gateway v.3.3 on-line portal (Miller et al., 2010)6. The phylogeny was rooted with Nymphaeales. The community phylogeny included 6,296 species from 209 families and 1,775 genera (Supplementary Figure 2). The relationships among them were consistent with the phylogeny of ﬂowering plants (The Angiosperm Phylogeny Group et al., 2016). A total of 3,457 species from 184 families and 1,325 genera in the phylogeny were also included in the SDMs so were retained for downstream analyses. of a trait, which leads to overestimation of functional similarity at broad spatial scales where almost all grid cells had at least one predicted species with every trait. Functional regionalization was carried out following the hierarchical clustering method described above under section “Floristic Delimitation.” No rogue grid cells were identiﬁed and removed. 6www.phylo.org Functional Delimitation To delimit clusters based upon functional distances between plant communities (functional regionalization), we scored seven independent plant traits for all 3,457 species for which SDMs were generated and which were included in the community phylogeny (100% coverage for 3,457 species for all seven traits). These included 931 species scored by Moonlight et al. (2020). Traits were chosen on the basis of: (i) that they were simple and unambiguous to score from herbarium specimens or literature; and (ii) had hypothesized links with environmental and functional diﬀerences among biomes in NE Brazil. The seven traits chosen were: (i) latex; (ii) corky bark; (iii) spines; (iv) compound leaves; (v) nitrogen nodulation; (vi) Crassulacean acid metabolism (CAM) photosynthesis; and (vii) C4 photosynthesis (Supplementary Table 2). The literature used to score traits is detailed in Appendix S7 of Moonlight et al. (2020). To investigate whether core biomes overlapped in environmental space, environmental comparison of biome delimitations was achieved by plotting clusters from diﬀerent analyses in environmental space based on mean annual temperature (bio1) and rainfall data (bio12) extracted for each grid cell. The bio1 and bio12 values for each biome grid cell from each analysis were summed. The mean annual temperature (bio1) from MODIS temperature data (Wan and Dozier, 1996; Wan, 2014) and mean annual rainfall (bio12) from CHIRPS rainfall data (Funk et al., 2014) were used. Functional distance matrices between all grid cells in NE Brazil were created based upon estimated species lists. Euclidean distances of grid cells in 6-dimensional trait space based on the proportion of species with each trait in each grid cell was used for measuring functional distance. Published functional diversity metrics (e.g., Ricotta et al., 2016) were found inappropriate for measuring functional distance between plant communities because they are based on the presence rather than the proportion RESULTS Major Biomes Across NE Brazil Major Biomes Across NE Brazil All three individual regionalizations identiﬁed two clusters of Caatinga SDTF, two clusters as Mata Atlantica, and three clusters as Cerrado (Figures 2A–C). The functional analysis resulted in the identiﬁcation of an additional cluster within both the Mata Atlantica and Cerrado, and an extra cluster that we were unable December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 6 Biologically Meaningful Biomes Cardoso et al. FIGURE 2 \| Cluster classiﬁcations across northeast Brazil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (A–C) and in combination (D–F): (A) functional data; (B) phylogenetic data; (C) ﬂoristic data; (D) functional and phylogenetic data; (E) phylogenetic and ﬂoristic data; (F) functional and ﬂoristic data; and (G) functional, phylogenetic, and ﬂoristic data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). azil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (A–C) and etic data; (C) ﬂoristic data; (D) functional and phylogenetic data; (E) phylogenetic and ﬂoristic data; (F) FIGURE 2 \| Cluster classiﬁcations across northeast Brazil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (A–C) and in combination (D–F): (A) functional data; (B) phylogenetic data; (C) ﬂoristic data; (D) functional and phylogenetic data; (E) phylogenetic and ﬂoristic data; (F) functional and ﬂoristic data; and (G) functional, phylogenetic, and ﬂoristic data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). to assign to any recognized biome (Figure 2A). The combined “total evidence” analyses of all three approaches suggested seven major clusters based on functional, phylogenetic, and ﬂoristic data (Figure 2G). identify most of this area as Mata Atlantica but the phylogenetic regionalization identiﬁes most of these areas as Cerrado (Figures 2, 3). Comparison of clusters delimited by single approaches shows that clusters delimited by ﬂoristic and phylogenetic approaches are highly similar both in spatial extent and number of major clusters where both analyses resolve seven major clusters in largely similar areas across NE Brazil (Figures 2, 3). Functional clusters show largest diﬀerences compared to phylogenetic and ﬂoristic clusters, and resolve ten major clusters indicating higher resolution of functional data for biome delimitation within NE Brazil, enabling further splitting of vegetation types as Differences Between Approaches The areas where diﬀerences between the three regionalizations are seen can be considered as transitional areas (Figure 3 and Supplementary Figure 4). Most disagreement between the three individual regionalizations is seen in the southern part of the Caatinga biogeographic domain in the Chapada Diamantina area (Figures 2, 3). Functional and ﬂoristic regionalizations December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org Biologically Meaningful Biomes Cardoso et al. FIGURE 3 \| Areas of agreement and disagreement among biome delimitations across northeast Brazil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (see Figures 2A–C). Areas of agreement are shown as “core” clusters (A–C) or biomes (D) and areas of disagreement are shown as “transitional zones.” (A) functional and phylogenetic data; (B) phylogenetic and ﬂoristic data; (C) functional and ﬂoristic data; and (D) functional, phylogenetic, and ﬂoristic data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). Alternative plots showing the identity of transitional zones are given in Supplementary Figure 4. FIGURE 3 \| Areas of agreement and disagreement among biome delimitations across northeast Brazil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (see Figures 2A–C). Areas of agreement are shown as “core” clusters (A–C) or biomes (D) and areas of disagreement are shown as “transitional zones.” (A) functional and phylogenetic data; (B) phylogenetic and ﬂoristic data; (C) functional and ﬂoristic data; and (D) functional, phylogenetic, and ﬂoristic data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). Alternative plots showing the identity of transitional zones are given in Supplementary Figure 4. Caatinga within the driest and hottest areas of NE Brazil (Figure 4A). These areas are dominated by a ﬂoristically, functionally, and phylogenetically distinct ﬂora adapted to dry conditions with seasonal rainfall. This “core” Caatinga area is found in areas with 353–1,271 mm annual rainfall and with a mean annual temperature from 19.6 to 28.0◦, and does not overlap with other core biomes in environmental space (Figure 4A). The ﬂora of the “core” Caatinga is characterized by a high proportion of species that are succulent, nodulating (Figure 4B), and with spines but without corky bark (Figure 4C). The low rainfall in the “core” Caatinga is notable. Differences Between Approaches Dry forests in the Americas are found in areas with up to 1800 mm rainfall (DRYFLOR, 2016; Dexter et al., 2018) but none of the “core” Caatinga dry forests approach this threshold, despite the prevalence of areas with higher rainfall in the wider study area of NE Brazil. functionally distinct clusters (Figures 2, 3 and Supplementary Figure 3). This is despite our functional classiﬁcation being based on only seven categorical traits. The “Core” Caatinga In terms of the SDTF biome, all three analyses identiﬁed two groups as Caatinga SDTF (Figures 2A–C). A large area within NE Brazil is identiﬁed here as the “core Caatinga” biome supported by all three regionalizations (functional, phylogenetic, and ﬂoristic) (Figure 4). Areas surrounding the “core” Caatinga are identiﬁed as “transitional Caatinga” (Figure 4): these include areas supported as Caatinga by one or two regionalizations but not all three. Transitional Caatinga is found across the western and southern borders of the “core” Caatinga, much less so along the eastern side along the boundary with the coastal Mata Atlantica domain (Figures 3, 4). Floristic and phylogenetic data support transitional Caatinga in the South and West of the “core” Caatinga, but functional data identify these areas as savanna-like Cerrado (e.g., Chapada do Araripe; Figures 2, 3). In common with the individual analyses, all mapped Caatinga groups were largely congruent with the Caatinga domain along its eastern border but included several diﬀerences along the eastern border, which are discussed in detail below. DISCUSSION Core Biomes and Transition Zones Frontiers in Ecology and Evolution \| www.frontiersin.org Core Biomes and Transition Zones Biomes have been delimited in various ways, including based on the spatial distribution of physiognomic, ﬂoristic or functional discontinuities amongst plant communities (Mucina, 2019) or even dynamic global vegetation biome modeling involving the Our integrated biome delimitation using a combination of ﬂoristic, functional, and phylogenetic data identiﬁes the “core” December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 8 Biologically Meaningful Biomes Cardoso et al. FIGURE 4 \| Distribution of the Caatinga seasonally dry forest biome within NE Brazil in geographic, environmental (A) and trait (B,C) space. Colors indicate areas delimited as Caatinga in no analyses (gray), one or two analyses (“transitional Caatinga,” yellow and orange), and three analyses (“core” Caatinga, red). The black line in the map indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). ombination of physical environment, plant functional types, hysiology, and biochemical ﬂuxes (Prentice et al., 1992; Kaplan growth form and canopy structure (Woodward et al., 2004) ﬂoristic maps have focused on dominant plant families, genera FIGURE 4 \| Distribution of the Caatinga seasonally dry forest biome within NE Brazil in geographic, environmental (A) and trait (B,C) space. Colors indicate areas delimited as Caatinga in no analyses (gray), one or two analyses (“transitional Caatinga,” yellow and orange), and three analyses (“core” Caatinga, red). The black line in the map indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). FIGURE 4 \| Distribution of the Caatinga seasonally dry forest biome within NE Brazil in geographic, environmental (A) and trait (B,C) space. Colors indicate areas delimited as Caatinga in no analyses (gray), one or two analyses (“transitional Caatinga,” yellow and orange), and three analyses (“core” Caatinga, red). The black line in the map indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística (IBGE) (2012). combination of physical environment, plant functional types, physiology, and biochemical ﬂuxes (Prentice et al., 1992; Kaplan et al., 2003). Physiognomic delimitations have followed plant growth form and canopy structure (Woodward et al., 2004); ﬂoristic maps have focused on dominant plant families, genera, and species and the associated ecological correlates (e.g., in December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 9 Biologically Meaningful Biomes Cardoso et al. Core Biomes and Transition Zones These diﬀerent approaches based upon diﬀerent types of data produce diﬀerent biome maps, but no map produced using a single type of data can possibly satisfy all aspects of the multifaceted deﬁnition of a biome expected to have shaped their biodiversity over evolutionary time, i.e., delimited biomes may not be ﬂoristically, functionally, or ecologically distinct. Our approach diﬀers in two ways from previous methods depicting global or regional distribution of biomes, including the Caatinga (e.g., Whittaker, 1970; White, 1983; Schrire et al., 2005; Instituto Brasileiro de Geograﬁa e Estatística [IBGE], 2012; Conradi et al., 2020; Ringelberg et al., 2020): ﬁrstly, we identify “core” biome areas supported by three distinct lines of evidence (ﬂoristic, functional, and phylogenetic), and secondly, we identify transition zones that are supported by distinct data sources that point to biologically important areas of transition previously neglected by most biome maps. Our integrated biome analyses are able to highlight “core” areas where all three data sources (ﬂoristic, functional, and phylogenetic) agree on the distribution of the same biome (Figure 4). We suggest that such areas of congruence based on multiple lines of evidence may be of particular interest to biology and earth system science. ( ) While comparative analyses have employed multiple taxa to interrogate biomes, other approaches seek to identify key taxa or functional groups that can be used to delimit biomes. One such technique was employed by Ringelberg et al. (2020), who used modeled distributions of a single functional group (i.e., stem succulents) to map the “succulent biome” (Schrire et al., 2005; Ringelberg et al., 2020). The results do not adequately capture the biome complexity in Brazil. For example, Ringelberg et al. (2020) delimit not just the Caatinga as succulent biome, but also parts of the Chaco and the campos rupestres of the Chapada Diamantina, which are ecologically, historically, and functionally distinct biomes (Pennington et al., 2000; DRYFLOR, 2016; Silva-de-Miranda et al., 2018; Rapini et al., 2021). Indeed, we demonstrate that not just the “core” Caatinga but also transitional and non-Caatinga areas have high proportions of stem succulents (Figure 4B). Such single-criterion approaches to biome delimitation do not diﬀer much from an operational view of biomes (Conradi et al., 2020). If biomes are to be deﬁned as evolutionary theaters, the distributions of as general group of taxa as possible should be examined in conjunction with their phylogenetic relationships and functional characteristics. Core Biomes and Transition Zones diversiﬁed under a regime of regular and prolonged drought and without the inﬂuences of ﬁre and other physical disturbances (Schrire et al., 2005; Pennington et al., 2009; Ringelberg et al., 2020). The climate space of the global succulent biome as recently modeled by the distribution of stem succulents involves a mean annual rainfall that closely matches that of South American SDTFs (Ringelberg et al., 2020), which also have a high proportion of stem succulents (DRYFLOR, 2016; Queiroz et al., 2017). Also at intercontinental scales, Segovia et al. (2020) underlines the structuring of evolutionary diversity of trees in the Neotropics along precipitation gradients. Precipitation- related bioclimatic variables were singled out as the most important precipitation measures predicting the succulent biome with a MaxEnt approach to large-scale biome distribution modeling of South American SDTFs (Särkinen et al., 2011). These same precipitation variables were found by Oliveira- Filho et al. (2013a) to be most important in distinguishing the succulent (including the Caatinga) and savanna (including the Cerrado) biomes. The analysis of community phylogenetic distances and the biome assignments of 466 ﬂoristic sites across eastern Brazil were best explained, of all the bioclimatic variables, by only annual precipitation at a threshold of <1,200 mm (Oliveira-Filho et al., 2013a). This ecological structure reﬂects the link between annual precipitation and phylogenetic niche conservatism (Oliveira-Filho et al., 2013a; Segovia et al., 2020). Substrate conditions were shown to be ecologically signiﬁcant (with or without water-holding capacity), but this ecological variable may not mask the more important inﬂuence of annual precipitation as explanatory of the community phylogenetic structure (Oliveira-Filho et al., 2013a). Africa, White, 1983; Power et al., 2017; Aleman et al., 2020; South America, Silva-de-Miranda et al., 2018); and functional systems on the presence of functional groups, such as evergreen trees found in the tropical rainforest biome, succulents in the seasonally dry tropical forest or succulent biome, and a continuous grass layer in savannas (Whittaker, 1970; Scholes and Archer, 1997; Schrire et al., 2005). Additionally, strictly environmental-based delimitations of biomes based on climate, edaphic composition or degree of ﬁre disturbance have provided important insights into our understanding of the ecological limits driving the assembly of plant communities (Archibald et al., 2013; Langan et al., 2017). Core Biomes and Transition Zones Our integrated ecological and evolutionary approach involving multiple taxa across all growth forms and associated measurements of functional and phylogenetic diversity seem to better describe “core” evolutionary arenas. Ultimately, the identiﬁcation of biologically relevant areas should depend upon its intended purpose. If you are interested in the response of the Caatinga to climate change, for example, you should focus on the areas that independent data sources agree are “core” Caatinga (Figure 4), not areas where data sources disagree (i.e., transition zones), where the species, ecological function, or phylogenetic diversity may overlap that of other biomes. Likewise, if you wish to measure the functional traits of a species, you measure the traits of an individual at the “core” of the species concept, not a hybrid of dubious identiﬁcation. Also, because the transition zones may be particularly dynamic or vulnerable to climate change (i.e., “zones of tension,” Clements, 1905), if your interest is in conserving the maximum species diversity and ecosystem function of Caatinga dry forests in the face of climate change, it may be best to model a set of species/geographic area/set of traits that all data agree are Caatinga. Transitional Biome Areas The identity of clusters identiﬁed in the functional analysis that could not be linked to any previously indicated biomes is unclear. They might reﬂect that the functional data is capturing the complexity with respect to the high within- biomes habitat heterogeneity across NE Brazil. For example, each of the most predominant savanna, rainforest, and the “core” Caatinga seasonally dry forest biomes are not physiognomically, ﬂoristically, and edaphically homogeneous, rather they also exhibit highly specialized habitats. Just in the “core” Caatinga, plant communities seem to have been evolutionarily structured by major soil types like karst, sand, and crystalline (Santos et al., 2012; Moro et al., 2015; Queiroz et al., 2017); the savanna here encompasses all the campos rupestres vegetation on mountaintops of the Chapada Diamantina, which mostly involve ﬁre-sensitive plant lineages, including succulents (Rapini et al., 2021); and the Mata Atlantica involves habitats as distinct as the more open coastal restingas (Scarano, 2002; Oliveira et al., 2014; Fernandes and Queiroz, 2015). These habitats may not be unique in their phylogenetic or ﬂoristic composition as potentially distinct biomes but are in their functional composition. One of the perhaps most important messages of our study relates to areas where the three regionalizations disagree in their cluster delimitation (Figure 3). These areas of disagreement between ﬂoristic, functional, and phylogenetic data indicate the presence of transition zones between two or more biomes, presumably along environmental gradients. An alternative method of visualizing transition zones is presented in Supplementary Figure 3. Most previous biome delimitation analyses have not included transition zones, treating biomes as categorical variables with no intermediates (e.g., Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012); see Figure 1). Here we highlight transition zones as biologically interesting areas in their own right that cover a large percentage of our study area. For example, the area we delimit as “core” Caatinga covers 420k km2 and the areas we delimit as transition zones cover 480k km2 (254k km2 delimited as Caatinga by two analyses; 227k km2 delimited as Caatinga by one analysis), or 14% larger than the “core” Caatinga itself. The existence and extent of transition zones raises an important question: what may underlie the diﬀerences among the results from our individual regionalizations and what may these diﬀerences tell us about biomes in NE Brazil? The “Core” Caatinga The Caatinga and other seasonally dry tropical forests have often been considered equivalent to the trans-continentally distributed “succulent biome,” typically characterized by nutrient-rich substrates with little water holding capacity, and by a highly seasonal, drought-prone climate in which succulent plant lineages (e.g., columnar and arborescent members of Agavaceae, Bromeliaceae, Cactaceae, and Euphorbiaceae) evolved and Our results show that the “core” Caatinga area is in fact narrower than the widely used deﬁnition of Caatinga by Instituto December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 10 Biologically Meaningful Biomes Cardoso et al. Brasileiro de Geograﬁa e Estatística [IBGE], 2012. It excludes the wetter end of dry forests and transitional areas between savanna and dry forests (Figure 4). Not only that, but other major biomes exist within the Caatinga region (Queiroz et al., 2017). Disagreement between the three regionalizations in the Chapada Diamantina region, for example, highlights the biological reality of complex variation across environmentally highly heterogeneous areas, and should not be ignored. Thus, areas delimited by an essentially geographic approach such as that used by Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012) should not be termed “biomes.” These approaches not only oversimplify the complexity of the interdigitating nature of biomes, but also disregard the global nature dimension of biomes, which is fundamental to a more biologically realistic use of biomes in ecology, conservation, and evolutionary biogeography. adaptations to successfully thrive in dry forests identiﬁed here as Caatinga). Functional clusters, however, show clear diﬀerences to ﬂoristic and phylogenetic clusters, both in the number of clusters and the geographic distribution of those clusters (Figure 3). The diﬀerences between the functional (Figure 3A) and phylogenetic (Figure 3B) regionalizations indicate that the functional traits included in our analyses are not conserved across the phylogeny of the angiosperms, or that phylogenetic trait conservatism acts diﬀerently for diﬀerent traits. This may relate to the speciﬁc traits used here (e.g., we do not expect spines or corky bark to be conserved) but our analyses do include several well conserved traits (e.g., nodulation, latex, and C4 photosynthesis). The diﬀerences between the ﬂoristic (Figure 3A) and functional (Figure 3C) regionalizations may indicate that the possession of certain trait combinations may allow limited sets of species to span borders created by ecological and evolutionary processes. Transitional Biome Areas Transition zones may reveal areas along environmental gradients between “core” biomes where sets of traits from more than one biome may permit survival, as suggested by the “environmental crossroads hypothesis” (Neves et al., 2020). Additionally, we predict that diﬀerences in the adaptations required to successfully persist under diﬀerent rainfall regimes lead to distinct ﬂoristic and functional compositions across biomes. The level of phylogenetic distinctiveness, on the other hand, will depend on the relative ease at which plants have acquired adaptations required for crossing environmental gradients, i.e., the level of phylogenetic niche and trait conservatism (Donoghue, 2008; Crisp et al., 2009). Agreement in clusters delimited by all three regionalizations in areas such as along the eastern border of the “core” Caatinga suggests ecological ﬁltering is acting in conjunction with phylogenetic niche conservatism along the same environmental gradient (Caatinga-Mata Atlantica border; Figure 4). Transitional Caatinga areas in the southern parts of the Caatinga biogeographic domain indicate that ecological ﬁltering is acting along diﬀerent environmental gradients to phylogenetic niche conservatism, at least for some of the traits included in our analyses. Frontiers in Ecology and Evolution \| www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2021. 723558/full#supplementary-material Supplementary Figure 1 \| Species richness across northeast Brazil showing (A) “raw” species richness from cleaned occurrence data for 9,134 species with ≥5 records; (B) “raw” species richness from cleaned occurrence data for 3,547 species included in all analyses; (C) modeled species richness for 3,547 species included in all analyses. ACKNOWLEDGMENTS We thank Centro de Referência em Informação Ambiental (CRIA) and Reﬂora for sharing their complete distribution datasets; Alexandre Brunello, Luiza Cosme, Moabe Fernandes, Raquel Miatto, Marcelo Mizushima, Tony Oliveira, Oliver Phillips, Desirée Ramos, Raphael Rocha, Rubens Santos, Valdemir Silva, and Elmar Veenendaal for helping collecting silica-gel dried leaf samples during ﬁeldwork; Glaucia Drummond (Fundação Biodiversitas, Canudos) and Eudes Velozo (Fazenda Esperança, Boa Vista do Tupim) for their support during ﬁeldwork; Matt Lavin and two reviewers for their valuable suggestions to improve the manuscript; and Associate Editor Danilo Neves for inviting us to contribute with the special issue “Temporal and large-scale spatial patterns of plant diversity and diversiﬁcation.” DC acknowledges Jon Lloyd for inviting him to be speaker of the 44th New Phytologist Symposium “Determinants of tropical vegetation structure and function” in Accra, Ghana, when most ideas of this article were originally presented. That lineages can transcend geographic barriers is also a product of the evolutionary accessibility of distinct biomes that create geographical opportunity (Edwards and Donoghue, 2013). So, by inferring biotic interchange across biogeographic regions (e.g., Antonelli et al., 2018), without considering the biotic complexity within them, it is impossible to uncover the true balance of phylogenetic biome conservatism versus evolutionary biome shifts. Fortunately, recent progress has been made toward developing more biogeographically realistic approaches that model how lineages shift between biomes depending on the temporal availability and geographical connectivity of biomes (Landis et al., 2021a,b). A deﬁnition that captures the most biologically meaningful nature of a biome in space and time is clearly critical for comparative analyses involving Landis et al.’s (2021a) phylogenetic biome shift model. Thus, we believe that our approach of combining the multiple dimensions describing the spatial distribution of biomes will help to more reliably map and understand the evolution and functioning of biodiversity. Biologically Meaningful Biomes in Evolutionary Biogeography Our results show that similar clusters are delimited using phylogenetic and ﬂoristic data across NE Brazil, some of which we suggest correspond to biomes that can be recognized at global scale, including SDTF, rainforest, and savanna. This indicates that phylogenetic niche conservatism is operating and preventing plant lineages from crossing environmental gradients across evolutionary time (e.g., extreme drought constrained species Comparative phylogenetic approaches of multiple taxa to understand the evolutionary history of the biodiversity in species-rich, yet geologically and climatically complex regions like the Neotropics have revealed important insights into how lineages and species interact with ecology and geography over evolutionary time. Such analyses often employ the reconstruction of ancestral areas across phylogenies to estimate December 2021 \| Volume 9 \| Article 723558 Frontiers in Ecology and Evolution \| www.frontiersin.org 11 Biologically Meaningful Biomes Cardoso et al. AUTHOR CONTRIBUTIONS Thus, such comparative approaches cannot allow us to deepen our understanding of whether the “core” Caatinga or the other putatively distinct biomes of NE Brazil (Figure 3) are more evolutionarily accessible to lineages from Amazonian tropical rainforests. In other words, some Amazon to Caatinga “switches” may be to “rainforests” within the Caatinga biogeographic region and thus represent geographic movements, not evolutionary switches. Likewise, most studies neglect that the grass-rich, ﬁre-prone savanna biome that predominates in the Cerrado domain is criss-crossed by a network of gallery forests (i.e., rainforests; Silva-de-Miranda et al., 2018), as well as ﬁre-sensitive seasonally dry, evergreen or semideciduous forests, depending on water availability, in patches of high fertility soils (Oliveira-Filho et al., 2013b; Bueno et al., 2018). DC, PWM, and TS designed the study and led the writing with signiﬁcant contributions from all co-authors. DC, GR, and PWM entered functional trait data. GO generated sequence data for community phylogeny. CD designed the pipeline for producing community phylogenies. PWM led all analyses. All authors commented and agreed on the last version of the manuscript. FUNDING This research was funded by the Natural Environment Research Council-Newton grant NE/N012526/1 and Fundação de Amparo à Pesquisa do Estado de São Paulo grant 2015/50488-5 “Nordeste: New Science for a Neglected Biome,” and Royal Society Advanced Fellowship grant NAF/R1/180331. DC’s research in plant biodiversity is also funded by Fundação de Amparo à Pesquisa do Estado da Bahia (Universal no. APP0037/2016) and Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico Research Productivity PQ-2 grant 308244/2018-4. AUTHOR CONTRIBUTIONS the rates of biotic interchange between biogeographic regions. For example, Antonelli et al. (2018) showed an impressively large number of dispersal events out of Amazonia to other major neotropical regions, where at least 85 species among angiosperms, birds, ferns, frogs, mammals, and squamates inhabiting the Caatinga region were inferred to have derived from an Amazonian ancestor. Biogeographic regions are often treated as homogeneous units (i.e., synonymous with biomes) in estimates of ancestral distributions. This approach cannot take into account the complexity of climatic, evolutionary, and functional spaces that conﬁne species ecologically as we demonstrate for NE Brazil here. As such, it risks conﬂating the primary roles of geography and ecology, such that biome switching is likely to be overestimated in ecologically conﬁned groups with broad distributions but underestimated in biogeographically conﬁned groups with broad ecological distributions. Thus, such comparative approaches cannot allow us to deepen our understanding of whether the “core” Caatinga or the other putatively distinct biomes of NE Brazil (Figure 3) are more evolutionarily accessible to lineages from Amazonian tropical rainforests. In other words, some Amazon to Caatinga “switches” may be to “rainforests” within the Caatinga biogeographic region and thus represent geographic movements, not evolutionary switches. Likewise, most studies neglect that the grass-rich, ﬁre-prone savanna biome that predominates in the Cerrado domain is criss-crossed by a network of gallery forests (i.e., rainforests; Silva-de-Miranda et al., 2018), as well as ﬁre-sensitive seasonally dry, evergreen or semideciduous forests, depending on water availability, in patches of high fertility soils (Oliveira-Filho et al., 2013b; Bueno et al., 2018). the rates of biotic interchange between biogeographic regions. For example, Antonelli et al. 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Supplementary Figure 4 \| Areas of agreement and disagreement among biome classiﬁcations across northeast Brazil based upon hierarchical clustering analyses of functional, phylogenetic, and ﬂoristic data alone (see Figures 2A–C). Areas of Supplementary Table 2 \| Species included in the analyses, including taxonomic family, functional traits, and model performance statistics. reement (“core” biomes) are highlighted as per the legend and are disagreement (“transitional zones”) are shown with intermediate colors between core biomes. (A) functional and phylogenetic data; (B) phylogenetic and ﬂoristic Supplementary Table 3 \| The relationship between Continuous Boyce Index (CBI) and the number of independent records by species. data; (C) functional and ﬂoristic data; and (D) functional, phylogenetic, and ﬂoristic DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Supplementary Figure 2 \| Community phylogeny for northeast Brazil. Supplementary Figure 3 \| Dendrograms showing the major clusters from the cluster analyses based upon: (A) phylogenetic data; (B) ﬂoristic data; (C) Frontiers in Ecology and Evolution \| www.frontiersin.org December 2021 \| Volume 9 \| Article 723558 12 Biologically Meaningful Biomes Cardoso et al. data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012). data. The black line indicates the limits of the Caatinga domain sensu Instituto Brasileiro de Geograﬁa e Estatística [IBGE] (2012). functional data; (D) taxonomic and functional data combined; (E) taxonomic and phylogenetic data combined; (F) functional and phylogenetic data combined; (G) taxonomic, functional, and phylogenetic data combined. 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https://openalex.org/W2972857783	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0222439&type=printable	English	null	Comparison between Aptima Assays (Hologic) and the Allplex STI Essential Assay (Seegene) for the diagnosis of Sexually transmitted infections	PloS one	2,019	cc-by	4,956	RESEARCH ARTICLE Editor: S.M. Bruisten, GGD Amsterdam, NETHERLANDS Editor: S.M. Bruisten, GGD Amsterdam, NETHERLANDS Received: June 3, 2019 Accepted: August 29, 2019 Published: September 12, 2019 Copyright: © 2019 de Salazar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: June 3, 2019 Accepted: August 29, 2019 Published: September 12, 2019 Copyright: © 2019 de Salazar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data underlying the findings described in this study have been deposited to Figshare and they are accessible via https://doi.org/10.6084/m9.figshare.9159746.v2. Funding: The authors received no specific funding for this work. Comparison between Aptima Assays (Hologic) and the Allplex STI Essential Assay (Seegene) for the diagnosis of Sexually transmitted infections Adolfo de Salazar1, Beatriz Espadafor2, Ana Fuentes-Lo´pez1, Antonio Barrientos-Dura´n1, Luis Salvador2, Marta A´ lvarez1, Federico Garcı´aID1* 1 Hospital Universitario San Cecilio, Servicio de Microbiologı´a, Instituto de Investigacio´n Ibs, Granada, Spain, 2 Hospital Universitario Virgen de las Nieves, Servicio de Dermatologı´a, Centro de ETS, Granada, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * fegarcia@ugr.es * fegarcia@ugr.es * fegarcia@ugr.es OPEN ACCESS Sexually transmitted infections (STIs) remain a worldwide problem and a severe threat to public health. The purpose of this study was to compare Aptima® Assays (Hologic®) and the Allplex™STI Essential Assay (Seegene®) for the simultaneous detection of Chlamydia tra- chomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium in clinical practice. The Aptima® assays (Hologic®) are based on a transcription-mediated amplification (TMA) method. The Allplex™STI Essential assay (Seegene®) is based on a multiplex Real-Time PCR (RT-PCR) method. A total of 622 clinical samples from different anatomical sites were tested using both methods. A total of 88 (14.1%) and 66 (10.6%) posi- tive samples were found for any of the TMA assays used and for the RT-PCR assay, respec- tively. Aptima® assays showed a slightly higher rate of positive results for all pathogens except for T. vaginalis, the results of which were similar to those obtained with Allplex™. The most commonly detected pathogen was C. trachomatis (37 samples; 5.9% using TMA assays) and the anatomical site with the highest prevalence of microorganisms was a non- urogenital site, the pharynx (27 positive samples; 4.3%). Using the Aptima® assays as refer- ence method, the comparison showed that the average specificity of multiplex RT-PCR was 100.0% for the four pathogens. However an average sensitivity of 74.5% was observed, showing 95.2% (CI95%; 93.6–96.9) of overall concordance (κ = 0.80). In conclusion, the Aptima® assays show a higher sensitivity on a wide range of sample types compared to the Allplex™assay. Citation: de Salazar A, Espadafor B, Fuentes-Lo´pez A, Barrientos-Dura´n A, Salvador L, A´lvarez M, et al. (2019) Comparison between Aptima Assays (Hologic) and the Allplex STI Essential Assay (Seegene) for the diagnosis of Sexually transmitted infections. PLoS ONE 14(9): e0222439. https://doi. org/10.1371/journal.pone.0222439 Aptima Assays vs Allplex STI Essential Assay for STI our adherence to PLOS ONE policies on sharing data and materials. The other authors declare no conflict of interest. estimated globally [1]. For Mycoplasma genitalium, prevalence has been estimated at 1.3% in high-income countries and 3.9% in low-income countries [2]. These microorganisms are responsible for a variety of clinical syndromes in women and men, such as urethritis, cervicitis, prostatitis and vaginitis [3–6], which may lead to severe complications and long-term sequelae, including pelvic inflammatory disease, infertility, chronic pelvic pain, ectopic pregnancy, neu- rological and cardiovascular disease in adults, premature delivery, neonatal death, severe dis- ability or blindness in infants, and increased risk of HIV acquisition and transmission [7, 8]. our adherence to PLOS ONE policies on sharing data and materials. The other authors declare no conflict of interest. Prompt recognition and appropriate treatment are essential for the control of transmission of STIs, and this requires sensitive and accurate laboratory diagnostic methods. The imple- mentation of nucleic acid amplification tests (NAATs) has revolutionized diagnostics in the detection of C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium, due to their numerous advantages over conventional methods [9]. However, these tests traditionally required separate processes for nucleic acid extraction and amplification/detection, which may increase the risk of errors (manipulation errors, contamination during the extraction step, etc.), especially in high-volume diagnostic laboratories. Another limitation is the need for batch samples, which may delay diagnosis and treatment initiation. The use of multiplex RT-PCR for the diagnosis of STI is widespread in Microbiology labo- ratories, because they offer a large panel of microorganisms in a simple reaction, at a low cost. However, these techniques may have a lower sensitivity than others commercially available. Currently there are assays for the diagnosis of STI based on transcription-mediated amplifi- cation (TMA) targeting directly ribosomal RNA (rRNA), with the advantage of a higher num- ber of copies per cell compared to DNA-based tests, which only target one copy. Assays based on TMA potentially increase sensitivity of detection compared to assays based on RT-PCR tar- geting single-copy genes [10–15]. To date, only a few studies compare these two methodologies for the simultaneous diagnosis of the 4 most important pathogens causing STIs [16, 17]. The objective of this study was to assess the performance of multiplex RT-PCR [Allplex™ STI Essential (Seegene1, Seoul, South Korea)]. Patients The study was conducted between May 2017 and November 2017 in Granada, Spain. A total of 375 patients from the Centre for Sexually Transmitted Diseases in Granada were enrolled in the study. The median age of males (n = 243; 65%) and females (n = 132; 35%) was 29 years [IQR: 23–37]. The study was designed and conducted according to the principles expressed in the Declaration of Helsinki and it was approved by the local Ethics Committee of Hospital Universitario San Cecilio. Verbal informed consent was obtained from all participants. Data underlying the findings described in this study have been deposited to Figshare and they are accessible via https://doi.org/10.6084/m9.figshare.9159746.v2. All other relevant data are shown in the present manuscript. This is an in vitro diagnostic (IVD) and CE- marked system for the simultaneous detection of C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium. The following FDA-cleared assays based on TMA were used as the refer- ence method: Aptima Combo 21 (for C. trachomatis and N. gonorrhoea), Aptima1 T. vagina- lis and Aptima1 M. genitalium (Hologic1, San Diego, USA)]. Introduction Competing interests: Federico Garcı´a has received honoraria from Werfen, Hologic and Roche for participation in advisory boards and lectures related to the topic of the paper. This does not alter Sexually transmitted infections (STIs) remain a worldwide problem and a severe threat to pub- lic health. In 2012, approximately 130.9, 78.3, and 142.6 million new cases of Chlamydia tra- chomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis infections, respectively, were 1 / 9 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Specimen collection A total of 622 prospective clinical specimens from different anatomical sites (urine and endo- cervical, pharyngeal and anal swabs) according to the reported type of sexual practices (vagi- nal, oral and/or anal intercourse) of 375 participants were collected in duplicate. Table 1 2 / 9 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Aptima Assays vs Allplex STI Essential Assay for STI Table 1. Distribution of the collected samples. Anatomical site Male n (%) of patients Female n (%) of patients Pharyngeal 16 (6.6%) 23 (17.4%) Endocervical - 22 (16.7%) Urine 130 (53.5%) 0 Pharyngeal and Urine 37 (15.2%) 0 Anal and Urine 1 (0.4%) 0 Anal and Pharyngeal 14 (5.8%) 2 (1.5%) Anal and Endocervical - 3 (2.3%) Endocervical and Urine - 1 (0.8%) Endocervical and Pharyngeal - 63 (47.7%) Endocervical and Pharyngeal and Urine - 1 (0.8%) Anal and Endocervical and Pharyngeal - 17 (12.9%) Anal and Urine and Pharyngeal 45 (18.5%) 0 Total number of Patients (375) 243 (64.8%) 132 (35.2%) Total number of Samples (622) 385 (61.9%) 237 (38.1%) https://doi.org/10.1371/journal.pone.0222439.t001 shows a detailed description of the anatomical location of the collected samples. Specimens for routine testing were collected with dry swabs. The swabs were suspended in 2 mL of 1X Phosphate Buffered Saline solution (PBS) as transport system. Urine samples for TMA-assay testing were collected using the Aptima1 Urine Collection Kit for Male and Female Speci- mens (Hologic, San Diego, USA). Female endocervical and male urethral samples were col- lected with the Aptima1 Unisex Swab Specimen collection kit (Hologic, San Diego, USA). The Aptima1 Multitest Swab Specimen Collection Kit (Hologic, San Diego, USA) was used for the collection of pharyngeal and anal specimens. Random sampling was performed by alternating the collection of specimens for routine testing and specimens for Aptima1 test- ing. The distribution of the types of clinical specimens (622) was the following: 218 (35%) pharyngeal swabs, 214 (35%) first-void urine samples, 107 (17%) endocervical swabs and 83 (13%) rectal swabs. After collection, specimens were stored at 4˚C until testing, generally, for two or three days after specimen collection. All NAATs were performed in parallel by the same technician. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Real-Time multiplex PCR assay Testing was performed using the multiplex RT-PCR Allplex™STI Essential Assay (Seegene, Seoul, Korea). This assay can simultaneously detect 7 STI pathogens (C. trachomatis, N. gonor- rhoeae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum and U. parvum) in a single tube by using dual priming oligonucleotide (DPO™) and multiple detection temperatures (MuDT™) technologies, providing individual Ct values for multiple pathogens in a single channel. The DPO system differs structurally and functionally from the conventional primer system by including a poly deoxyinosine (I) linker, between two segments of primer sequences. This poly (I) linker allows dividing the DPO primer into two perfectly functional segments with different hybridization temperatures [18]. The elongation will be conducted when the two segments hybridize correctly giving rise to a high specificity between similar or related sequences. Previ- ous nucleic acid extraction was performed (400 μL sample volume) using MagNA Pure 96 Sys- tem (Roche); nucleic acids were eluted in 50 μL (final volume). Real-time PCR was performed in a CFX-96 real-time thermocycler (Bio-Rad, CA, USA), according to the manufacturer’s instructions. To maximize cost-efficiency without a significant delay in reporting, routine 3 / 9 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Aptima Assays vs Allplex STI Essential Assay for STI testing was performed in batches every Tuesday and Friday. For this study, only the results for C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium were analyzed. Data analysis Sensitivity (SE), specificity (SP) and kappa coefficient (κ) of the multiplex RT-PCR were calculated and compared with TMA assays for detection of C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis using the statistical package SPSS, version 23 (IBM, Chicago, IL, USA). The corresponding two-tailed 95% score (Wilson) confidence intervals (CIs) were also estimated. Transcription-mediated amplification assay The Aptima1 assays comprise three main steps: target capture, TMA of the species-specific targets in the rRNA, and target detection by hybridization with complementary probes linked to chemiluminescent labels. The TMA step consists of a target nucleic acid amplification method using RNA transcription (RNA polymerase) and DNA synthesis (reverse transcrip- tase) to produce a RNA amplicon from a target nucleic acid; TMA can be used to target both RNA and DNA [19]. Aptima1 M. genitalium (MG), Aptima1 Combo 2 (detecting both C. trachomatis, N. gonorrhoeae in one sample) and Aptima1 T. vaginalis assays were used on the Panther1 sys- tem (Hologic, San Diego). This system is a fully automated testing platform with true sample- to-result automation allowing sample testing with Aptima1 assays, therefore avoiding the sep- arate DNA extraction step. For this evaluation, samples collected for the Aptima1 assays were stored at 4˚C and run on the same day as the RT-PCR. https://doi.org/10.1371/journal.pone.0222439.t002 genitalium identified using the Allplex™STI Essential assay (n = 10); ten of these Aptima-positive samples were collected from pharyngeal swabs. The diagnostic performance of the Allplex assay was evaluated by the calculation of SE and SP parameters. Concordance between the Aptima1 and the Allplex assays was determined through the calculation of the Cohen’s Kappa index, κ coefficient of the Allplex assay in rela- tion to the Aptima1 assays (reference method) for the detection of C. trachomatis, N. gonor- rhoeae, M. genitalium and T. vaginalis (Table 2). A specificity of 100.0% was found for the four pathogens, however an average sensitivity of 74.5% was observed for the Allplex assay. Both methods (TMA and multiplex RT-PCR) have shown a variable consistency depending on the microorganism detected, with κ values ranging between 0.58 for M. genitalium and 0.91 for C. trachomatis. In general, a concordance of 95.2% (CI95%; 93.6–96.9) and κ = 0.80 were obtained between both methods. Among the 622 samples, there were 28 discrepancies: 4 anal, 3 endocervical, 13 pharyngeal and 8 urine samples. Co-infections were observed in 7 samples from 7 different patients and inconsistent results were detected in 6 of these patients. A detailed description of these results is shown in Table 5. Results A total of 622 samples were tested using both methods. Positive results were found in 88 (14.1%) out of 622 samples for all the Aptima1 assays used. Regarding the Allplex™assay, only 66 (10.6%) out of 622 samples showed positive results. Table 2 and S1 Table show the diagnos- tic performance of the systems used for detection of the different pathogens. The TMA-based assays performed on the Panther1 platform showed slightly higher positive results for C. tra- chomatis, N. gonorrhoeae and M. genitalium, while results for the T. vaginalis samples were similar for both RT-PCR (Allplex™) and TMA (Aptima1) assays. The most frequently detected pathogen was C. trachomatis (37 samples; 5.9%). The TMA-positive samples were further analyzed according to their anatomical site. Results revealed that a non-urogenital sample, the pharynx, was the most frequent site with a positive result (29 positive samples; 4.7%) followed by urine (27 positive samples; 4.3%), endocervical Table 2. Diagnostic performance of Allplex™STI Essential assays in relation to the Aptima1 assays for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis. Aptima1 Allplex™ DIAGNOSTIC PERFORMANCE PATHOGENS Positive n (%) Positive n (%) SENSITIVITY % SE (C.I. 95%) SPECIFICITY % SP (C.I. 95%) Kappa (k) C. trachomatis 37 (5.9%) 31 (5.0%) 83.8% (67.3–93.2) 100.0% (99.2–100) 0.91 N. gonorrhoeae 29 (4.7%) 21 (3.4%) 72.4% (52.5–86.6) 100.0% (99.2–100) 0.83 M. genitalium 24 (3.9%) 10 (1.6%) 41.7% (22.8–63.1) 100.0% (99.2–100) 0.58 T. vaginalis 5 (0.8%) 5 (0.8%) 100.0% (46.3–100) 100.0% (99.2–100) 1 CI, confidence interval. formance of Allplex™STI Essential assays in relation to the Aptima1 assays for C. trachomatis, N. gonorrhoeae, M. genita I Essential assays in relation to the Aptima1 assays for C. trachomatis, N. gonorrhoeae, M. genitalium and T. le 2. Diagnostic performance of Allplex™STI Essential assays in relation to the Aptima1 assays for C. trachomatis, N. go i li Table 2. Diagnostic performance of Allplex™STI Essential assays in relation to the Aptima1 assays for C. trachomat vaginalis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 4 / 9 Aptima Assays vs Allplex STI Essential Assay for STI Table 3. Anatomical distribution and prevalence of the Aptima1-positive samples. Site n C. trachomatis n (%) N. gonorrhoeae n (%) M. genitalium n (%) T. vaginalis n (%) Anal 83 6 (7.2%) 6 (7.2%) 4 (4.8%) 1 (1.2%) Endocervical 107 12 (11.2%) 2 (1.9%) 4 (3.7%) 4 (3.7%) Pharyngeal 218 9 (4.1%) 10 (4.6%) 10 (4.6%) 0.0% Urine 214 10 (4.7%) 11 (5.1%) 6 (2.8%) 0.0% https://doi.org/10.1371/journal.pone.0222439.t003 Table 4. Anatomical distribution and prevalence of the Allplex™-positive samples. Site n C. trachomatis n (%) N. gonorrhoeae n (%) M. genitalium n (%) T. vaginalis n (%) Anal 83 5 (6.0%) 6 (7.2%) 1 (1.2%) 1 (1.2%) Endocervical 107 11 (10.3%) 1 (0.9%) 3 (2.8%) 4 (3.7%) Pharyngeal 218 6 (2.8%) 8 (3.7%) 2 (0.9%) 0.0% Urine 214 9 (4.2%) 6 (2.8%) 4 (1.9%) 0.0% https://doi.org/10.1371/journal.pone.0222439.t004 Table 3. Anatomical distribution and prevalence of the Aptima1-positive samples. Site n C. trachomatis n (%) N. gonorrhoeae n (%) M. genitalium n (%) T. vaginalis n (%) Anal 83 6 (7.2%) 6 (7.2%) 4 (4.8%) 1 (1.2%) Endocervical 107 12 (11.2%) 2 (1.9%) 4 (3.7%) 4 (3.7%) Pharyngeal 218 9 (4.1%) 10 (4.6%) 10 (4.6%) 0.0% Urine 214 10 (4.7%) 11 (5.1%) 6 (2.8%) 0.0% https://doi.org/10.1371/journal.pone.0222439.t003 Table 3. Anatomical distribution and prevalence of the Aptima1-positive samples. Table 4. Anatomical distribution and prevalence of the Allplex™-positive samples. Site n C. trachomatis n (%) N. gonorrhoeae n (%) M. genitalium n (%) T. vaginalis n (%) Anal 83 5 (6.0%) 6 (7.2%) 1 (1.2%) 1 (1.2%) Endocervical 107 11 (10.3%) 1 (0.9%) 3 (2.8%) 4 (3.7%) Pharyngeal 218 6 (2.8%) 8 (3.7%) 2 (0.9%) 0.0% Urine 214 9 (4.2%) 6 (2.8%) 4 (1.9%) 0.0% https://doi.org/10.1371/journal.pone.0222439.t004 Table 4. Anatomical distribution and prevalence of the Allplex™-positive samples. https://doi.org/10.1371/journal.pone.0222439.t004 (22 positive samples; 3.5%) and anal samples (17 positive samples; 2.7%) (Tables 3 and 4). A total of 14 more samples were found positive for M. genitalium using Aptima1 assays (n = 24) than positive samples for M. genitalium identified using the Allplex™STI Essential assay (n = 10); ten of these Aptima-positive samples were collected from pharyngeal swabs. (22 positive samples; 3.5%) and anal samples (17 positive samples; 2.7%) (Tables 3 and 4). A total of 14 more samples were found positive for M. genitalium using Aptima1 assays (n = 24) than positive samples for M. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Discussion The relatively high prevalence of STIs and the need for a rapid and accurate diagnostic tool for their detection justifies that any new methodology must be thoroughly evaluated before its implementation in routine laboratory practice. In this study, we evaluated the performance of TMA-based assays (Aptima Combo 21 (for C. trachomatis and N. gonorrhoea), Aptima1 T. vaginalis and Aptima1 M. genitalium) on the Panther1 platform for simultaneous detection of C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium in comparison with a multi- plex RT-PCR assay (Allplex™STI Essential, Seegene1). Although Aptima1 assays have previ- ously shown good performances in the diagnosis of these pathogens [10–15], this is, to our knowledge, the first time that the detection of these four pathogens together has been com- pared to Allplex™STI Essential RT-PCR-based assay. The effectiveness of the Allplex™assay has also been previously proven [20–22]. 5 / 9 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Aptima Assays vs Allplex STI Essential Assay for STI Table 5. Discordant results between Aptima1 and Allplex™assays regarding the detection of C. trachomatis, M. genitalium and N. gonorrhoeae. Sample (n) Aptima1 Allplex™ Anal (1) CT GC GC Anal (2) MG Negative Anal (1) MG CT CT Endocervical (1) GC Negative Endocervical (1) MG Negative Endocervical (1) MG CT MG Pharyngeal (3) CT Negative Pharyngeal (2) GC Negative Pharyngeal (6) MG Negative Pharyngeal (2) MG CT CT Urine (1) CT Negative Urine (5) GC Negative Urine (1) MG Negative Urine (1) MG GC GC CT, C. trachomatis; MG, M. genitalium; NG, N. gonorrhoeae. https://doi.org/10.1371/journal.pone.0222439.t005 Table 5. Discordant results between Aptima1 and Allplex™assays regarding the detection of C. trachomatis, M. genitalium and N. gonorrhoeae. https://doi.org/10.1371/journal.pone.0222439.t005 Both systems showed the highest prevalence of C. trachomatis in the analyzed samples, which is in line with this pathogen being the most frequently reported STI in Europe [23]. The sensitivity of the TMA assays was higher for most pathogens compared to multiplex RT-PCR. It is very remarkable that most of the samples responsible for this higher sensitivity were pha- ryngeal swab samples, and that M. genitalium was more frequently detected in this location with TMA assays. For most bacterial STI, the throat is the anatomical location with the lowest number of positive samples [24] however our findings at least in the case of M. genitalium, confirm the results of recent reports in the literature [25]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Author Contributions Conceptualization: Adolfo de Salazar, Beatriz Espadafor, Federico Garcı´a. Conceptualization: Adolfo de Salazar, Beatriz Espadafor, Federico Garcı´a. Data curation: Beatriz Espadafor, Luis Salvador. Data curation: Beatriz Espadafor, Luis Salvador. Methodology: Adolfo de Salazar, Ana Fuentes-Lo´pez, Antonio Barrientos-Dura´n, Marta A´lvarez. Writing – original draft: Adolfo de Salazar, Federico Garcı´a. Writing – review & editing: Adolfo de Salazar, Beatriz Espadafor, Federico Garcı´a. Writing – review & editing: Adolfo de Salazar, Beatriz Espadafor, Federico Garcı´a. Discussion Our results confirm that other sites should always be considered based on patient’s sexual habits [26]. In fact, using urogenital samples alone overlooks many infections, resulting in many patients failing to receive proper treatment [27]. The sensitivity and specificity of these TMA assays have been evaluated previously, with sensitivities and specificities > 90% [13, 14, 28], however recently, false-negative Chlamydia trachomatis have been reported using Aptima Combo21 assays in Finland and Sweden due to a 23S rRNA C1515T mutation [29, 30]. It is important to mention that a single genetic target region should not be trusted in molecular diagnosis of infections to avoid underdiagnosis of possible mutants, this could be a limitation of this assay, that only amplify chlamydial 23S rRNA. Our study has two important limitations. First, we could not resolve all discordant results with a third test because our sample volume was low and we did not want to dilute the sample to prevent losing sensitivity. Thereof, we could not calculate the positive predictive value and negative predictive value of this test, and we could not rule out that the higher sensitivity of the TMA assays could be due to false positive detections. Second, T. vaginalis specimens found in pharyngeal swabs with the RT-PCR test were not included in the final analysis. In fact, we found 19 pharyngeal samples that were scored positive for T. vaginalis only with the Allplex™ test; it is well documented that DNA-based methods may lack specificity for discriminating T. vaginalis from some other oral Trichomonas species existing in the pharyngeal microbiota, such as Trichomonas tenax [31, 32]. 6 / 9 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222439 September 12, 2019 Aptima Assays vs Allplex STI Essential Assay for STI Our results confirm the effectiveness of the Aptima1 assays for STI diagnosis, providing additional evidence supporting the implementation of this methodology for routine testing in clinical diagnostics. Importantly, this TMA technology provides important features for labora- tory automation and daily clinical practice. Thus, patients might benefit highly from this tech- nology, as they can be evaluated and diagnosed at the same medical visit. Acknowledgments The results published here are part of the PhD thesis of candidate Adolfo de Salazar, in the Bio- medicine Doctoral Program of the University of Granada. g y We thank Nutraceutical Translations for English language editing of this manuscript. Supporting information S1 Table. Details regarding the number of positive and negative samples identified with Aptima1 and Allplex™assays. (DOCX) References 1. World Health Organization. Report on global sexually transmitted infection surveillance 2015. WHO; 2016. https://apps.who.int/iris/handle/10665/249553 2. Baumann L, Cina M, Egli-Gany D, Goutaki M, Halbeisen FS, Lohrer G-R, et al. Prevalence of Myco- plasma genitalium in different population groups: systematic review andmeta-analysis. Sex Transm Infect. 2018; 94(4):255–62. https://doi.org/10.1136/sextrans-2017-053384 PMID: 29440466 3. 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https://openalex.org/W3163142079	https://link.springer.com/content/pdf/10.1007/JHEP12(2021)190.pdf	English	null	Non-equilibrium dynamics of a scalar field with quantum backreaction	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	16,524	Open Access, c⃝The Authors. Article funded by SCOAP3. Non-equilibrium dynamics of a scalar ﬁeld with quantum backreaction JHEP12(2021)190 Published for SISSA by Springer Received: May 25, 2021 Revised: December 13, 2021 Accepted: December 20, 2021 Published: December 27, 2021 Received: May 25, 2021 Revised: December 13, 2021 Accepted: December 20, 2021 Published: December 27, 2021 Received: May 25, 2021 Revised: December 13, 2021 Accepted: December 20, 2021 Published: December 27, 2021 Published for SISSA by Springer Keywords: Nonperturbative Eﬀects, Thermal Field Theory, Quantum Dissipative Sys- tems Kimmo Kainulainen and Olli Koskivaara Kimmo Kainulainen and Olli Koskivaara Department of Physics, University of Jyväskylä, P.O. Box 35 (YFL), FI-40014 Jyväskylä, Finland Helsinki Institute of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland E-mail: kimmo.kainulainen@jyu.fi, olli.a.koskivaara@student.jyu.fi Abstract: We study the dynamical evolution of coupled one- and two-point functions of a scalar ﬁeld in the 2PI framework at the Hartree approximation, including backreaction from out-of-equilibrium modes. We renormalize the 2PI equations of motion in an on-shell scheme in terms of physical parameters. We present the Hartree-resummed renormalized eﬀective potential at ﬁnite temperature and critically discuss the role of the eﬀective poten- tial in a non-equilibrium system. We follow the decay and thermalization of a scalar ﬁeld from an initial cold state with all energy stored in the potential, into a fully thermalized system with a ﬁnite temperature. We identify the non-perturbative processes of parametric resonance and spinodal instability taking place during the reheating stage. In particular we study the unstable modes in the region where the vacuum 1PI eﬀective action becomes complex and show that such spinodal modes can have a dramatic eﬀect on the evolution of the one-point function. Our methods can be easily adapted to simulate reheating at the end of inﬂation. Keywords: Nonperturbative Eﬀects, Thermal Field Theory, Quantum Dissipative Sys- tems ArXiv ePrint: 2105.09598 ArXiv ePrint: 2105.09598 Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP12(2021)190 Contents 1 Introduction 1 2 2PI eﬀective action and equations of motion 2 2.1 Hartree approximation 4 3 Renormalization 5 3.1 Renormalized equations of motion 8 3.2 Eﬀective potential and physical parameters 9 3.3 Finite temperature eﬀective potential 10 4 Wigner space equations 13 5 Numerical results 15 5.1 Particle production and reheating via parametric resonance 16 5.2 Strong spinodal instability 18 5.3 Self-thermalization 21 6 Conclusions 25 A Numerical implementation 26 Contents 1 Introduction 1 2 2PI eﬀective action and equations of motion 2 2.1 Hartree approximation 4 3 Renormalization 5 3.1 Renormalized equations of motion 8 3.2 Eﬀective potential and physical parameters 9 3.3 Finite temperature eﬀective potential 10 4 Wigner space equations 13 5 Numerical results 15 5.1 Particle production and reheating via parametric resonance 16 5.2 Strong spinodal instability 18 5.3 Self-thermalization 21 6 Conclusions 25 A Numerical implementation 26 JHEP12(2021)190 A Numerical implementation 1 Introduction Classical scalar ﬁelds coupled to quantum matter play an important role in various settings in cosmology. They are used to study the creation of seed perturbations for structure formation, reheating processes, particle production and the creation of baryon asymmetry. Almost exclusively in these treatments it is assumed that the scalar ﬁeld evolves in some classical, possibly quantum corrected but ﬁxed eﬀective potential. One rarely accounts for the backreaction of the non-equilibrium quanta that may be created during the dynamical process. However, such quanta may be produced copiously during out-of-equilibrium phase transitions [1, 2] by parametric resonance [3–7] or by spinodal instability [6, 8–13], and they could signiﬁcantly aﬀect the evolution of the system [14–18]. In this paper we study the eﬀects of quantum backreaction on the scalar ﬁeld evolution using two-particle irreducible (2PI) eﬀective action methods. A crucial step in the rigorous analysis of the problem is performing a consistent renor- malization of the equations of motion derived from the 2PI eﬀective action. This is a highly non-trivial task, because in any ﬁnite truncation of the 2PI expansion, a number of auxiliary vertex and self-energy functions appear that require setting up consistent renor- malization conditions [19]. Other works on the renormalization of 2PI-truncated theories – 1 – include for example references [20–22]. In this paper we carefully go through the renor- malization of our model using the method of cancellation of the sub-divergences [23–26]. We emphasize that while the renormalization counterterms are constants, the divergences that get subtracted, and hence also the vacuum state of the system, depend on the infrared physics, such as temperature, or even the shape of the non-equilibrium particle spectrum. To be speciﬁc, we study a simple λφ4-model with a spontaneous symmetry breaking tree-level potential. We work in the Hartree approximation and perform the auxiliary renormalizations using the MS subtraction scheme. The renormalized equations of motion and the 2PI eﬀective action are however scale independent and completely speciﬁed in terms of physical parameters. We present explicit results for the vacuum and ﬁnite temperature eﬀective potentials as well as for the vacuum potential in the presence of non-equilibrium ﬂuctuations. We stress that in the non-equilibrium case the eﬀective potential can only be constructed a posteriori and it is not in general a useful quantity for solving the equations of motion. 1 Introduction JHEP12(2021)190 With our renormalized equations we can follow in real time how the potential energy of the classical ﬁeld is transferred into quantum ﬂuctuations by the non-perturbative pro- cesses. We identify a strong parametric resonance, even though our self-coupled system is too complicated to admit a comprehensive analytical stability analysis. We also show that due to backreaction from spinodal instability the ﬁeld can pass through a potential barrier even when starting with less energy than the initial barrier height. We also follow the full thermal history of a system that starts with pure potential energy, until it is fully thermalized with nearly all of its energy stored in thermal plasma. We also show that at the initial stages of reheating the quantum system is highly coherent, but the coherence is gradually erased by interactions as the system thermalizes. This paper is organized as follows. In section 2 we review the 2PI eﬀective action techniques and introduce our truncation scheme, the Hartree approximation. In section 3 we show how to self-consistently renormalize the 2PI equations of motion and express them in terms of physical quantities. We also study both resummed vacuum and thermal eﬀective potentials in the Hartree case and compare them with other approximations. In section 4 we write our equations of motion in the Wigner space in terms of moment functions following references [27, 28], and also complement the equations with phenomenological friction terms. Section 5 is dedicated to numerical results. We compute the evolution of various quantities, such as the classical ﬁeld, particle number and coherence functions using the fully coupled 2PI equations. Finally, section 6 contains our conclusions. 2 2PI eﬀective action and equations of motion We will study the non-equilibrium dynamics of a scalar ﬁeld theory with the potential V (φ) = −1 2µ2φ2 + 1 4!λφ4 using the two-particle irreducible (2PI) eﬀective action technique of non-equilibrium quantum ﬁeld theory [29, 30]. The 2PI eﬀective action for this theory is Γ2PI[ϕ, ∆] = S[ϕ] −i 2TrC ln(∆) + i 2TrC ∆−1 0 ∆ + Γ2[ϕ, ∆], (2.1) (2.1) – 2 – Figure 1. The Keldysh contour in the complex time plane, running from some initial time to an arbitrary future time and back again. Figure 1. The Keldysh contour in the complex time plane, running from some initial time to an arbitrary future time and back again. JHEP12(2021)190 where ϕ(x) is the classical ﬁeld and ∆(x, y) is the classical connected two-point function and the trace contains integration over the Keldysh contour [31] C of ﬁgure 1. Moving to a real-time representation the classical action can be written as S[ϕ] = P a=± aδabS[ϕb], where a and b indicate the branch on the complex time-contour, and S[ϕb] = Z d4x 1 2(∂µϕb)2 + 1 2µ2ϕ2 b −1 4!λϕ4 b . (2.2) (2.2) Similarly, the inverse classical propagator is given by i∆−1 0,ab(x, y; ϕ) = − □x −µ2 + 1 2λϕ2 a δ(4)(x −y)δab. (2.3) (2.3) Finally, Γ2 consists of all 2PI vacuum graphs with lines corresponding to the full propagator ∆and interactions inferred from the shifted action Sint ϕ, φq = − X a=± aδab Z d4x 1 3!λϕbφ3 qb + 1 4!λφ4 qb , (2.4) (2.4) where φ = ϕ + φq and φq is the quantum ﬁeld. where φ = ϕ + φq and φq is the quantum ﬁeld. The stationarity conditions of Γ2PI will give the equations of motion for the one- and two-point functions: The stationarity conditions of Γ2PI will give the equations of motion for the one- an two-point functions: δΓ2PI δϕa = 0 and δΓ2PI δ∆ab = 0. (2.5) (2.5) When the classical solution to the latter equation, parametrized in terms of ϕ, is reinserted back into the eﬀective action, we formally recover the 1PI action ˆΓ1PI[ϕ] = Γ2PI[ϕ, ∆[ϕ]]. In the full dynamical case the two equations are however strongly coupled and should be solved simultaneously, as we will do in our study. 2 2PI eﬀective action and equations of motion For the classical ﬁeld ϕ+(x) = ϕ−(x) and we may drop the branch index and ﬁnd: When the classical solution to the latter equation, parametrized in terms of ϕ, is reinserted back into the eﬀective action, we formally recover the 1PI action ˆΓ1PI[ϕ] = Γ2PI[ϕ, ∆[ϕ]]. In the full dynamical case the two equations are however strongly coupled and should be solved simultaneously, as we will do in our study. For the classical ﬁeld ϕ+(x) = ϕ−(x) and we may drop the branch index and ﬁnd: □x −µ2 + 1 6λϕ2(x) + 1 2λ∆(x, x) ϕ(x) = δΓ2 δϕ(x). (2.6) (2.6) We also left the branch indices out from the local correlation function ∆(x, x), which is the same for all components of the two-point function ∆ab(x, y). The stationarity condition We also left the branch indices out from the local correlation function ∆(x, x), which is the same for all components of the two-point function ∆ab(x, y). The stationarity condition – 3 – ∼λ (0) R + δ (0) λ + ∼λ (1) R + δ (1) λ 2 + ∼λ (0) R + δ (0) λ 2 + ∼λ (1) R + δ (1) λ 4 + ∼λ (0) R + δ (0) λ λ (1) R + δ (1) λ 2 + · · · ∼λ (0) R + δ (0) λ + ∼λ (1) R + δ (1) λ 2 + ∼λ (0) R + δ (0) λ 2 + ∼λ (1) R + δ (1) λ 4 + ∼λ (0) R + δ (0) λ λ (1) R + δ (1) λ 2 + · · · Figure 2. The ﬁrst few terms contributing to Γ2, including their precise coupling constant depen- dences. JHEP12(2021)190 JHEP12(2021)190 for ∆ab(x, y) leads to the Schwinger-Dyson equation for ∆ab(x, y) leads to the Schwinger-Dyson equation □x −µ2 + 1 2λϕ2(x) i∆ac(x, y) = aδacδ(4)(x −y) + b Z d4z Πab(x, z)∆bc(z, y), (2.7) (2.7) where summation over b is implied and the self-energy function is given by Πab(x, y) = 2iab δΓ2[ϕ, ∆] δ∆ba(y, x) = aδabδ(4)(x −y)Πsg(x) + Πab nsg(x, y). (2.8) (2.8) To proceed we also have to specify an approximation for the interaction term Γ2. 2.1 Hartree approximation The ﬁrst few terms contributing to Γ2, arising from the action (2.4), are shown in ﬁgure 2 (the role of the indices in the couplings is related to renormalization and will be explained in the next section). In this work we shall work in the Hartree approximation, which includes only the ﬁrst term in the series, given by ΓH 2 = −λ 8 Z d4x ∆2(x, x). (2.9) (2.9) In this case the self-energy has only a singular or local part: In this case the self-energy has only a singular or local part: Πsg(x) = −iλ 2 ∆(x, x), (2.10) (2.10) while Πab nsg(x, y) = 0. Obviously ∂ΓH 2 /∂ϕ = 0 as well, so there is no contribution to equation (2.6) in the Hartree approximation. We can now write the non-renormalized equations of motion compactly as □x −µ2 + 1 6λϕ2(x) + 1 2λ∆(x, x) ϕ(x) = 0, (2.11a) □x −µ2 + 1 2λϕ2(x) + 1 2λ∆(x, x) i∆ab(x, y) = aδabδ(4)(x −y), (2.11b) (2.11a) (2.11b) Eventually we will move to the Wigner space deﬁned in section 4 and solve these equations numerically in some example cases for homogeneous systems, but before we can do that, we have to address the divergences in ∆ab and in particular in the local correlation function ∆(x, x). – 4 – 3 Renormalization Systematic renormalization in the context of the 2PI expansion was thoroughly discussed in reference [19]. Here we use the method introduced in reference [23], and later used in refer- ences [24, 26], and we include also a connection to physical parameters. The key issue is that any ﬁnite order truncation of Γ2[ϕ, ∆] leads to an approximation for ˆΓ1PI[ϕ] that contains inﬁnite resummations of 1PI diagrams and the associated counterterms. This gives rise to a number of auxiliary n-point functions which need independent renormalization conditions. These conditions can be deﬁned by requiring that all sub-divergences cancel [23], but one needs to introduce a diﬀerent renormalized parameter for each diﬀerent operator. To be precise, all n-point functions can be classiﬁed in terms of the number of classical ﬁelds that connect to them, and all functions that are connected also to propagator lines are auxiliary. JHEP12(2021)190 Below we shall ﬁrst renormalize the auxiliary n-point functions in the MS-scheme and show that the resulting 1PI action is independent of the renormalization scale. We start by deﬁning the renormalized ﬁelds, propagators, couplings and masses: φ ≡Z1/2 (2) φR, λ ≡λ (I) R + δλ(I), ∆≡Z(0)∆R, µ2 ≡µ2 R(I) −δµ2 (I), (3.1) (3.1) where the index, I = 0, 1, 2, 4, follows the power of the classical ﬁeld associated with the n-point function. Written in terms of the renormalized quantities, the 2PI eﬀective action becomes: Γ2PI[ϕR, ∆R] = S[ϕR] −i 2TrC ln(Z(0)∆R) + i 2TrC h ∆−1 0R∆R i + δS[ϕR] + i 2TrC δ∆−1 0 ∆R + Γ2 h ϕR, ∆R; λ (I) R + δ (I) λ i , (3.2) (3.2) where S[ϕR] is the same as in equation (2.2) with ϕ →ϕR, µ2 →µ2 R(2) and λ →λ (4) R , and ∆−1 0R is the same as (2.3) with ϕ →ϕR, µ2 →µ2 R(0) and λ →λ (2) R . Moreover we deﬁned the classical counterterm action where S[ϕR] is the same as in equation (2.2) with ϕ →ϕR, µ2 →µ2 R(2) and λ →λ (4) R , and ∆−1 0R is the same as (2.3) with ϕ →ϕR, µ2 →µ2 R(0) and λ →λ (2) R . 3 Renormalization Moreover we deﬁned the classical counterterm action δS[ϕRb] ≡ Z d4x " δ(2) ϕ 2 (∂µϕRb)2 −1 2δ(2) µ ϕ2 Rb −1 4!δ (4) λ ϕ4 Rb # (3.3) (3.3) and the inverse classical counterterm propagator and the inverse classical counterterm propagator iδ∆−1 0,ab(x, y; ϕR) ≡− δ(0) ϕ □x + δ(0) µ + 1 2δ (2) λ ϕ2 Ra δ(4)(x −y)δab, (3.4) (3.4) where δ(I) ϕ ≡Z(I) −1 and the other eﬀective counterterms are deﬁned as: δ (0) λ ≡Z2 (0) λ (0) R + δλ(0) −λ (0) R , (3.5a) δ (2) λ ≡Z(0)Z(2) λ (2) R + δλ(2) −λ (2) R , (3.5b) δ (4) λ ≡Z2 (2) λ (4) R + δλ(4) −λ (4) R , (3.5c) δ(I) µ ≡Z(I) −µ2 R(I) + δµ2 (I) + µ2 R(I). (3.5d) δ (0) λ ≡Z2 (0) λ (0) R + δλ(0) −λ (0) R , (3.5a) δ (2) λ ≡Z(0)Z(2) λ (2) R + δλ(2) −λ (2) R , (3.5b) δ (4) λ ≡Z2 (2) λ (4) R + δλ(4) −λ (4) R , (3.5c) δ(I) µ ≡Z(I) −µ2 R(I) + δµ2 (I) + µ2 R(I). (3.5d) – 5 – – 5 – Also in the interaction term in (3.2) the renormalized couplings in the combination λ (I) R +δ (I) λ follow the power of the classical ﬁeld in the interaction term (2.4), rewritten in terms of the renormalized quantities. Also in the interaction term in (3.2) the renormalized couplings in the combination λ (I) R +δ (I) λ follow the power of the classical ﬁeld in the interaction term (2.4), rewritten in terms of the renormalized quantities. The renormalized equations of motion can now be derived from the renormalized ef- fective action, or more directly from (2.11a) and (2.11b), by writing the non-renormalized quantities in terms of the renormalized ones: Z(2)□x −µ2 R(2) + δ(2) µ + 1 6 λ (4) R + δ (4) λ ϕ2 R + 1 2 λ (2) R + δ (2) λ ∆R ϕR = 0, (3.6a) Z(0)□x −µ2 R(0) + δ(0) µ + 1 2 λ (2) R + δ (2) λ ϕ2 R + 1 2 λ (0) R + δ (0) λ ∆R i∆ab R (x, y) = aδabδ(4). 1These choices are partly speciﬁc for the Hartree approximation, where the self-energy Πab is propor- tional to the local correlation function. In any higher order 2PI truncation λ (0) R and λ (2) R would need to be renormalized separately. 3 Renormalization (3.6b) (3.6a) (3.6b) JHEP12(2021)190 Here we suppressed the arguments in the local functions ϕR(x) and ∆R(x, x), as well as in δ(4)(x −y), for brevity. We now proceed to determine the various counterterms appearing in these equations and in the end ﬁnd the renormalized equations of motion that include the eﬀects of quantum corrections. Auxiliary renormalization conditions. Because the operator acting on ∆ab R in (3.6b) is independent of branch indices, we can concentrate on the time ordered component ∆11 R of the two-point function. We choose the mass-shell renormalization condition in the vacuum conﬁguration ϕR = vR, which simultaneously minimizes the eﬀective action. That is, we set i ∆11 R −1 = p2 −m2 R, d dp2 i ∆11 R −1 = 1, and δΓ2PI δϕR ϕR=vR = 0. (3.7) (3.7) These conditions imply that Z(0) = 1 in the Hartree approximation, and in our current scheme we can also set Z(2) = 1 (see footnote 2 below). The renormalization conditions (3.7) then become: −µ2 R(2) + δ(2) µ + 1 6 λ (4) R + δ (4) λ v2 R + 1 2 λ (2) R + δ (2) λ ∆R(vR) = 0, (3.8a) −µ2 R(0) + δ(0) µ + 1 2 λ (2) R + δ (2) λ v2 R + 1 2 λ (0) R + δ (0) λ ∆R(vR = m2 R, (3.8b) (3.8a) (3.8b) where ∆R(vR) refers to the still divergent local correlator computed at the renormaliza- tion point. It should be noted that ∆ab R is an auxiliary function and the parameter m2 R is not yet related to any physical mass. Similarly, none of the couplings are yet related to observables, and there is considerable amount of choice related to their deﬁnition. We will choose the following conditions:1 δ (0) λ = δ (2) λ , (3.9a) −µ2 R(0) + δ(0) µ = −µ2 R(2) + δ(2) µ , (3.9b) λ (4) R = λ (2) R −1 3δ (4) λ + δ (2) λ . (3.9c) (3.9a) (3.9c) 1These choices are partly speciﬁc for the Hartree approximation, where the self-energy Πab is propor- tional to the local correlation function. In any higher order 2PI truncation λ (0) R and λ (2) R would need to be renormalized separately. 3 Renormalization 1These choices are partly speciﬁc for the Hartree approximation, where the self-energy Πab is propor- tional to the local correlation function. In any higher order 2PI truncation λ (0) R and λ (2) R would need to be renormalized separately. – 6 – Because Z(0,2) = 1 here, equation (3.9), together with eqs. (3.1) and (3.5) implies that λ (0) R = λ (2) R . Equation (3.9b) is less restrictive: it merely states that both renormalized mass terms are related to the same bare mass term. Conditions (3.9b) and (3.9c) still allow us to choose δ(2) µ and δ (4) λ such that m2 R and λ (4) R can be matched to a physical mass parame- ter and a physical coupling. Using the conditions (3.9) and equation (3.8b) we can write equation (3.8a) simply as 1 m2 R −1 3λ (4) R v2 R = 0. (3.10) (3.10) That is, we are able to keep the tree-level relation between the coupling λ (4) R , the background ﬁeld vR and the mass parameter m2 R. That is, we are able to keep the tree-level relation between the coupling λ (4) R , the background ﬁeld vR and the mass parameter m2 R. JHEP12(2021)190 Cancelling the sub-divergences. In order to proceed, we need to ﬁnd out the diver- gence structure of the local correlation function. Using dimensional regularization we can write ∆R(vR) = Qϵ Z ddp (2π)d ∆11 R (p) = −m2 R 16π2 2 ϵ + 1 −ln m2 R Q2 , (3.11) ∆R(vR) = Qϵ Z ddp (2π)d ∆11 R (p) = −m2 R 16π2 2 ϵ + 1 −ln m2 R Q2 , (3.11) where ϵ ≡4 −d and 2/ϵ ≡2/ϵ −γE + ln(4π) and Q is an arbitrary renormalization scale. We now separate ∆R into divergent and ﬁnite parts as follows: ∆R(vR) = Qϵ Z ddp (2π)d ∆11 R (p) = −m2 R 16π2 2 ϵ + 1 −ln m2 R Q2 , (3.11) where ϵ ≡4 −d and 2/ϵ ≡2/ϵ −γE + ln(4π) and Q is an arbitrary renormalization scale. We now separate ∆R into divergent and ﬁnite parts as follows: (3.11) ∆R(vR) Q Z (2π)d ∆R (p) 16π2 ϵ + 1 ln Q2 , (3.11) where ϵ ≡4 −d and 2/ϵ ≡2/ϵ −γE + ln(4π) and Q is an arbitrary renormalization scale. 3 Renormalization We now separate ∆R into divergent and ﬁnite parts as follows: where ϵ ≡4 −d and 2/ϵ ≡2/ϵ −γE + ln(4π) and Q is an arbitrary renormalization scal We now separate ∆R into divergent and ﬁnite parts as follows: ∆R(vR) ≡m2 R∆ϵ + ∆F0 m2 R, Q , (3.12) (3.12) where ∆ϵ ≡−1/ 8π2ϵ . In what follows we will suppress the Q-dependence of the function ∆F0 for brevity. Next we insert the decomposition (3.12) back into equation (3.8b), use relations (3.9) and let the leading order terms deﬁne the renormalized mass independently from the terms containing divergences or counterterms. In this way we get two equations out of the equation (3.8b): m2 R ≡−µ2 R(2) + 1 2λ (2) R h v2 R + ∆F0 m2 R i , (3.13) 0 = δ(2) µ + 1 2δ (2) λ h v2 R + ∆F0 m2 R i + 1 2 λ (2) R + δ (2) λ m2 R∆ϵ. (3.14) (3.13) Using deﬁnition (3.13) again in equation (3.14) and rearranging we get δ(2) µ −1 2 λ (2) R + δ (2) λ µ2 R(2)∆ϵ + 1 2 h v2 R + ∆F0 m2 R i δ (2) λ + 1 2 λ (2) R + δ (2) λ λ (2) R ∆ϵ = 0. (3.1 This equation has a consistent solution where the leading constant terms and the terms multiplying the combination (the sub-divergence part) v2 R+∆F0 cancel independently. This gives two constraint equations, δ (2) λ + 1 2 λ (2) R + δ (2) λ λ (2) R ∆ϵ = 0, (3.16a) δ(2) µ −1 2 λ (2) R + δ (2) λ µ2 R(2)∆ϵ = 0, (3.16b) (3.16a) (3.16b) from which we can ﬁnally solve the counterterms δ (2) λ and δ(2) µ : δ (2) λ = − 1 2 λ (2) R 2∆ϵ 1 + 1 2λ (2) R ∆ϵ and δ(2) µ = µ2 R(2) 1 2λ (2) R ∆ϵ 1 + 1 2λ (2) R ∆ϵ . (3.17) δ (2) λ = − 1 2 λ (2) R 2∆ϵ 1 + 1 2λ (2) R ∆ϵ (3.17) – 7 – Scale dependence. 3 Renormalization The scale dependence of the auxiliary couplings and the mass pa- rameters can now be worked out as usual by requiring that the bare parameters do not run: ∂Q Qϵλ (2) R + δ (2) λ = 0 and ∂Q Qϵµ2 R(I) −δ(I) µ = 0. Using equations (3.17) one then immediately ﬁnds: Q∂λ (2) R ∂Q = λ (2) R 2 16π2 and Q ∂µ2 R(2) ∂Q = λ (2) R µ2 R(2) 16π2 . (3.18) (3.18) The latter equation applies for both mass parameters, assuming that δ(0) µ and δ(2) µ diﬀer by at most a ﬁnite and Q-independent term, which is the case in the Hartree approximation. Equations (3.18) can be easily integrated: The latter equation applies for both mass parameters, assuming that δ(0) µ and δ(2) µ diﬀer by at most a ﬁnite and Q-independent term, which is the case in the Hartree approximation. Equations (3.18) can be easily integrated: JHEP12(2021)190 JHEP12(2021)190 λ (2) R (Q) = λ (2) R (Q0) 1 + λ(2) R (Q0) 32π2 ln Q2 0 Q2 and µ2 R(I)(Q) = µ2 R(I)(Q0) 1 + λ(2) R (Q0) 32π2 ln Q2 0 Q2 . (3.19) (3.19) Remember that as a result of equation (3.9a) λ (0) R = λ (2) R . On the other hand, the coupling λ (4) R does not run at all; indeed, to keep λ (4) R ﬁnite, we must have δ (4) λ = 3δ (2) λ up to ﬁnite terms according to equation (3.9c), which implies Q∂λ (4) R ∂Q = 0 ⇒ λ (4) R = constant. (3.20) (3.20) We shall see below that λ (4) R can be further ﬁxed by some physical condition. 3.2 Eﬀective potential and physical parameters Let us now consider the adiabatic limit of the evolution equations, where ∆F is given purely by vacuum ﬂuctuations with no physical excitations. In this case deﬁnition (3.21) reduces to m2(ϕR) ≡−µ2 R(2) + 1 2λ (2) R h ϕ2 R + ∆F0 m2(ϕR) i , (3.25) (3.25) and correspondingly ∆R(ϕR) ≡m2(ϕR)∆ϵ + ∆F0 m2(ϕR) . (3.26) (3.26) Note that m2(ϕR) and ∆R diﬀer from deﬁnitions (3.21) and (3.22) only through a diﬀerent value of the background ﬁeld ϕR. Using the equation of motion (3.6b) in the renormalized 2PI action (3.2) we can write down the 1PI eﬀective potential in the Hartree approximation as follows: Note that m2(ϕR) and ∆R diﬀer from deﬁnitions (3.21) and (3.22) only through a diﬀerent value of the background ﬁeld ϕR. Using the equation of motion (3.6b) in the renormalized 2PI action (3.2) we can write down the 1PI eﬀective potential in the Hartree approximation as follows: VH(ϕR) = −1 V ΓH 2PI ϕR, ∆ = V0(ϕR) + i 2V Tr h ln ∆R i −1 8 λ (2) R + δ (2) λ ∆ 2 R(ϕR), (3.27) VH(ϕR) = −1 V ΓH 2PI ϕR, ∆ = V0(ϕR) + i 2V Tr h ln ∆R i −1 8 λ (2) R + δ (2) λ ∆ 2 R(ϕR), (3.27) (3.27) where V is the space-time volume and the tree-level eﬀective potential is V0(ϕR) = 1 2 −µ2 R(2) + δ(2) µ ϕ2 R + 1 4! λ (4) R + δ (4) λ ϕ4 R = −λ (4) R 12 ϕ4 R, (3.28) (3.28) where in the last step we dropped all terms of order ϵ. Writing iTr h ln ∆R i = V R dm2 ∆R and using equation (3.26) one ﬁnds that the divergences cancel between the two last terms in equation (3.27) and the ﬁnite part of Tr h ln ∆R i creates the one-loop correction to the eﬀective potential. After a little algebra one ﬁnds the result: VH(ϕR) = −λ (4) R 12 ϕ4 R + m4(ϕR) 2λ (2) R −m4(ϕR) 64π2 ln m2(ϕR) Q2 −1 2 . (3.29) (3.29) This is the vacuum eﬀective potential in the Hartree approximation, found for example in reference [32]. Despite the apparent Q-dependence, VH(ϕR) is in fact scale-independent. 3.1 Renormalized equations of motion It is essential to impose a correct treatment of the local correlation function away from the renormalization point in the equations of motion (3.6a) and (3.6b). Analogously to (3.13), we ﬁrst deﬁne a leading order mass function that contains all ﬁnite terms in equation (3.6b): m2(ϕR, ∆F) ≡−µ2 R(2) + 1 2λ (2) R ϕ2 R + ∆F . (3.21) (3.21) Here ∆F is the ﬁnite part of the local correlation function, which must be deﬁned analo- gously to equation (3.12): Here ∆F is the ﬁnite part of the local correlation function, which must be deﬁned analo- gously to equation (3.12): 2 ∆R ≡m2(ϕR, ∆F)∆ϵ + ∆F. (3.22) (3.22) Note that both the ﬁnite part and the divergence contain non-trivial contributions from both the vacuum and the non-equilibrium ﬂuctuations. Using deﬁnitions (3.21) and (3.22) we can write equation (3.6b) as follows: Note that both the ﬁnite part and the divergence contain non-trivial contributions from both the vacuum and the non-equilibrium ﬂuctuations. Using deﬁnitions (3.21) and (3.22) we can write equation (3.6b) as follows: □x + m2(ϕR, ∆F) + δ(2) µ + 1 2δ (2) λ ϕ2 R + ∆F +1 2 λ (2) R + δ (2) λ m2(ϕR, ∆F)∆ϵ i∆ab R (x, y) = aδabδ(4)(x −y). (3.23) (3.23) Using deﬁnition (3.21) again one can show that all divergent terms in equation (3.23) arrange as in equation (3.15) and cancel as a result of the renormalization conditions (3.16). Using deﬁnition (3.21) again one can show that all divergent terms in equation (3.23) arrange as in equation (3.15) and cancel as a result of the renormalization conditions (3.16). – 8 – Then, noting that λ (4) R + δ (4) λ = −2λ (4) R + O(ϵ), the same manipulations can be done also in equation (3.6a). This results in the ﬁnal renormalized equations of motion: h □x + m2(ϕR, ∆F) i ϕR = 1 3λ (4) R ϕ3 R, (3.24a) h □x + m2(ϕR, ∆F) i i∆ab R (x, y) = aδabδ(4)(x −y). 3.2 Eﬀective potential and physical parameters 3.2 Eﬀective potential and physical parameters 3.1 Renormalized equations of motion (3.24b) (3.24a) (3.24b) These equations appear deceivingly simple: when written for the Wightman function ∆< R = ∆+− R , equation (3.24b) takes the form of a wave equation with a time-dependent mass and, as we shall see in the next section, equation (3.24a) describes the motion of the one- point function in a quantum corrected eﬀective potential including backreaction from non- equilibrium modes. However, despite their apparent simplicity, the equations are strongly coupled through the local correlator in the gap equation (3.21) for the mass term. JHEP12(2021)190 3.2 Eﬀective potential and physical parameters Indeed, one can ﬁrst show from deﬁnition (3.25), using (3.18), that ∂Qm2(ϕR) = 0. Then by a direct diﬀerentiation and using equations (3.18) and (3.20) one ﬁnds that ∂QVH(ϕR) = 0. – 9 – Physical parameters. Diﬀerentiating (3.25) with respect to ϕR one can ﬁrst derive the identity Physical parameters. Diﬀerentiating (3.25) with respect to ϕR one can ﬁrst derive the identity ∂m2 ∂ϕR 1 −λ (2) R 32π2 ln m2 Q2 = λ (2) R ϕR. (3.30) (3.30) Using (3.30) it is simple to show that the ﬁrst derivative of the potential can be written as ∂VH ∂ϕR = −1 3λ (4) R ϕ3 R + m2(ϕR)ϕR. (3.31) (3.31) Comparing equation (3.31) with equation (3.24a) we can see that in the case of pure vacuum ﬂuctuations the equation of motion can be written as ∂2 t ϕR+∂VH/∂ϕR = 0. Diﬀerentiating equation (3.31) once more with respect to ϕR one ﬁnds Comparing equation (3.31) with equation (3.24a) we can see that in the case of pure vacuum ﬂuctuations the equation of motion can be written as ∂2 t ϕR+∂VH/∂ϕR = 0. Diﬀerentiating equation (3.31) once more with respect to ϕR one ﬁnds JHEP12(2021)190 ∂2VH ∂ϕ2 R = m2(ϕR) + h λ (2) R m2(ϕR) −λ (4) R i ϕ2 R. (3.32) (3.32) Because m2(vR) = m2 R, we now see that the on-shell mass parameter mR of the auxiliary propagator can be identiﬁed with a physical mass,2 if we at the same time deﬁne λ (2) R m2 R ≡λ (4) R . (3.33) (3.33) This is the choice of parameters we shall use in the rest of this paper. So far we have deﬁned the counterterm δ (4) λ only up to a ﬁnite constant. This, and other remaining freedom in choosing the counterterms (see footnote 2) would allow us to further match λ (4) R to some observable. Given that λ (4) R does not run, equations (3.33) and (3.32) are enough to ﬁx the parameters of the theory. Going beyond the Hartree approximation would lead to more complicated calculations and relations between the auxiliary parameters, but would not change the derivation conceptually. 2In fact these relations imply that mR corresponds to a mass deﬁned at p2 = 0, but in the Hartree case this is the same as the physical pole mass. Going beyond Hartree approximation, one can still make mR agree with the physical on-shell mass using the remaining freedom in deﬁnitions (3.9) and in the deﬁnition of the wave-function counterterm Z(2), which allow adding ﬁnite parts to δ (2) ϕ , δ (2) µ and δ (4) λ . 3.3 Finite temperature eﬀective potential In our derivation in section 3.1 we did not specify the ﬁnite part of the local correlation function ∆F, and in what follows we will compute it numerically from the equations of mo- tion. Before that it is useful to make one more observation concerning thermal corrections. Indeed, we can include thermal corrections by a simple generalization of equations (3.25) and (3.26): ( ) m2(ϕR, T) ≡−µ2 R(2) + 1 2λ (2) R h ϕ2 R + ∆F(ϕR, T) i , (3.34) with ∆R(ϕR, T) ≡m2(ϕR, T)∆ϵ + ∆F(ϕR, T) and ∆F(ϕR, T) ≡∆F0 m2(ϕR, T) + T 2I m2(ϕR, T)/T 2, (3.35) (3.34) ∆F(ϕR, T) ≡∆F0 m2(ϕR, T) + T 2I m2(ϕR, T)/T 2, (3.35) (3.35) 2In fact these relations imply that mR corresponds to a mass deﬁned at p2 = 0, but in the Hartree case this is the same as the physical pole mass. Going beyond Hartree approximation, one can still make mR agree with the physical on-shell mass using the remaining freedom in deﬁnitions (3.9) and in the deﬁnition of the wave-function counterterm Z(2), which allow adding ﬁnite parts to δ (2) ϕ , δ (2) µ and δ (4) λ . 2In fact these relations imply that mR corresponds to a mass deﬁned at p2 = 0, but in the Hartree case this is the same as the physical pole mass. Going beyond Hartree approximation, one can still make mR agree with the physical on-shell mass using the remaining freedom in deﬁnitions (3.9) and in the deﬁnition of the wave-function counterterm Z(2), which allow adding ﬁnite parts to δ (2) ϕ , δ (2) µ and δ (4) λ . – 10 – where I x = 2∂xJ x and J x is the dimensionless bosonic one-loop thermal integral J (x) ≡ 1 2π2 Re Z ∞ 0 dy y2 ln 1 −e−√ y2+x−iε . (3.36) (3.36) Here the inﬁnitesimal imaginary part iε deﬁnes the correct branch of the logarithm for a negative m2. 3.3 Finite temperature eﬀective potential With these deﬁnitions one can go through the analysis of the previ- ous paragraph and show that the equation of motion of the homogeneous ﬁeld is now ∂2 t ϕR + ∂VH(ϕR, T)/∂ϕR = 0, where VH(ϕR, T) is the thermally corrected, scale indepen- dent eﬀective potential in the Hartree approximation: VH(ϕR, T) = VH ϕR m→mT −1 2m2 T T 2I m2 T /T 2 + T 4J m2 T /T 2, (3.37) JHEP12(2021)190 (3.37) where m2 T ≡m2(ϕR, T). Note that in the 2PI approach also the vacuum part VH(ϕR) of the potential is computed with the thermally corrected mass, which is the solution to equations (3.34) and (3.35). It is worth mentioning that in each special case considered above, from the vacuum renormalization (3.12) to the quantum corrected eﬀective action with (3.35) and without (3.26) thermal corrections, and ﬁnally to the general case (3.22), the divergence that gets removed by counterterms is diﬀerent and depends on the value of the background ﬁeld, the temperature and the particle distribution. This is an unavoidable feature of the 2PI equations with a ﬁnite order truncation. However, in all cases the counterterms themselves remain the same, uniquely deﬁned constants. Comparison to ring-resummed potentials. Equations (3.34), (3.35) and (3.37) pro- vide a consistent resummation of the thermal potential to super-daisy level. They can be seen as a consistent generalization of the Parwani resummation method [33]. In these ap- proaches the thermal corrections aﬀect all modes on equal footing, while in the usual ring re- summation method [34, 35] only the long wavelength modes are screened by the short wave- length modes in a thermal plasma. The advantage of the potential (3.37) is that it provides a consistently renormalized, smooth continuation between the non-relativistic and relativis- tic regimes. In all other ring-resummed potentials this behaviour has to be put in by hand. To eﬀect a fair comparison of diﬀerent approximations, we write all potentials using the same renormalization conditions. To be concrete, we use the conditions ∂2 ϕV (vR) = m2 R and ∂4 ϕV (vR) = λR. 3.3 Finite temperature eﬀective potential The critical temperatures are Tc ≈169.20 GeV in the ring, Tc ≈153.29 GeV in the Parwani and Tc ≈155.67 GeV in the Hartree approximation. We used mR = 100 GeV and λR = 6 (which implies λ (4) R ≃5.2). The vertical line in the left panel shows T0 = p 12/λRmR, where the high-temperature limit approximated thermal mass vanishes at ϕR = 0. where only the zero-mode is dressed by thermal corrections, one ﬁnds: VRing(ϕR, T) ≡V1L(ϕR, T) + T 12πRe m3 0(ϕR) −m3 0(ϕR, T) . (3.40) (3.40) Above we wrote the Hartree potential in terms of the scale dependent variables. However, since the potential is actually scale independent, we can rewrite it at the scale Q = mR, explicitly in terms of the physical parameters: VH(ϕR, T)=−λ (4) R 12 ϕ4 R + m4 T 2λ (4) R −m4 T 64π2 ln m2 T m2 R −1 2 −m2 T T 2 2 I m2 T T 2 +T 4J m2 T T 2 , (3.41) where m2 T is the solution to the gap equation, which now becomes m2 T ≡m2 R + 1 2λ (4) R (ϕ2 R −v2 R) + λ (4) R 32π2 m2 T ln m2 T m2 R + m2 T −m2 R + T 2I m2 T T 2 , (3.42) m2 T ≡m2 R + 1 2λ (4) R (ϕ2 R −v2 R) + λ (4) R 32π2 m2 T ln m2 T m2 R + m2 T −m2 R + T 2I m2 T T 2 , (3.42) (3.42) with m2 R = 1 3λ (4) R v2 R and where ﬁnally λ (4) R is related to the renormalized coupling λR ≡ ∂4 ϕVH(vR, 0) by λR = λ (4) R 1 + 3 3λ (4) R 32π2 + 3 3λ (4) R 32π2 2 , (3.43) (3.43) as can be shown by direct diﬀerentiation of equation (3.41). as can be shown by direct diﬀerentiation of equation (3.41). In the left panel of ﬁgure 3 we show the evolution of the second ϕ-derivatives of the potentials near the critical temperature at ϕR = 0. 3.3 Finite temperature eﬀective potential With these conditions the standard one-loop corrected potential without the ring-corrections becomes V1L(ϕR, T) ≡−1 2µ2 Rϕ2 R + 1 4!λRϕ4 R + V1−loop(ϕR) + T 4J m2 0(ϕR) T 2 , (3.38) (3.38) where m2 0(ϕR) = −µ2 R + 1 2λRϕ2 R and the standard one-loop vacuum potential is (this potential also satisﬁes the condition ∂ϕV1−loop(vR) = 0) V1−loop(ϕR) = 1 64π2 m4 0(ϕR) ln m2 0(ϕR) m2 R −3 2 + 2m2 Rm2 0(ϕR) . (3.39) (3.39) In the Parwani approximation [33] one replaces m2 0(ϕR) with the lowest order thermal mass m2 0(ϕR, T) = m2 0(ϕR) + 1 24λRT 2 in equation (3.38) and in the ring approximation [34, 35], – 11 – Figure 3. Left: the evolution of the second ϕ-derivatives of the diﬀerent potentials at ϕR = 0. Middle: the evolution of the ratio v(T)/T, where v(T) is the position of the second minimum. Right: the potential at critical temperature in each approximation. The critical temperatures are Tc ≈169.20 GeV in the ring, Tc ≈153.29 GeV in the Parwani and Tc ≈155.67 GeV in the Hartree approximation. We used mR = 100 GeV and λR = 6 (which implies λ (4) R ≃5.2). The vertical line in the left panel shows T0 = p 12/λRmR, where the high-temperature limit approximated thermal mass vanishes at ϕR = 0. JHEP12(2021)190 gure 3. Left: the evolution of the second ϕ-derivatives of the diﬀerent potentials at ϕR = 0. Figure 3. Left: the evolution of the second ϕ-derivatives of the diﬀerent potentials at ϕR = 0. Middle: the evolution of the ratio v(T)/T, where v(T) is the position of the second minimum. Right: the potential at critical temperature in each approximation. The critical temperatures are Tc ≈169.20 GeV in the ring, Tc ≈153.29 GeV in the Parwani and Tc ≈155.67 GeV in the Hartree approximation. We used mR = 100 GeV and λR = 6 (which implies λ (4) R ≃5.2). The vertical line in the left panel shows T0 = p 12/λRmR, where the high-temperature limit approximated thermal mass vanishes at ϕR = 0. Figure 3. Left: the evolution of the second ϕ-derivatives of the diﬀerent potentials at ϕR = 0. Middle: the evolution of the ratio v(T)/T, where v(T) is the position of the second minimum. Right: the potential at critical temperature in each approximation. 3.3 Finite temperature eﬀective potential The sharp kinks seen in the ring (green dashed line) and Parwani (red dash-dotted line) cases at T = T0 result from the non-analytic dependence of the resummed potentials on the thermally corrected mass term (we are using the high-temperature expansion for m2(ϕ, T) in these schemes). The one- loop result (blue dotted line) does not share this feature, because there we are using the – 12 – non-resummed mass term. Interestingly, the Hartree result (black line) does not show signs of similar non-analyticity. In the middle panel we show the evolution of the ratio v(T)/T, where v(T) is the position of the asymmetric minimum as a function of T. There are signiﬁcant diﬀerences between the approximations: in all resummed potentials a metastable minimum emerges, and it has the largest jump in the Hartree case. In the one-loop case the metastability does not develop, but there is a jump in v(T)/T at T ≈120 GeV due to the non-analytic behaviour, now of the vacuum mass term as a function of ϕ. In the right panel we show the potentials at the critical temperature (whose value for each model is given in the ﬁgure caption). The transition strength is dramatically diﬀerent in the diﬀerent approximations and it is by far the strongest in the Hartree case. Of course one should keep in mind that this is a very simple model, with only a single scalar ﬁeld. However, when one compares the one-loop results with lattice calculations, one typically ﬁnds that both ring and Parwani approximations give weaker transitions than the lattice or 3d-perturbation theory calculations [36]. It would be interesting to see if the Hartree approximation was in better agreement with these schemes when applied in more complex models. JHEP12(2021)190 4 Wigner space equations (4.3) (4.3) Then taking the real and imaginary parts of equation (4.2) integrated over k0 and the imaginary part of the same equation integrated over k0 and weighted by k0, we get three Then taking the real and imaginary parts of equation (4.2) integrated over k0 and the imaginary part of the same equation integrated over k0 and weighted by k0, we get three – 13 – equations coupling the moments ρnk with n = 0, 1, 2 to the ﬁeld equation for a homogeneous ﬁeld ϕR(t): equations coupling the moments ρnk with n = 0, 1, 2 to the ﬁeld equation for a homogeneous ﬁeld ϕR(t): 1 4∂2 t ρ0k −ρ2k + ω2 k(t) ρ0k = 0, (4.4a) ∂tρ1k = 0, (4.4b) ∂tρ2k −1 2 h ∂t m2 eﬀ(t) i ρ0k = 0, (4.4c) h ∂2 t + m2 eﬀ(t) i ϕR = 1 3λ (2) R ϕ3 R. (4.4d) (4.4d) JHEP12(2021)190 We used the shorthand m2 eﬀ(t) ≡m2(ϕR, ∆R) for the mass deﬁned in (3.22) and (3.21) and deﬁned ω2 k(t) ≡k2 + m2 eﬀ(t). The gap equation (3.21), including the out-of-equilibrium (or thermal) modes, can be written explicitly as We used the shorthand m2 eﬀ(t) ≡m2(ϕR, ∆R) for the mass deﬁned in (3.22) and (3.21) and deﬁned ω2 k(t) ≡k2 + m2 eﬀ(t). The gap equation (3.21), including the out-of-equilibrium (or thermal) modes, can be written explicitly as m2 eﬀ(t) = −µ2 R(2) + 1 2λ (2) R ( ϕ2 R + ∆F0 m2 eﬀ(t) + Z k " ρ0k(t) −θ ω2 k(t) 2ωk(t) #) , (4.5) (4.5) where we deﬁned R k ≡ 1 2π2 R ∞ 0 d\|k\|\|k\|2, and the Heaviside theta-function θ ω2 k(t) ensures that the vacuum does not contain the unstable spinodal modes.3 If ρ0k(t) is identiﬁed with a thermal distribution (including the vacuum part), equa- tion (4.5) clearly reduces to (3.34). After discretizing the momentum variable, equa- tions (4.4c), (4.4a), (4.4d) and (4.5) can be written as a closed set of coupled ﬁrst order diﬀerential equations, which include backreaction from the non-equilibrium modes into the evolution of the homogeneous ﬁeld ϕR. The gap equation (4.5) must be solved ﬁrst at the entry to the routine, after which the solution is advanced using (4.4c), (4.4a) and (4.4d). 3Spinodal modes are the unstable modes that appear when the eﬀective mass function is negative. We deﬁne them explicitly in equation (5.1) below. Note that the vacuum energy integral in the spinodal region, computed with the Heaviside function, is identical with the integral computed taking the absolute value of the mass squared function and integrating over all momenta. 4 Wigner space equations We now proceed to solving the general equations (3.24b) and (3.24a) for homogeneous non- equilibrium systems. Of these, equation (3.24a) is already in the desired form, when we assume that ﬁeld ϕR is homogeneous, but equation (3.24b) for the correlation function will be easier to handle in the mixed representation. Because of the homogeneity an ordinary Fourier transformation is suﬃcient for the spatial coordinates, but for the time variable we need the Wigner transformation: ∆ab Rk(k0, t) = Z dr0 ∆ab Rk t + r0 2 , t −r0 2 eik0r0, (4.1) (4.1) where t ≡1 2(x0 + y0) and r0 ≡x0 −y0. Because all correlation functions ∆ab(x, y) have the same local limit, it suﬃces to consider the equation for the lesser Wightman function ∆+−≡∆<. Starting from equation (3.24b), we ﬁnd that in the Wigner representation it satisﬁes the equation, 1 4∂2 t −k2 −ik0∂t + e−i 2 ∂m t ∂k0m2ϕR, ∆R ∆< Rk(k0, t) = 0. (4.2) (4.2) Here the index m in the derivative ∂m t signals that the time-derivative acts only on the mass function and not on the propagator. Equation (4.2) is still equivalent to (3.24b) and highly complicated because of the inﬁnite tower of t- and k0-derivatives involved. It can be recast into a simpler form by introducing a moment expansion. Following reference [27] we ﬁrst introduce the moment functions: Here the index m in the derivative ∂m t signals that the time-derivative acts only on the mass function and not on the propagator. Equation (4.2) is still equivalent to (3.24b) and highly complicated because of the inﬁnite tower of t- and k0-derivatives involved. It can be recast into a simpler form by introducing a moment expansion. Following reference [27] we ﬁrst introduce the moment functions: ρkn(t) = Z dk0 2π kn 0 ∆< Rk(k0, t). 4 Wigner space equations The former can be further related to the particle and antiparticle number densities nk and nk, so that one eventually ﬁnds [27, 28]: nk = 1 ωk ρ2k + ρ1k, (4.7a) nk = 1 ωk ρ2k −ρ1k −1, (4.7b) fc± k = ωkρ0k −1 ωk ρ2k ± i 2∂tρ0k. (4.7c) (4.7a) (4.7b) (4.7c) The coherence functions fc± k measure the degree of quantum coherence, or squeezing, between particle-antiparticle pairs with opposite 3-momenta [39]. A non-coherent vacuum state must then be deﬁned as a state with no squeezing in addition to having no particles. This corresponds to setting nk = nk = fc± k ≡0, which is equivalent to: ρvac 0k = Θk 2ωk , ∂tρvac 0k = 0, ρvac 1k = −1 2 and ρvac 2k = ωk 2 Θk, (4.8) (4.8) where Θk ≡θ ω2 k(t) . Because we are assuming that ϕR is a real scalar ﬁeld we also have nk = nk, which implies that ρ1k = −1/2 at all times, so that the equation for ρ1k is actually redundant. This is indeed a consistent solution even with the friction terms included. 4 Wigner space equations In practice one must introduce a UV-cutoﬀfor the magnitude of the momentum \|k\|, but results should not depend on its precise value, because all non-trivial physics results from gradient terms acting in the infrared region. We have indeed shown that this is the case in our numerical examples. Friction. Our main goal is to study the dynamical evolution of ϕR including the backre- action from the modes excited during the zero-crossings (parametric resonance) and from the unstable modes (spinodal, or tachyonic, instability). We would also like to study dissipative interactions in our system. To do this correctly, we should go beyond the Hartree approximation. This would be in principle a straightforward but very laborious task. Some formal results can be found for example in [37]. Here we will instead add phenomenological friction terms to our equations. Following references [27] and [28] we – 14 – generalize equations (4.4a), (4.4b) and (4.4c) as follows: 1 4∂2 t ρ0k −ρ2k + ω2 k(t)ρ0k = −c1∂tρ0k, (4.6a) ∂tρ1 = −c2 δρ1k −δρeq 1k , (4.6b) ∂tρ2k −1 2 h ∂t m2 eﬀ(t) i ρ0k = −c2 δρ2k −δρeq 2k , (4.6c) (4.6a) (4.6b) (4.6c) where δρnk ≡ρnk −ρvac nk with ρvac nk being the vacuum moments deﬁned in equations (4.8) below, and the explicit forms for the equilibrium distributions ρeq nk have to be provided externally depending on the problem. Collision integrals could be computed accurately in the context of the cQPA formalism following reference [28] (see also [38]), but here we are only interested in qualitative eﬀects, for which the above phenomenological approach is suﬃcient. Even then the coeﬃcients ci could be some momentum dependent functions, but for simplicity we will assume that they are constants. Note that ρnk and ρeq nk in general have diﬀerent vacuum distributions due to diﬀerent respective solutions to mass gap equations. JHEP12(2021)190 Number densities and coherence function. We can get a better understanding of the physical meaning of the moments by comparing them with the spectral cQPA solutions found in reference [27]. As explained in section 4.2 of reference [27], the moments are in a one-to-one correspondence with the cQPA mass-shell functions fm k± and the coherence function fc k. 5.1 Particle production and reheating via parametric resonance JHEP12(2021)190 We ﬁrst consider a case where the ﬁeld starts from a relatively large value and oscillates several times between positive and negative ﬁeld values. Because we are also interested in the spinodal instability, we consider a tree-level potential with a negative mass term. As physical parameters we use mR = 100 GeV and λ (4) R = 1, given which, µ2 R(2)(Q0) can be solved from (3.13), while the running couplings and masses are deﬁned in (3.19). We compute the initial value for the eﬀective mass function m2(ϕR, ∆R) using the vacuum Hartree approximation (3.25). We used running parameters everywhere in our calculations. This served as a useful consistency check, since all ﬁnal results must be (and indeed were) scale independent. In this example we also set the friction terms to zero, ci = 0. The essential results for this run are shown in ﬁgures 4 and 5. In the left panel of ﬁgure 4 we show the evolution of the classical ﬁeld ϕR, which here displays an orderly oscillation pattern with a slowly decaying amplitude. The middle panel of ﬁgure 4 shows the evolution of the ﬂuctuations in the zeroth moment integrated over the 3-momentum, which is the non-equilibrium contribution to the local correlation function: R k δρ0k = R k ρ0k −ρvac 0k ≡ δ∆F(t, t). These results are in good agreement with reference [16], where this problem was studied earlier using the mode equation approach. The rapid increase of δ∆F(t, t) at early times is caused by two non-perturbative processes, the spinodal instability and the parametric resonance. Spinodal instability. The presence of a spinodal instability is manifest in the right panel of ﬁgure 4, where the eﬀective mass term m2(ϕR, ∆R) is seen to become periodically negative in the region t ≲0.25. Indeed, whenever the mass-function is negative, all k-modes satisfying k2 + m2(ϕR, ∆R) < 0 (5.1) (5.1) 4The use of the term phase transition is not very accurate here, as we do not have a phase transition in the same sense as for example in the electroweak transition. Rather, we have a situation where the universe evolves from a cold initial state to a hot ﬁnal state. It is a common practice however to refer to this phenomenon as a phase transition as well, and we will also do so in what follows. 5 Numerical results We shall now solve the coupled dynamical equations (4.6a), (4.6b), (4.6c), (4.5) and (4.4d) in a few examples chosen to illustrate the rich physics of the strongly coupled system – 15 – including the quantum backreaction. We will uncover some known results and ﬁnd new phenomena associated with spinodal and resonant particle production at phase transitions.4 We will show that a strong spinodal instability can cause a quantum assisted barrier pene- tration without tunneling, and we emphasize the diﬃculty of giving any sensible deﬁnition for the eﬀective potential in a non-equilibrium system. Eventually, we will follow the full thermalization process of a scalar ﬁeld starting at rest in the vacuum potential until the end, when the energy in the ﬁeld is almost completely transformed into thermal ﬂuctuations.5 5.1 Particle production and reheating via parametric resonance 5Let us make a note on units: in section 3.3, when discussing the thermal eﬀective potentials, we gave the mass parameter a value characteristic for the electroweak phase transition, mR = 100 GeV. Below we continue to use the same value as a benchmark, and we shall be measuring all dimensionful quantities in the GeV-units. In particular, we will be measuring time in units GeV−1, while we will be suppressing time-units in all plots. However, in all examples that we will consider below, the physical mass mR is the only mass scale in the problem. Thus, all results are in fact valid as such for an arbitrary mass value, if only one rescales all dimensionful parameters by a suitable power of mR/GeV. – 16 – 0 0.5 1 1.5 2 -400 -200 0 200 400 0 0.5 1 1.5 2 0.0 0.5 1.0 1.5 104 0 0.5 1 1.5 2 0 1 2 3 4 104 Figure 4. Shown is the evolution of the classical ﬁeld as a function of time (left), evolution of the integrated non-equilibrium part of the local correlation function (middle), and the eﬀective mass function m2(ϕR, ∆R) (right). We used λ (4) R = 1, mR = 100 GeV, ϕR,in = 300 GeV and ∂tϕR,in = 0. The moment functions were initialized to the non-coherent vacuum values (4.8). We also assumed no friction, setting ci to zero. 0 0.5 1 1.5 2 0.0 0.5 1.0 1.5 104 0 0.5 1 1.5 2 0 1 2 3 4 104 0 0.5 1 1.5 2 -400 -200 0 200 400 Figure 4. Shown is the evolution of the classical ﬁeld as a function of time (left), evolution of the integrated non-equilibrium part of the local correlation function (middle), and the eﬀective mass function m2(ϕR, ∆R) (right). We used λ (4) R = 1, mR = 100 GeV, ϕR,in = 300 GeV and ∂tϕR,in = 0. The moment functions were initialized to the non-coherent vacuum values (4.8). We also assumed no friction, setting ci to zero. JHEP12(2021)190 are unstable and can grow exponentially. This is the spinodal or tachyonic instability. One might then be tempted to associate the growth in ﬂuctuations in the period t ≲0.25 fully to the spinodal instability. If this was true, the excited modes should satisfy the condition (5.1), which here translates to \|k\| ≲60 GeV. 5.1 Particle production and reheating via parametric resonance However, from ﬁgure 5 we see that this is not the case. The fast production of modes is clearly visible in the upper panels which show the integrated particle number (left) and the integrated modulus of the coherence functions (right). But from the lower panels, showing time-momentum heat plots of the same quantities, we see that the excited modes are concentrated on a frequency band which lies entirely above the spinodal region (5.1). Parametric resonance. While our equations are highly non-linear and strongly self- coupled, it is apparent that the structures seen in the heat plots in ﬁgure 5 correspond to Mathieu instabilities associated with parametric resonance, familiar from the studies of inﬂationary reheating [4]. This problem was also studied using 2PI methods in ref- erence [7], albeit with a diﬀerent set of approximations and a diﬀerent potential. If we identify the mass squared of the mode function in the Mathieu equation with our mass function m2(ϕR, ∆R), and follow the analysis of section V in reference [4], we can (very roughly) estimate the Mathieu equation q-parameter in our case to be q ∼2 ∆m2 eﬀ (2πν)2 ≈2, (5.2) (5.2) where ∆m2 eﬀ≈2 × 104 GeV2 is the instantaneous amplitude and ν ≈21 GeV is the local frequency of oscillations of the eﬀective mass term m2(ϕR, ∆R), shown in ﬁgure 4. The value of the q-parameter, which remains roughly the same throughout the calculation, suggests an intermediate resonance between the narrow and broad regimes. Similarly, the expected position of the ﬁrst resonance band is by and large estimated to be \|k\|rb ∼πν 4√ 2 ≈60 GeV. (5.3) (5.3) This result, and the expected width of the resonance [4] ∆\|k\| ∼\|k\|rb ≈60 GeV are in quali- tative agreement with our results. In ﬁgure 5 we can even observe a second, much narrower This result, and the expected width of the resonance [4] ∆\|k\| ∼\|k\|rb ≈60 GeV are in quali- tative agreement with our results. In ﬁgure 5 we can even observe a second, much narrower – 17 – Figure 5. Shown is the evolution of the integrated number density (top left) and the absolute value of the integrated coherence function f c± k (top right), deﬁned in equations (4.7), for the same parameters as in ﬁgure 4. 5.1 Particle production and reheating via parametric resonance The bottom row shows the heat plots in the momentum and time variables for the unintegrated distributions multiplied by the phase space factors: k2 2π2 nk (lower left) and k2 2π2 f c± k (lower right). JHEP12(2021)190 Figure 5. Shown is the evolution of the integrated number density (top left) and the absolute value of the integrated coherence function f c± k (top right), deﬁned in equations (4.7), for the same parameters as in ﬁgure 4. The bottom row shows the heat plots in the momentum and time variables for the unintegrated distributions multiplied by the phase space factors: k2 2π2 nk (lower left) and k2 2π2 f c± k (lower right). band below the ﬁrst one, which dominates the particle production at t ≈1. While this is again in agreement with the qualitative expectations, its interpretation via Mathieu equa- tion methods becomes even more tenuous. At late times t ≳0.3 the shape of the growth pattern ﬁts well in the standard picture [4], but in the spinodal region the resonant pro- duction appears to be more eﬃcient than usual: upon spinodal zero-crossings the resonant production that normally shows (as it indeed does at later times also in our example) a pe- riod of anti-correlation, is here always positively correlated. While individual growth bursts are not enhanced, this positive correlation leads to particularly strong particle production. Because we did not include interactions in this run, the ﬂuctuation band structure remains stable at all times. The system also remains highly coherent, as is evident both from the increase of the integrated coherence function and the stability of the heat plot of the coherence function shown in the right panels of ﬁgure 5. 5.2 Strong spinodal instability In the above analysis we made little reference to the eﬀective potential. Indeed, the one- particle irreducible eﬀective action is not a very useful quantity in an out-of-equilibrium setting and it can even be deﬁned only after the equations of motion have been solved. Even then one cannot deﬁne it universally, but only as a quantity evaluated locally in time. We will now study this question in the case of a very strong spinodal instability. To be speciﬁc, we still use the values mR = 100 GeV, λ (4) R = 1 and ∂tϕR,in = 0, but we take ϕR,in = 243.5 GeV and include also friction. We assume that collisions drive the system to the vacuum state, i.e. we take δρeq nk ≡0, and we specify the coeﬃcients to be – 18 – 0 0.5 1 1.5 -200 0 200 -200 -100 0 100 200 -1 0 1 2 3 4 5 107 0 0.5 1 1.5 -1 0 1 2 104 Figure 6. The upper left panel shows the time evolution of ϕR (in units GeV) and the lower left panel that of the eﬀective mass function m2(ϕR, ∆R) (in units GeV2) in the case of a strong spinodal instability. In the right panel we show the time-evolution of the instantaneous eﬀective potential (5.4) (dashed black line), embedded in a plot of the vacuum Hartree potential (dashed red line). The colored dots indicate select times at which the instantaneous potential was evaluated as indicated in the left panels. The solid blue line shows the instantaneous value of the non-equilibrium vacuum potential (5.5). -200 -100 0 100 200 -1 0 1 2 3 4 5 107 0 0.5 1 1.5 -200 0 200 0 0.5 1 1.5 -1 0 1 2 104 JHEP12(2021)190 Figure 6. The upper left panel shows the time evolution of ϕR (in units GeV) and the lower left panel that of the eﬀective mass function m2(ϕR, ∆R) (in units GeV2) in the case of a strong spinodal instability. In the right panel we show the time-evolution of the instantaneous eﬀective potential (5.4) (dashed black line), embedded in a plot of the vacuum Hartree potential (dashed red line). The colored dots indicate select times at which the instantaneous potential was evaluated as indicated in the left panels. The solid blue line shows the instantaneous value of the non-equilibrium vacuum potential (5.5). 6Although we gave the friction terms only in a qualitative form, we can provide an estimate for the magnitude of the ci-coeﬃcients. From equations (4.6) it is clear that ci have the dimensions of mass. The lowest order contribution to the collision integrals arises at the second order in coupling in the 2PI expansion. Hence the naïve scale of the coeﬃcients ci is given by λ 4π 2m, which for λ (4) R = 1 and mR = 100 GeV gives ci ≃0.6 GeV. 5.2 Strong spinodal instability c1,2 = 0.6 GeV.6 In this case the initial potential energy of the ﬁeld is lower than the peak of the vacuum potential at ϕR = 0. This can be seen in the right panel of ﬁgure 6, where we plot the Hartree-resummed vacuum potential (red dashed line) and indicate the initial ﬁeld value by the black dot. Obviously, if the potential was held ﬁxed, the ﬁeld would simply oscillate around the positive minimum with a decaying amplitude. However, when backreaction is included, the picture changes dramatically. The actual ﬁeld evolution is shown in the upper left panel of ﬁgure 6. Curiously, the ﬁeld stays around the positive minimum during only one oscillation cycle, after which it apparently passes through the potential barrier, spending a rather long time near the middle of the potential with the eﬀective mass function close to zero. Of course what happens is that in the ﬁrst passage of the ﬁeld into the spinodal region, an explosive creation of ﬂuctuations takes place. This is clearly demonstrated in ﬁgure 7, which shows the integrated ﬂuctuations in the moment functions (upper panels) and the associated heat plots in the time-momentum plane (lower panels). These ﬂuctuations absorb a large amount of entropy, which decreases the free energy in the system and lowers the barrier between the minima allowing the ﬁeld to pass to the negative side. The key issue is to not confuse the total internal energy of the system and the free energy, which may vary strongly depending on the entropy production. – 19 – 0 0.5 1 1.5 0.0 0.5 1.0 104 0 0.5 1 1.5 0 1 2 107 0 0.5 1 1.5 20 40 60 80 100 0 200 400 600 0 0.5 1 1.5 20 40 60 80 100 0 2 4 6 8 105 Figure 7. The upper panels: shown are the integrated non-equilibrium ﬂuctuations of the moment functions, R k δρ0,2k. The colored dots have the same interpretation as in ﬁgure 6. The lower panels: heat plots showing the momentum distributions 1 2π2 k2δρnk corresponding to the upper panels. The left panels show the zeroth moment n = 0 and the right panels the second moment n = 2. 7In reference [16] yet another dynamical potential was deﬁned as the diﬀerence between the total ener of the system and the kinetic energy of the classical ﬁeld. 5.2 Strong spinodal instability 0 0.5 1 1.5 0 1 2 107 0 0.5 1 1.5 20 40 60 80 100 0 2 4 6 8 105 0 0.5 1 1.5 0.0 0.5 1.0 104 JHEP12(2021)190 Figure 7. The upper panels: shown are the integrated non-equilibrium ﬂuctuations of the moment functions, R k δρ0,2k. The colored dots have the same interpretation as in ﬁgure 6. The lower panels: heat plots showing the momentum distributions 1 2π2 k2δρnk corresponding to the upper panels. The left panels show the zeroth moment n = 0 and the right panels the second moment n = 2. Non-equilibrium eﬀective potentials. While the eﬀective potential cannot be deﬁned a priori, it is illustrative to construct it a posteriori as a time dependent potential that reproduces the equation of motion (4.4d) at all times. This potential can be constructed as the deﬁnite integral V1PI(t; ϕR) ≡ Z t tin −1 3λ (2) R ϕ3 R + m2(ϕR, ∆R)ϕR (∂˜tϕR)d˜t, (5.4) (5.4) where ϕR and ∆R are the solutions of the equations of motion. We show this potential as the dashed black line in ﬁgure 6. After the crossing to the negative side, the shape of the potential function settles and the ﬁeld oscillates around the negative minimum with a decaying amplitude. We stress that V1PI is only useful for the visualization and interpretation of results and there is no unique deﬁnition of the eﬀective potential in the non-equilibrium case. As was already mentioned in section 3.3, in any ﬁnite truncation the renormalized 2PI vacuum becomes dependent on the IR-physics. Another interesting potential7 function then is the equivalent of the vacuum Hartree potential in the presence of ﬂuctuations. This potential is deﬁned as VH∆(ϕR, ∆R) ≡VH(ϕR, ∆R) −1 2m2(ϕR, ∆R) Z k δρ0k, (5.5) (5.5) where VH(ϕR, ∆R) is the 2PI vacuum potential (3.29) evaluated replacing the vacuum mass function m2(ϕR) with the general mass function m2(ϕR, ∆R). Note that the integral term 7In reference [16] yet another dynamical potential was deﬁned as the diﬀerence between the total energy of the system and the kinetic energy of the classical ﬁeld. – 20 – 0 1 2 3 4 5 6 -400 -200 0 200 400 0 1 2 3 4 5 6 0 5 10 15 107 Figure 8. 5.2 Strong spinodal instability Shown is the time-evolution of the classical ﬁeld (left panel) and that of the total energy in the ﬂuctuations and the classical ﬁeld (right panel). Hϕ(t) is the energy in the classical ﬁeld and H∆(t) is the energy in the ﬂuctuations. The physical parameters and the speciﬁc form of the collision integrals used in this run are described in the text. 0 1 2 3 4 5 6 0 5 10 15 107 0 1 2 3 4 5 6 -400 -200 0 200 400 Figure 8. Shown is the time-evolution of the classical ﬁeld (left panel) and that of the total energy in the ﬂuctuations and the classical ﬁeld (right panel). Hϕ(t) is the energy in the classical ﬁeld and H∆(t) is the energy in the ﬂuctuations. The physical parameters and the speciﬁc form of the collision integrals used in this run are described in the text. JHEP12(2021)190 over the ﬂuctuations of the zeroth moment is a part of the vacuum Hartree potential, similarly to the case with the thermal potential (3.37). The potential (5.5) is shown with the blue solid line in the right panel of ﬁgure 6. It represents changes in the 2PI Hartree vacuum energy including the backreaction eﬀects, and like the instantaneous V1PI-potential, its barrier around ϕR = 0 is temporarily lowered by the backreaction. This example demonstrates that the ﬁnal stages of a phase transition may involve very complicated quantum dynamics, where classical expectations and constraints do not hold. We conclude this subsection by stressing on the diﬀerence of the ﬂuctuation spectra in the present case, shown in the lower panels of ﬁgure 7, and in the parametric resonance case shown in ﬁgure 5. Even though we used the same mass and coupling parameters, essentially all ﬂuctuations are here created by the spinodal instability. Indeed, they occupy a region in the phase space which is consistent with the instability constraint (5.1), continues all the way to zero momentum and lies entirely below the parametric resonance band. 5.3 Self-thermalization As our ﬁnal example we study thermalization of the scalar ﬁeld energy in a self-interacting system. We use the same physical parameters and initial conditions as in section 5.1 but include collision terms with the friction coeﬃcients c0,1 = 0.6 GeV, and assume that the collisions drive the system to thermal equilibrium, i.e. we take δρeq nk ≡δρth nk. With rigorously computed collision terms the thermal state would emerge automatically as an attractor solution, but in our phenomenological approach we need to give a deﬁnition for the instantaneous temperature. In thermal equilibrium a general moment can be written as ρth nk = 1 2 ωn−1 k h nBE(ωk) + (−1)n1 + nBE(ωk) i , (5.6) (5.6) where nBE(k0) = (ek0/T −1)−1 is the Bose-Einstein distribution function. In particular k0/T −1)−1 is the Bose-Einstein distribution function. In particular δρth 0k = 1 ωk nBE(ωk) and δρth 2k = ωknBE(ωk). (5.7) (5.7) – 21 – – 21 – 0 2 4 6 0.0 0.5 1.0 106 0 2 4 6 0 2 4 6 105 0 2 4 6 50 100 150 200 0 1 2 3 4 104 0 2 4 6 50 100 150 200 1 2 3 4 104 Figure 9. Shown are the evolution of the number density (left) and the modulus of the coherence functions (right). In the upper panels the quantities are integrated over momentum. We used the same parameters as in ﬁgure 5, except for non-zero friction coeﬃcients ci = 0.6 GeV in the collision integrals with thermal equilibrium solutions. 0 2 4 6 0 2 4 6 105 0 2 4 6 50 100 150 200 1 2 3 4 104 0 2 4 6 0 2 4 6 105 0 2 4 6 0.0 0.5 1.0 106 0 2 4 6 50 100 150 200 0 1 2 3 4 104 0 2 4 6 50 100 150 200 1 2 3 4 104 JHEP12(2021)190 Figure 9. Shown are the evolution of the number density (left) and the modulus of the coherence functions (right). In the upper panels the quantities are integrated over momentum. We used the same parameters as in ﬁgure 5, except for non-zero friction coeﬃcients ci = 0.6 GeV in the collision integrals with thermal equilibrium solutions. while δρth 1k = 0. 5.3 Self-thermalization We deﬁne the equivalent temperature T = T(t) by requiring that the thermal state has the same energy as what is stored in the ﬂuctuations: while δρth 1k = 0. We deﬁne the equivalent temperature T = T(t) by requiring that the thermal state has the same energy as what is stored in the ﬂuctuations: H∆(t) ≡ Z k δρ2k(t) ≡ Z k ωknBE(ωk). (5.8) (5.8) In all these equations ω2 k = k2 + m2(ϕR, ∆R) is a function of time. The energy stored in the classical ﬁeld is Hϕ(t) ≡1 2 ∂tϕR(t) 2 + VH∆(ϕR(t), ∆R(t)). (5.9) (5.9) With our deﬁnitions of the temperature and the collision integrals the total energy H = Hϕ + H∆should be conserved, and we checked that this is indeed the case to a high accu- racy in our calculations. For more details on this, and on the numerical setup in general, see appendix A. Spinodal slowing. In the left panel of ﬁgure 8 we show the evolution of the classical ﬁeld ϕR. Initially ϕR evolves as in the collisionless case, oscillating with a nearly constant frequency and a large amplitude, but around t ∼2 the frequency starts to decrease until it reaches a minimum around t ∼3. After this the ﬁeld gets trapped around the positive minimum while the oscillation frequency increases again. This spinodal slowing eﬀect was already seen in connection with the barrier crossing in section 5.2. The bearing of the spinodal modes is revealed in the inset in the left panel of ﬁgure 11, which shows that the eﬀective mass term m2(ϕR, ∆R) repeatedly becomes negative in this region. In the right panel of ﬁgure 8 we show the energy components Hϕ and H∆. Initially all energy is stored in the classical ﬁeld, but the fraction of energy in the ﬂuctuations increases until the system is reheated, with almost all of the energy contained in the ﬂuctuations. – 22 – 0 200 400 600 800 0 0.5 1 1.5 2 2.5 3 106 0 500 1000 1500 0 0.5 1 1.5 2 105 0 200 400 600 800 0.0 0.5 1.0 1.5 104 0 500 1000 1500 0.0 0.1 0.1 103 Figure 10. 5.3 Self-thermalization Shown are the momentum distributions k2 2π2 δρ2k (left) and k2 2π2 f c± k (right) for three diﬀerent times: t = 0.2 (solid blue lines) t = 1.3 (red dotted lines) and t = 6 (black dashed lines). Also shown in the left plot is the weighted thermal distribution k2 2π2 ωknBE(ωk) for the equivalent temperature T(t = 6) = 144.9 GeV (black dotted line). 0 200 400 600 800 0.0 0.5 1.0 1.5 104 0 500 1000 1500 0.0 0.1 0.1 103 0 200 400 600 800 0 0.5 1 1.5 2 2.5 3 106 0 500 1000 1500 0 0.5 1 1.5 2 105 JHEP12(2021)190 Figure 10. Shown are the momentum distributions k2 2π2 δρ2k (left) and k2 2π2 f c± k (right) for three diﬀerent times: t = 0.2 (solid blue lines) t = 1.3 (red dotted lines) and t = 6 (black dashed lines). Also shown in the left plot is the weighted thermal distribution k2 2π2 ωknBE(ωk) for the equivalent temperature T(t = 6) = 144.9 GeV (black dotted line). Mode transfer and decoherence. In ﬁgure 9 we again show the evolution of the number density and coherence functions, including both the integrated quantities and the time-momentum heat plots. There are striking, but expected diﬀerences between these plots and the corresponding non-interacting results shown in ﬁgure 5. First, the number density stops growing already at t ∼1 and eventually starts to decrease for t ≳2. As is seen from ﬁgure 8, ﬂuctuations dominate the total energy already for t ≳1, and the subsequent decrease of particle number results from a transfer of modes to higher energies. Thermalization process should also lead to decoherence, and this is indeed clearly visible in the upper right panel of ﬁgure 9, which shows the integrated function fc± k . From the heat plots we see that particle production gets progressively less eﬃcient and moves to smaller frequencies, as less and less energy is left in the classical ﬁeld. From the heat plot in the lower right panel we see that coherence is erased throughout the phase space at late times. Thermalization. In ﬁgure 10 we show the \|k\|-distributions of δρ2k (left panel) and the coherence function fc± k (right panel) weighted by the phase space factor, for selected times during the evolution. 5.3 Self-thermalization The black arrows indicate the limiting cases of vacuum (w = −1) and kinetic (w = 1) energy dominance as well as matter (w = 0) and radiation (w = 1/3) EOS’s, shown by horizontal lines. In all graphs shown the red arrow points the region of maximal spinodal slowing. JHEP12(2021)190 Also the ﬂuctuations in the equivalent temperature have but a small residual amplitude left. For the ﬁnal time we also plotted (black dotted line in the left panel of ﬁgure 10) the equivalent thermal spectrum k2 2π2 ωknBE(ωk) with T = 144.9 GeV, corresponding to the equivalent temperature at t = 6. The close agreement between the actual and thermal distributions shows that the system has indeed thermalized to a very high accuracy. Also the ﬂuctuations in the equivalent temperature have but a small residual amplitude left. For the ﬁnal time we also plotted (black dotted line in the left panel of ﬁgure 10) the equivalent thermal spectrum k2 2π2 ωknBE(ωk) with T = 144.9 GeV, corresponding to the equivalent temperature at t = 6. The close agreement between the actual and thermal distributions shows that the system has indeed thermalized to a very high accuracy. Equation of state. Let us ﬁnally study the evolution of the equation of state (EOS) in the system. The EOS-parameter is deﬁned as w ≡P H, (5.10) (5.10) where H = Hϕ+H∆is the total energy and the total pressure P = Pϕ+P∆is similarly the sum of the pressures in the classical ﬁeld and in the ﬂuctuations. The former is given by Pϕ = 1 2(∂tϕR)2 −VH∆(ϕR, ∆R), (5.11) (5.11) where VH∆was deﬁned in (5.5). The pressure contained in the ﬂuctuations can be com- puted as the spatial component of the energy-momentum tensor [27], and it can be written in terms of the moment functions as follows: where VH∆was deﬁned in (5.5). The pressure contained in the ﬂuctuations can be com- puted as the spatial component of the energy-momentum tensor [27], and it can be written in terms of the moment functions as follows: P∆(ϕR, ∆R) = Z k δρ2k(t) + 1 3k2 −ω2 k δρ0k(t) . (5.12) (5.12) It is easy to see that in the thermal limit (5.12) reduces to the negative of the thermal part of the eﬀective potential in the Hartree approximation: P∆= −T 4J m2 T /T 2. 5.3 Self-thermalization At a relatively early time t = 0.2 the distributions shown in solid blue still display a clear parametric resonance band structure. At a later time t = 1.3 (red dotted lines) the resonant spectrum is already much more complex, apparently with contributions from many narrow bands. Also a signiﬁcant mode-transfer to the thermal region has already taken place. Indeed, from the main plot in the left panel of ﬁgure 11 we see that the equivalent temperature at t = 1.3 is roughly 140 GeV, and as the ﬁeld is relatively light, ⟨m2 eﬀ⟩1/2/T ≲1 with ⟨m2 eﬀ⟩being the local average of the oscillating eﬀective mass function, the expected maximum of the thermal spectrum is located at ⟨\|k\|⟩≈3T ≈400 GeV. At the end of the simulation, t = 6 (black dashed curve), the system has essentially thermalized. Almost all energy is in the ﬂuctuations and very little particle production activity remains. The particle number in the resonance bands is small and the coherence is almost vanishing everywhere and in particular in the thermal region. – 23 – 6 0 1 2 3 4 5 6 -1 -0.5 0 0.5 1 0 1 2 3 4 5 6 0 50 100 150 2 3 4 5 6 0 0.5 1 0 1 2 3 4 5 6 -1 -0.5 0 0.5 1 Figure 11. In the left panel we show the equivalent temperature deﬁned through equation (5.8) as a function of time. The inset shows the parameter xeﬀ≡sgn m2 eﬀ m2 eﬀ 1/2/T. In the right panel we show the EOS-parameter of the system deﬁned in equation (5.10). The black arrows indicate the limiting cases of vacuum (w = −1) and kinetic (w = 1) energy dominance as well as matter (w = 0) and radiation (w = 1/3) EOS’s, shown by horizontal lines. In all graphs shown the red arrow points the region of maximal spinodal slowing. 0 1 2 3 4 5 6 0 50 100 150 2 3 4 5 6 0 0.5 1 Figure 11. In the left panel we show the equivalent temperature deﬁned through equation (5.8) as a function of time. The inset shows the parameter xeﬀ≡sgn m2 eﬀ m2 eﬀ 1/2/T. In the right panel we show the EOS-parameter of the system deﬁned in equation (5.10). 6 Conclusions JHEP12(2021)190 We have studied the non-equilibrium evolution of a system consisting of a classical scalar ﬁeld coupled to the two-point function describing quantum ﬂuctuations. We derived renor- malized evolution equations for the system using 2PI methods in the Hartree approxima- tion. We derived the eﬀective potential for this system in vacuum and in thermal equi- librium and compared the latter with the known one-loop-resummed eﬀective potentials. We showed that the Parwani-resummed thermal potential [33] is closest in spirit to the Hartree-resummed eﬀective potential. We showed that in a non-equilibrium situation the 2PI method, in any ﬁnite truncation, leads to an eﬀective vacuum potential (the vacuum state) that depends on the infrared physics. Indeed, even though the renormalization pro- cedure provides unique and constant counterterms, the split of the system into divergent and non-divergent parts depends on the IR-physics. We wrote our renormalized evolution equations as a set of coupled moment-equations for the correlation function and a ﬁeld equation for the one-point function in the mixed representation and included phenomenological collision integrals describing friction. We used this system to study the non-perturbative particle production and spinodal instabil- ity at the end of phase transitions. We found out that quantum backreaction can have signiﬁcant eﬀects on the evolution of the system and addressed the problems in trying to deﬁne any practical eﬀective potential for such dynamical systems. In particular we were able to follow the full thermal history of a self-interacting system starting from a cold initial state where all energy in the system was stored in the classical potential, until the end when the system was reheated and thermalized and the ﬁeld stayed at the minimum of the thermal (Hartree) eﬀective potential. In this work we assumed that the quantum system lived in the Minkowski space-time. Generalization to an expanding FRLW space-time is straightforward by a simple transform to conformal coordinates [40]. Moreover, in many realistic systems the time scales involved in the phase transition are much faster than the Hubble expansion. In those cases our results are representative of the physics as such. Also, we used only a phenomenological form for the collision integrals. It would be interesting to derive more realistic collision terms using the methods developed in [28, 39]. Also it would be interesting to couple the scalar ﬁeld also to other quantum ﬁelds. 5.3 Self-thermalization We plot the EOS-parameter w in the right panel of ﬁgure 11. The EOS-parameter starts from w = −1 and initially oscillates between w = −1, corresponding to total vac- uum energy dominance, and w = 1, corresponding to kinetic energy dominance (kina- tion) in the classical ﬁeld sector. However, as the energy is moved out from the ﬁeld and the system thermalizes, the EOS-parameter moves to the band 0 < w < 1/3 corre- sponding to normal matter. From the inset of the left panel we see that the average value – 24 – ⟨\|xeﬀ\|⟩= ⟨\|m2 eﬀ\|1/2/T⟩≈0.6 at late times. This indicates that the reheated thermal plasma is almost relativistic and indeed, the EOS-parameter is asymptoting close to w = 1/3 at late times. (In a purely thermal plasma with xeﬀ= 0.6 one would get w ≈0.315.) The periodic deviation below this value seen in ﬁgure 11 is due to the ﬁeld contributions to energy and pressure. 6 Conclusions This should be straightforward by combining the current results with the quantum transport equations for fermions developed in [41]. In this way one should be able to study reheating at the end of inﬂation in a realistic setup. – 25 – Acknowledgments This work was supported by the Academy of Finland grant 318319. OK was in addition supported by a grant from the Magnus Ehrnrooth Foundation. We wish to thank Alexandre Alvarez, Amitayus Banik, Haye Hinrichsen, Sami Nurmi, Werner Porod and Anna Tokareva for discussions and comments on the manuscript. A Numerical implementation In this appendix we discuss some technical points that are relevant for an accurate and eﬃcient solution of the evolution equations. The ﬁrst one concerns identifying a conserved quantity in the non-interacting limit. The equations rewritten using this variable are much more stable than the original equations. The second point concerns discretization. In a naïve binning of the momentum variable, the discrete integral of the vacuum term in equation (4.5) is badly behaved numerically near the edges of the spinodal regions. This problem can be avoided by a more careful deﬁnition of the binned variables. Finally, we show how our numerical setup conserves the total energy of the solved system to a high accuracy with the self-thermalizing system as a case study. JHEP12(2021)190 Stabilized equations. It was noted already in reference [27] that the moment equa- tions (4.4a), (4.4b) and (4.4c) can be written in a form that is more resistant to numerical instabilities, using the variable Xk ≡2ρ0kρ2k −ω2 k(t)ρ2 0k −1 4(∂tρ0k)2. (A.1) (A.1) Indeed, if we multiply (4.4a) by 2∂tρ0k and (4.4c) by 2ρ0k and subtract the resulting equations, we can show that Xk is conserved in the collisionless limit: ∂tXk = 0. With non- vanishing friction terms Xk is no longer conserved, but the derivation with equations (4.6) including friction proceeds analogously, and one ﬁnds: 1 4∂2 t ρ0k −ρ2k + ω2 k(t)ρ0k = −c1∂tρ0k, (A.2a) ∂tρ1k = −c2 δρ1k −δρeq 1k , (A.2b) ∂tXk = 2c1 ∂tρ0k 2 −2c2ρ0k δρ2k −δρeq 2k . (A.2c) (A.2b) (A.2c) We have thus replaced ρ2k by Xk as a dynamical variable. We will use (A.1) to set the initial condition for Xk in terms of the initial values for ρ0k, ∂tρ0k and ρ2k, and at any point during and at the end of the calculation we can compute ρ2k from Xk using the inverse relation 1 1 We have thus replaced ρ2k by Xk as a dynamical variable. We will use (A.1) to set the initial condition for Xk in terms of the initial values for ρ0k, ∂tρ0k and ρ2k, and at any point during and at the end of the calculation we can compute ρ2k from Xk using the inverse relation ρ2k = 1 2ρ0k Xk + 1 4(∂tρ0k)2 + ω2 k(t)ρ2 0k . (A.3) (A.3) (A.3) Coarse-grained binning. A Numerical implementation Whenever the eﬀective mass term is negative there is a mo- mentum for which m2(ϕR, ∆R) = −k2 and at this point the zeroth momentum vacuum function ρvac 0k = Θk/(2ωk) diverges. This is a mild, integrable singularity that does not Coarse-grained binning. Whenever the eﬀective mass term is negative there is a mo- mentum for which m2(ϕR, ∆R) = −k2 and at this point the zeroth momentum vacuum function ρvac 0k = Θk/(2ωk) diverges. This is a mild, integrable singularity that does not – 26 – Figure 12. Shown is the relative change in energy δH = H/H0 −1 during calculation in the self-thermalization case studied in section 5.3. Inset shows a close-up on the ﬁrst spinodal instability region. JHEP12(2021)190 Figure 12. Shown is the relative change in energy δH = H/H0 −1 during calculation in the self-thermalization case studied in section 5.3. Inset shows a close-up on the ﬁrst spinodal instability region. aﬀect the continuum limit, but it can cause overﬂows and numerical inaccuracy in a sys- tem with a ﬁnite discretization. This problem can be avoided by a careful choice of binned variables for the vacuum distribution. That is, we replace the vacuum distribution by a coarse-grained distribution deﬁned by an integration over each momentum bin q ∈[qi, qi+1]: aﬀect the continuum limit, but it can cause overﬂows and numerical inaccuracy in a sys- tem with a ﬁnite discretization. This problem can be avoided by a careful choice of binned variables for the vacuum distribution. That is, we replace the vacuum distribution by a coarse-grained distribution deﬁned by an integration over each momentum bin q ∈[qi, qi+1]: 1 2ωqci → 1 2q2 ci∆qi i0(qi+1) −i0(qi) , (A.4) where qci ≡1 2(qi + qi+1), ∆qi ≡qi+1 −qi and i0(q) ≡1 2 qωq −m2artanh q ωq . (A.5) 1 2ωqci → 1 2q2 ci∆qi i0(qi+1) −i0(qi) , (A.4) (A.4) where qci ≡1 2(qi + qi+1), ∆qi ≡qi+1 −qi and i0(q) ≡1 2 qωq −m2artanh q ωq . (A.5) (A.5) When the bin width goes to zero, the replacement (A.4) does not make any diﬀerence. However, for a ﬁnite discretization it avoids the singularity that would occur in the spinodal region when the eﬀective mass function coincides with one of the bin-momenta squared, m2(ϕR, ∆R) = −q2 ci. When the bin width goes to zero, the replacement (A.4) does not make any diﬀerence. A Numerical implementation However, for a ﬁnite discretization it avoids the singularity that would occur in the spinodal region when the eﬀective mass function coincides with one of the bin-momenta squared, m2(ϕR, ∆R) = −q2 ci. Energy conservation. In ﬁgure 12 we show the relative change in the total energy δH ≡H/H0−1 in the example we studied in section 5.3. The total energy is H = Hϕ+H∆, where partial energies in the ﬂuctuations H∆and in the classical ﬁeld Hϕ were deﬁned in equations (5.8) and (5.9). In this example the total energy should be conserved, and this is indeed true to a very high accuracy. In this run we used a discretized momentum \|k\| ∈[0, 2000] GeV with 1000 grid points. As can be seen in the ﬁgure, the error is essentially negligible between the spinodal regions. Within the spinodal regions there is some residual noise at early times. This arises from the integrable singularity near m2(ϕR, ∆R) = 0, even with the coarse grained binning, but even this error is small and can be further reduced by reducing the bin width. We conclude that numerical errors are well under control in our calculations. – 27 – Open Access. 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https://openalex.org/W2751147978	https://www.ajol.info/index.php/tjpr/article/download/160745/150314	English	null	Anti-osteoporotic effect of Gengnian Jianshen decoction in rats	Tropical journal of pharmaceutical research	2,017	cc-by	2,947	Anti-osteoporotic effect of Gengnian Jianshen decoction Wang Wang1, Qi-Bin Lu2, Qiang Fu3, Jie Mao1 and Jun Li3 1Department of Gynecology, The Second Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210017, 2Department of Gynecology, Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu Province, 3Department of Orthopedics, Changhai Hospital Affiliated to The Second Military Medical University, Shanghai 200433, China For correspondence: Email: luqibin494@126.com; Tel: +86 025-85811926 For correspondence: Email: luqibin494@126.com; Tel: +86 025-85811926 Sent for review: 31 October 2016 Revised accepted: 15 July 2017 Abstract Purpose: To investigate the therapeutic effect and mechanism of action of Gengnian Jianshen Decoction (GJD) on ovariectomy-induced osteoporosis in rats. ( ) y p Methods: Female Sprague-Dawley rats were randomly assigned to a sham-operated group (control), and five ovariectomy (OVX) sub-groups, that is, OVX with vehicle (OVX), OVX with Xianling Gubao capsule (positive control drug, 120 mg/kg/day), and OVX with GJD doses (70, 140, and 280 mg/kg/day). The treatments were given orally daily for 16 weeks (starting 4 weeks after the rats were subjected to ovariectomy. Bone mineral density (BMD) of the L4 vertebrae and right femur of each rat was estimated. Serum levels of estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), as well as interleukin-6 (IL-6) and insulin-like growth factor 1(IGF-1) of rats in all the groups were determined using ELISA. g Results: The results showed that GJD dose-dependently inhibited BMD reduction in L4 vertebrae and emur, and significantly increased serum E2, FSH and LH levels (p < 0.05) in the osteoporotic rats. Moreover, GJD significantly decreased serum IL-6 levels and increased levels of IGF-1 (p < 0.05). g Results: The results showed that GJD dose-dependently inhibited BMD reduction in L4 vertebrae and femur, and significantly increased serum E2, FSH and LH levels (p < 0.05) in the osteoporotic rats. Moreover, GJD significantly decreased serum IL-6 levels and increased levels of IGF-1 (p < 0.05). Conclusion: These findings indicate that GJD prevents OVX-induced osteoporosis in rats without hyperplastic effects on the uterus. Thus, GJD has potential for use in the treatment of post-menopausal osteoporosis. , g y (p ) Conclusion: These findings indicate that GJD prevents OVX-induced osteoporosis in rats without hyperplastic effects on the uterus. Thus, GJD has potential for use in the treatment of post-menopausal osteoporosis. Keywords: Gengnian Jianshen decoction, Osteoporosis, Ovariectomy, Bone mineral density Keywords: Gengnian Jianshen decoction, Osteoporosis, Ovariectomy, Bone mineral density Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i8.24 Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i8.24 Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i8.24 Original Research Article Original Research Article INTRODUCTION associated costs are rising rapidly due to increases in aging populations [3]. In the elderly, hip fractures are closely associated with mortality [4]. Hormone deficiency is known to impair cancellous metaphyseal and reduce BMD in humans and animals. Therefore, estrogen deficiency in post-menopausal women has been regarded as a critical factor in the susceptibility of this population to osteoporosis [5]. Osteoporosis is twice as common in women as in men [6], and approximately one in three women over 50 years old experiences an osteoporotic fracture in her life time [7]. Osteoporosis is a systemic skeletal disease characterized by reduced bone mass and microarchitectural deterioration of bone tissue with concomitant increase in bone fragility and susceptibility to fractures [1]. According to data released by the World Health Organization (WHO), osteoporosis affects several million people throughout Europe, USA, and Japan [2]. The incidence of osteoporosis increases dramatically with life expectancy. Accordingly, the risk of osteoporotic fractures and their Trop J Pharm Res, August 2017; 16(8): 1925 Wang et al Committee of Second Affiliated Hospital of Nanjing University of Chinese Medicine (approval ref no. 20100308), and was carried out in compliance with the Directive 2010/63/EU on the handling of animals used for scientific purposes [16]. Committee of Second Affiliated Hospital of Nanjing University of Chinese Medicine (approval ref no. 20100308), and was carried out in compliance with the Directive 2010/63/EU on the handling of animals used for scientific purposes [16]. Clinically, hormone replacement therapy (HRT) has been a popular therapeutic strategy for post- menopausal osteoporosis [8, 9]. However, long- term application of HRT has potential malignant effects on reproductive tissues [10-13]. Other medicines that stimulate bone formation (e.g., growth hormone, sodium fluoride, and parathyroid hormone), or inhibit bone resorption (e.g., bisphosphonates and calcitonin) may prevent progression of bone loss in established osteoporosis. However, these drugs are not effective for a large proportion of the world population, especially in developing countries. In addition, they have side effects such as gastrointestinal reactions, cancers, osteonecrosis of the jaw, and reduced skeletal strength [14,15]. Consequently, there are efforts to develop new drugs with improved therapeutic efficacies, fewer undesirable side effects, and lower costs, so as to substitute or reduce the usage of medicines currently in use. BMD measurement The BMDs of the L1-5 vertebrae and right femurs were estimated using dual-energy X-ray absorptiometry scanning (DEXA, GE Healthcare, USA) with small animal measurement. The measurements were expressed as grams of mineral contents per cm2 of surface area. Scans were performed by the same blinded technician. y Gengnian Jianshen Decoction (GJD) is an empirical formula that has been used for the treatment of osteoporosis in China for many years. This study was aimed at investigating the therapeutic effects and mechanism of action of GJD on ovariectomy-induced osteoporosis in rats. Determination of serum E2, FSH, LH, IL-6 and IGF-1 levels Determination of serum E2, FSH, LH, IL-6 and IGF-1 levels After the rats were sacrificed by cervical dislocation, and serum levels of E2, FSH and LH, and IL-6 and IGF-1 levels were determined by ELISA. BMD of L1-5 vertebrae and femurs Values of BMD of the L1-5 vertebrae and femurs are presented in Table 1. These results demonstrate that OVX significantly decreased BMD in the L4 vertebrae and femurs, when compared with the control group (p < 0.05). Compared to the OVX group, GJD treatment significantly and dose-dependently inhibited decreases in BMD in OVX-induced L4 vertebrae and femurs (p < 0.05). The positive control drug Xianling Gubao capsule also significantly increased the BMD of the L4 vertebrae and femurs (p < 0.05), in a manner similar to that observed in the H-GJD group (p > 0.05). INTRODUCTION Sixty rats were randomly divided into six groups of ten rats: a sham-operated group (control) and five ovariectomy (OVX) sub-groups, that is, OVX with vehicle (OVX), OVX with positive control drug (Xianling Gubao capsule, 120 mg/kg/day), and OVX with GJD doses (70, 140 and 280 mg/kg/day). Statistical analysis Gengnian Jianshen decoction was composed of Uncaria rhynchophylla（Miq.）Miq. ex Hav. (15g), Poria cocos (Schw.) Wolf (10g), Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm. (15g), Rehmannia glutinosa (10g), Dioscorea opposita Thunb. (10g), Cornus officinalis Sieb. et Zucc. (10g), Psoralea corylifolia Linn. (15g), Dipsacales (10g), Achyranthes bidentata Blume. (10g), Eucommia ulmoides (10g), Triticum aestivum L (30g), Apatite (20g) and Nelumbo nucifera Gaertn. (5g). These herbs were mixed, and decocted with 1700 mL of water for 45 min. The decoction was performed twice to maximize GJD extract yield. The extract was concentrated and filtered through a membrane filter to obtain GJD with a concentration of 1200 µg/mL. Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA combined with Bonferroni’s multiple comparison test, using SPSS version 16.0. Differences were considered statistically significant at p < 0.05. Trop J Pharm Res, August 2017; 16(8): 1926 Animals and treatments XGC: Xianling Gubao capsule, L- EBME: low dose of GJD, M-EBME: middle dose of GJD, H-EBME: high dose of GJD Effect of GJD on serum levels of E2, FSH and LH Effect of GJD on serum levels of E2, FSH and pharmacological and clinical advantages of HRT as a widely accepted therapeutic strategy for osteoporosis, serious side effects of long-term application have also been reported. Therefore, development of new preventive and therapeutic drugs for osteoporosis is of critical importance. Interestingly, Chinese medicinal herbal extracts have been extensively investigated for their pharmacological effects, especially as they relate to preservation of bone integrity [17]. Serum E2, FSH and LH levels decreased significantly in OVX group rats relative to the control group (p < 0.01). Compared to the OVX group, GJD treatment significantly and dose- dependently increased serum E2, FSH and LH levels (p < 0.05) in the osteoporotic rats. Effect of GJD on serum IL-6 and IGF-1 levels Decreased BMD is one of the major factors that jeopardize the strength of the bone, thereby resulting in increased susceptibility to fractures. Thus, BMD measurement can best predict risk of fracture [18]. The results obtained in the present study showed that OVX reduced BMD in the right femurs and L4 vertebrae which are rich in trabecular bone, while treatment with GJD dose- dependently and significantly blocked decreases in BMD. Although BMD is among the strongest predictors of resistance to facture, empirical observations and theoretical analyses have shown that the biomechanical properties of bone Serum IL-6 level increased and IGF-1 level decreased significantly in OVX group rats, relative to the control group (p < 0.05). In addition, GJD treatment significantly and dose- dependently decreased serum IL-6 levels and increased IGF-1 levels (p < 0.05) in the rats. Animals and treatments Healthy, six-month-old female Sprague-Dawley rats (weighing 220 ± 20 g) were provided by Jiangsu Animal center (certificate no. SYXK 2003-0007). The animals had free access to feed and water, and were allowed to acclimatize for at least one week before use. The rat experiment was approved by Animal Care and Use Trop J Pharm Res, August 2017; 16(8): 1926 Wang et al Wang et al Table 1: Effect of GJD on BMD of L4 vertebrae and femurs (n = 10) Table 1: Effect of GJD on BMD of L4 vertebrae and femurs (n = 10) Group Dose (mg/kg) BMD of vertebra (g/cm2) BMD of femur (g/cm2) Control - 0.61 ± 0.07 0.38 ± 0.04* OVX - 0.23 ± 0.04 0.13 ± 0.04 XGC 120 0.35 ± 0.05* 0.26 ± 0.04* L-EBME 70 0.26 ± 0.03* 0.16 ± 0.04* M-EBME 140 0.38 ± 0.04* 0.25 ± 0.04* H-EBME 280 0.58 ± 0.03* 0.34 ± 0.03* P < 0.05 and p < 0.01 versus OVX group. XGC: Xianling Gubao capsule, L-EBME: low dose of GJD, M-EBME: middle dose of GJD, H-EBME: high dose of GJD Table 2: Effect of GJD on serum hormone levels (n = 10) ( ) Group Dose (mg/kg) E2 (pmol/L) FSH (IU/L) LH (mIU/mL) Control - 5.68 ± 0.21* 1.96 ± 0.07** 2.88 ± 0.12* OVX - 1.45 ± 0.16 0.54 ± 0.06 1.24 ± 0.08 XGC 120 3.96 ± 0.25** 1.36 ± 0.06* 2.34 ± 0.05* L-EBME 70 2.36 ± 0.37 0.87 ± 0.05 1.78 ± 0.14 M-EBME 140 3.26 ± 0.35* 1.24 ± 0.04* 2.32 ± 0.08* H-EBME 280 4.89± 0.14 1.56 ± 0.04 2.67 ± 0.06* P < 0.05 and p < 0.01 versus OVX group. XGC: Xianling Gubao capsule, L-EBME: low dose of GJD, M-EBME: middle dose of GJD, H-EBME: high dose of GJD Table 3: Effect of GJD on serum IL-6 and IGF-1 levels (n = 10) Group Dose (mg/kg) IL-6 (pmol/L) IGF-1 (pmol/L) Control - 27.25 ± 2.21 14.33 ± 1.18 OVX - 49.33 ± 3.46 3.42 ± 0.94** XGC 120 32.43 ± 2.87* 7.59 ± 1.24* L-EBME 70 46.37 ± 3.12 4.93 ± 1.16 M-EBME 140 40.42 ± 2.36* 7.13 ± 1.35* H-EBME 280 27.36 ± 2.57* 9.25 ± 1.04** P < 0.05 and *p < 0.01 versus OVX group. Conflict of Interest 11. Orija IB, Mehta A. Hormone replacement therapy: current controversies. Clinical Endocrinol 2013; 59: 657-658. No conflict of interest associated with this work. 12. Lacey JV, Mink PJ, Lubin JH. Menopausal hormone replacement therapy and risk of ovarian cancer. J Ame Med Asso 2012; 288: 334-341. DISCUSSION The high incidence, serious complications and dramatically decreased quality of life associated with osteoporosis are evidence of the severe effects of this disease in humans. Despite the Trop J Pharm Res, August 2017; 16(8): 1927 Wang et al and trabecular microarchitecture influence trabecular bone strength as well [19]. Attribution License (http://creativecommons.org/ licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopena ccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Attribution License (http://creativecommons.org/ licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopena ccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Estradiol plays an important role in human sclerotin. When female serum estradiol level decreases significantly, osteoporosis occurs [20]. Compared to the OVX group, GJD treatment significantly increased serum E2, FSH and LH levels in a dose-dependent manner in the osteoporotic rats. Studies have shown that high doses or chronic administration of IL-6 in rats or mice caused increased degradation of proteins in skeletal muscle, although the normal levels of IL- 6 have been proven hypertrophic [21]. Indirect effects of IL-6 on IGF-1 signaling have also been reported. Increased circulating levels of IL-6 are associated with significantly reduced serum IGF- 1 levels and elevated expression of muscle SOCS3 mRNA [22]. These suggest the role of IL- 6 as a negative regulator of IGF-1 signaling. Compared with the OVX group, GJD treatment significantly decreased serum IL-6 level and increased IGF-1 level in a dose-dependent manner in the rats. CONCLUSION 5. Marcus R. An expanded overview of postmenopausal osteoporosis. J Mus Neuronal Int 2002; 2: 195-197. The results obtained in the present study indicate that GJD can prevent OVX-induced osteoporosis in rats without hyperplastic effects on the uterus. This suggests that GJD has promising potential for future use in treating post-menopausal osteoporosis. The results obtained in the present study indicate that GJD can prevent OVX-induced osteoporosis in rats without hyperplastic effects on the uterus. 6. Sugerman DT. JAMA patient page. Osteoporosis JAMA 2014; 311: 104-105. yp p This suggests that GJD has promising potential for future use in treating post-menopausal osteoporosis. 7. Johnell O, Kanis JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporosis Inter 2006; 17: 1726-1733. 8. Stevenson JC. Justification for the use of HRT in the long-term prevention of osteoporosis. Maturitas 2005; 51: 113-126. REFERENCES 1. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and therapy. Osteoporosis prevention, diagnosis, and therapy. J Am Med Ass 2001; 285: 785-795. 2. Eastell R, Black DM, Boonen S. Effect of once-yearly zoledronic acid five milligrams on fracture risk and change in femoral neck bone mineral density. J Clin Endo Metabol 2009; 94: 3215-3225. 3. Burge R, Dawson-Hughes B, Solomon DH. Incidence and economic burden of osteoporosis-related fractures in the United States 2005-2025. J Bone Min Res 2007; 22: 465-475. 4. Omsland TK, Emaus N, Tell GS. Mortality following the first hip fracture in Norwegian women and men. A NOREPOS study. Bone 2014; 63: 81-86. Contribution of Authors 13. Rossouw JE, Anderson GL, Prentice RL. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. J Ame Med Assoc 2012; 288: 321-333. 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https://openalex.org/W2053175777	https://journal-inflammation.biomedcentral.com/counter/pdf/10.1186/s12950-015-0065-4	English	null	Functional activity of peripheral blood eosinophils in allergen-induced late-phase airway inflammation in asthma patients	Journal of inflammation	2,015	cc-by	8,194	Lavinskiene et al. Journal of Inflammation (2015) 12:25 DOI 10.1186/s12950-015-0065-4 Lavinskiene et al. Journal of Inflammation (2015) 12:25 DOI 10.1186/s12950-015-0065-4 RESEARCH Open Access Open Access © 2015 Lavinskiene et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Objective: We aimed to investigate peripheral blood eosinophil chemotaxis, generation of spontaneous reactive oxygen species (ROS), and apoptosis in patients with allergic asthma after bronchial allergen challenge. Material and methods: A total of 18 patients with allergic asthma (AA), 14 with allergic rhinitis (AR), and 10 healthy subjects (HS) underwent bronchial challenge with a specific allergen extract. Eosinophils from peripheral blood were isolated 24 h before as well as 7 and 24 h after bronchial allergen challenge. Chemotaxis, spontaneous ROS production in eosinophils, and apoptosis were analyzed by flow cytometry. Serum and induced sputum IL-5 levels were measured by ELISA; the cell count in sputum was analyzed by the May-Grünwald-Giemsa method. Results: Before bronchial allergen challenge, peripheral blood eosinophil chemotaxis, spontaneous ROS production was enhanced and eosinophil apoptosis was reduced in the patients with AA as compared with AR patients and HS (P < 0.05). Meanwhile, eosinophil chemotaxis and ROS generation markedly increased in the patients with AA 7 h and 24 h after challenge compared with other groups and baseline values (P < 0.05). The percentage of apoptotic eosinophils in the patients with AA decreased at 7 h as well as 24 h after challenge when compared with other groups and the baseline values (P < 0.05). There was a significant correlation between the migrated peripheral blood eosinophil count and the sputum eosinophil count (Rs = 0.89, P < 0.0001) and the sputum IL-5 level (Rs = 0.68, P = 0.002) at 24 h after bronchial challenge only in the patients with AA. Furthermore, the percentage of peripheral blood apoptotic eosinophils significantly correlated with eosinophil count in sputum (Rs = −0.53, P = 0.02), and ROS production correlated with the serum IL-5 levels (Rs = 0.71, P = 0.01). Conclusion: During allergen-induced late-phase airway inflammation, peripheral blood eosinophils demonstrated further alterations of their functional activity manifested by enhanced spontaneous ROS production, increased chemotaxis, and diminished apoptosis in patients with AA. Keywords: Eosinophils, Airway inflammation, Allergic asthma, Apoptosis, Chemotaxis, ROS Keywords: Eosinophils, Airway inflammation, Allergic asthma, Apoptosis, Chemotaxis, ROS IL-5 in luminal fluid and serum, and a heightened cap- acity of airway cells for ex vivo generation of IL-5 [3-5]. IL-5 in luminal fluid and serum, and a heightened cap- acity of airway cells for ex vivo generation of IL-5 [3-5]. Functional activity of peripheral blood eosinophils in allergen-induced late-phase airway inflammation in asthma patients Simona Lavinskiene, Kestutis Malakauskas, Jolanta Jeroch, Deimante Hoppenot and Raimunda Simona Lavinskiene, Kestutis Malakauskas, Jolanta Jeroch, Deimante Hoppenot and Raimundas Sakalauskas * Correspondence: lavinskiene.simona@gmail.com Department of Pulmonology and Immunology, Lithuanian University of Health Sciences, Kaunas, Lithuanian Introduction Asthma is an inflammatory disorder of the airways in- volving T‐cells, mast cells, and eosinophils [1]. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction [2]. Following airway allergen exposure, the development of airway eosinophilia is associated with increased IL-5 expression in the sputum, elevated concentrations of IL-5 plays a key role in eosinophil proliferation, differ- entiation, maturation, migration to tissue sites and sur- vival, as well as prevention of eosinophil apoptosis [6,7]. Eosinophil chemotaxis to the lungs during allergic air- way inflammation represents a major part of the inflam- mation process [8]. Transmigration of the eosinophil through the vascular endothelium is a multistep process; rolling, tethering, firm adhesion, and transendothelial migration are regulated by the coordinated interaction between networks involving chemokine, cytokine and * Correspondence: lavinskiene.simona@gmail.com Department of Pulmonology and Immunology, Lithuanian University of Health Sciences, Kaunas, Lithuanian Lavinskiene et al. Journal of Inflammation (2015) 12:25 Page 2 of 9 adhesion molecules [9,10]. Experiments with in vitro allergen as well as endobronchial allergen challenge have shown that blood and bronchoalveolar lavage eosinophils from subjects with asthma have a greater responsiveness to chemoattractants and enhanced chemotaxis [11,12]. During the process of allergic inflammation, eosinophils release not only toxic granule proteins but also reactive oxygen species (ROS), which are known to cause tissue damage [13]. It has been demonstrated that allergic pa- tients have upregulated oxidative metabolism in blood eosinophils when compared with healthy subjects [14,15]. It leads to the observations that eosinophils isolated from allergic patients might be already activated in peripheral blood streams before they infiltrate the tissue. Patients with allergic asthma and rhinitis had a clinical history of the disease for ≥1 year, current symptoms, and positive results of skin prick test (≥3 mm) with the fol- lowing allergens: Dermatophagoides pteronyssinus (D. pteronyssinus), birch pollen, or mixture of 5 grasses. All the patients were not using inhaled, nasal, or oral ste- roids at least 1 month before visits; short-acting β2 ago- nists, at least 12 h; long-acting β2 agonists, at least 48 h prior the lung function test, and antihistamines and antileukotrienes, 7 days before the skin prick test and the lung function test. None of the patients had a history of smoking. Baseline forced expiratory volume in one second (FEV1) was more than 70% of the predicted value in all patients. Skin prick and lung function testing All h i d f ll All the patients were screened for allergy by the skin prick test using standardized allergen extracts (Stallergenes S.A., France) for the following allergens: D. pteronyssinus, D. farinae, cat and dog dander, mixture of pollen of 5 grasses, birch pollen, mugwort, Alternaria, Aspergillus, and Cla- dosporium. Histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 min after application. The results of the skin prick test were considered positive if the mean wheal diameter was ≥3 mm [21]. There is no doubt that eosinophils are important cells which participate in allergic airway inflammation. Mean- while, associations between eosinophil infiltration in the airways and peripheral blood eosinophil chemotaxis, ROS production, and apoptosis have not been com- pletely elucidated yet. Therefore, the regulation of blood eosinophil activity in asthmatic patients especially after allergen challenge needs to be investigated. Pulmonary function was tested using a pneumotacho- metric spirometer “CustovitM” (Custo Med, Germany). Baseline FEV1, forced vital capacity (FVC), and FEV1/ FVC ratio were recorded as the highest of three repro- ducible measurements. The results were compared with the predicted values matched for age, body height, and sex according to the standard methodology [22]. We hypothesized that evaluating peripheral blood eo- sinophils functional activity (chemotaxis, apoptosis, and ROS production) during allergen-induced late-phase air- way inflammation is important for understanding the pathogenesis of eosinophilic inflammation in the airways. Introduction All the healthy subjects were nonsmokers, without symptoms of asthma or rhinitis, with normal findings of spirometry, and all showed negative results of the skin prick test. adhesion molecules [9,10]. Experiments with in vitro allergen as well as endobronchial allergen challenge have shown that blood and bronchoalveolar lavage eosinophils from subjects with asthma have a greater responsiveness to chemoattractants and enhanced chemotaxis [11,12]. During the process of allergic inflammation, eosinophils release not only toxic granule proteins but also reactive oxygen species (ROS), which are known to cause tissue damage [13]. It has been demonstrated that allergic pa- tients have upregulated oxidative metabolism in blood eosinophils when compared with healthy subjects [14,15]. It leads to the observations that eosinophils isolated from allergic patients might be already activated in peripheral blood streams before they infiltrate the tissue. In the absence of any inflammatory survival-prolonging factors, eosinophils die by apoptosis in a few days, but in inflamed airways, eosinophils survival is thought to be prolonged due to the surrounding proinflammatory fac- tors such as IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor [16,17]. There are some data about impaired peripheral blood eosinophil apoptosis in allergic patients [18], and this might contribute to greater airway eosinophilia. Methods Subjects Measurement of airway responsiveness to methacholine Airway responsiveness was assessed as changes in the airway function after challenge with inhaled methacho- line using a reservoir method [23]. Methacholine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation I; Pari, Stanberg, Germany). Aerolized methacholine was inhaled through a one-way valve at 5- min intervals starting with 15-μg methacholine dose and doubling it until a 20% decrease in FEV1 from the baseline or the total cumulative dose of 3.87 mg was achieved. The bronchoconstricting effect of each dose of methacholine was expressed as a percentage of decrease in FEV1 from the baseline value. The provocative dose of methacholine causing a ≥20% fall in FEV1 (PD20) was calculated from the log dose–response curve by linear interpolation of two adjacent data points. A total of 42 nonsmoking adults (13 men and 29 women) were examined: 18 patients with intermittent or mild-to- moderate persistent allergic asthma, defined according to the GINA criteria [19], 14 patients with mild-to-moderate persistent allergic rhinitis, defined according to the ARIA criteria [20], and 10 healthy subjects who comprised the control group. The patients were recruited from the De- partment of Pulmonology and Immunology, Hospital of the Lithuanian University of Health Sciences, Kaunas. The study protocol was approved by the Regional Biomedical Research Ethics Committee of the Lithuanian University of Health Sciences (BE-2-23), and each participant gave his/ her informed written consent. The study was registered in the U.S. National Institutes of Health trial registry Clinical- Trials.gov with identifier NCT02214303. Page 3 of 9 Page 3 of 9 Lavinskiene et al. Journal of Inflammation (2015) 12:25 Analysis of ROS production y p Spontaneous ROS production in peripheral blood eo- sinophils was performed in sterile 96-well microplates (Falcon, BD, USA). For the detection of generated ROS, dihydrorhodamine-123 (DHR-123, 750 ng/mL final, Invitrogen, USA), a nonfluorescent dye, was added. DHR-123, interacting with intracellular ROS, is oxidized to the green-fluorescent rhodamine-123.The plates were filled with eosinophil cultures and incubated for 45 min (37°C, 5% CO2). The relative amount of generated ROS was measured flow cytometrically by determination of mean green fluorescence intensity in the eosinophil population. Sputum induction and processing The subjects inhaled 10 mL of sterile hypertonic saline solution (3%, 4%, or 5% NaCl, Ivex Pharmaceuticals, USA) at room temperature from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each in- halation was 7 min, and it was stopped after expector- ation an adequate amount of sputum. In order to detect a possible decrease in FEV1, spirometry was performed after each inhalation. Sputum was poured into a Petri dish and separated from saliva. A 4-fold volume of freshly prepared 0.1% dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at room temperature. Next, an equal volume of phosphate-buffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-μm cell stainer (Becton Dickinson, USA). The mixture was centrifuged for 10 min at 4°C; the supernatant was aspirated and stored at −70°C for later analysis. The total cell counts, percentage of epi- thelial cells, and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz, Germany) under a microscope (B5 Professional, Motic, China) by employing the Trypan blue exclusion method. The cytospin samples of induced sputum were pre- pared using a cytofuge instrument (Shandon Southern Instruments, USA). The number of migrated eosinophils was calculated by flow cytometry using Liquid Counting Beads (BD Biosci- ences, USA) according to the manufacturer’s recommen- dations. The amount of migrated eosinophils was expressed in percentages. Peripheral blood collection and isolation of eosinophils Eosinophil chemotaxis in vitro was performed in a 10- well cell transmigration chamber (Neuro Probe, USA). The lower and upper wells of chamber were isolated by a polyvinylpyrrolidone (PVP)-treated polycarbonate track- etch membrane, containing 2 × 106 3 μm/mm2 pores (Neuro Probe, USA). The lower wells were pre-filled with isotonic Percoll (GE Healthcare, Finland) and eotaxin, a chemotactic factor, at different concentrations (10, 100, or 1000 ng/mL). RPMI 1640 was used as a negative control. The upper wells were filled with eo- sinophil culture suspension (1 × 103/mL) and incubated for 2 h (37°C, 5% CO2). After the incubation, the suspensions of upper and lower wells were resuspended in tubes for flow cytome- try. Nonmigrated eosinophils remained in the upper wells. The migration rate was calculated from the total number of eosinophils harvested from the lower well and expressed as percentage of the total input of eosino- phils into the upper well. Apoptosis l d Isolated eosinophils were resuspended in the annexin- binding buffer (pH 7.4) containing 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2 (Invitrogen, USA) and incubated with fluorescein isothiocyanate-labeled (FITC)- annexin V (Invitrogen, USA) and propidium iodide (PI) for 15 min at room temperature in the dark. After the in- cubation, apoptosis was analyzed by flow cytometry using the CellQuest software (BD Biosciences, USA). Apoptotic cells were quantified as the percentage of the total popula- tion that was positive for FITC, but negative for PI. Nec- rotic cells were positive for PI. Peripheral blood eosinophil chemotaxis, apoptosis and ROS production assay Chemotaxis in vitro Peripheral blood collection and isolation of eosinophils Peripheral blood samples for eosinophil isolation were collected into sterile vacutainers with ethylenediamine- tetraacetic acid (EDTA). Polymorphonuclear leukocytes (PMNs) were isolated by high density gradient centrifuga- tion. The whole blood was layered on Ficoll-Paque PLUS (GE Healthcare, Finland) and centrifuged at 1000 g for 30 min at room temperature. PMNs were separated by hypotonic lysis of erythrocytes and eosinophils were sepa- rated using a magnetic eosinophil isolation kit (Miltenyi Biotek, USA). Isolated eosinophils were diluted in cell cul- ture RPMI 1640 media (Biological Industries, Israel) at a final concentration of 2 × 106/mL. The viability of eo- sinophil was checked flow cytometrically using propi- dium iodide (2 mg/mL) and it always was > 95 %. Statistical analysis Statistical analysis was performed by using the Statistical Package for Social Sciences, version 17.0 for Windows (SPSS 17.0). The normality assumption of data was veri- fied with the Kolmogorov-Smirnov test. The data were expressed as a median and a range. The results of methacholine PD20 measurements are expressed as a geometric mean. PD20 values were log-transformed for analysis to fit a normal distribution. Induced sputum cell analysis The prepared sputum cytospins were stained by the May-Grünwald-Giemsa method for differential cell counts. Cell differentiation was determined by counting approxi- mately 400 cells in random fields of view under a light microscope, excluding squamous epithelial cells. The cells were identified by standard morphological criteria, nuclear morphology, and cytoplasmic granulation. Cell counts were expressed as percentages of total cells and absolute values (106/L). Page 4 of 9 Page 4 of 9 Lavinskiene et al. Journal of Inflammation (2015) 12:25 Detection of cytokine in serum and induced sputum supernatant Detection of cytokine in serum and induced sputum supernatant age and gender differences comparing the groups. Twenty- one patients were sensitized to D. pteronyssinus; 5 patients, to birch pollen; and 6 patients, to a mixture of pollen of 5 grasses. The mean wheal diameter induced by an allergen was similar in both groups of patients. The demographic and clinical data of the study subjects are presented in Table 1. There were no significant difference in the baseline FEV1 (% of predicted) the comparing all groups. A provoca- tive dose of methacholine causing a 20% decrease in FEV1 (PD20) was documented in 18 patients with allergic asthma and 1 patient with allergic rhinitis. The serum and sputum IL-5 levels were measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Abcam, USA). The minimum detectable concentration was 5 pg/mL. The peripheral blood cell analysis was performed on an automated hematology analyzer (Sysmex XE-5000, Japan). Eosinophil composition of peripheral blood and induced sputum The eosinophil count in the peripheral blood 24 h before bronchial allergen challenge was significantly higher in the patients with allergic asthma compared with the patients with allergic rhinitis and healthy subjects (0.32 × 109/L [range, 0.09–0.65] vs. 0.16 × 109/L [0.04– 0.88] and 0.13 × 109/L [0.04–0.94], P <0.05). At 24 hours after bronchial challenge, the peripheral blood eosinophil count was significantly increased in the patients with allergic asthma compared with the baseline values and the healthy subjects (Figure 1A). The increased peripheral blood eosinophil count also was recorded 24 h after bronchial challenge in the pa- tients with allergic rhinitis compared with the baseline values (0.22 × 109/L [range, 0.04–1.19] vs. 0.16 × 109/L [range, 0.04–0.88], P < 0.05). Meanwhile, at the base- line as well as 7 h and 24 h after bronchial challenge, the total eosinophil count in the sputum was significantly higher in the patients with allergic asthma than those with allergic rhinitis and the healthy subjects (P < 0.05) (Figure 1B). At the baseline as well as 7 h and 24 h after bronchial challenge, the patients with allergic rhinitis showed a significantly greater eosinophil count in the sputum than the healthy subjects (P < 0.05). Bronchial allergen challenge had no impact on eosinophil count in healthy subjects. Due to a skewed distribution of the variable, nonpara- metric tests were applied. The Kruskal-Wallis test was used to evaluate differences between the groups of patients and the control group. Differences between 2 independent groups were determined by the Mann– Whitney U test. Differences among 3 and more paired samples were evaluated by the Friedman test. Differ- ences between 2 dependent samples were evaluated by the Wilcoxon test. The Spearmen rank test was used to assess relationships between measurements. Statistical significance was assumed at a P value of <0.05. Characteristics of studied subjects a a o ud d ubj A total of 42 nonsmoking adults (17 men and 25 women; mean age 31 ± 9 years) were examined: 18 pa- tients with intermittent or mild-to-moderate persistent allergic asthma, 14 patients with mild-to-moderate per- sistent allergic rhinitis, and 10 healthy subjects who comprised the control group. There were no significant Table 1 Demographic and clinical characteristics of study subjects Characteristics Patients with allergic asthma N = 18 Patients with allergic rhinitis N = 14 Healthy subjects N = 10 Age (years), median (range) 31 (21–50) 30 (18–49) 28 (22–45) Sex (male/female), n 10/8 4/10 3/7 Wheal diameter induced by allergen (mm), median (range) 6.2 (4–11) 7.8 ± 1.8 (4–13) 0 Sensitization to D. pteronyssinus/birch/5 grass mixture allergen, n 13/3/2 9/3/2 0 PD20 (mg), geometric mean (range) 0.38 (0.25–0.54) 0.52* 0/0/0 FEV1 (% of predicted), mean ± SD 98 ± 15 105 ± 10 102 ± 11 N = 1 because methacholine challenge provoked bronchoconstriction only to one allergic rhinitis patient. PD20 - a provocative dose of methacholine causing a 20% decrease in FEV1; FEV1 - forced expiratory volume in one second. Table 1 Demographic and clinical characteristics of study subjects Functional activity of peripheral blood eosinophils Peripheral blood eosinophil chemotaxis in vitro Functional activity of peripheral blood eosinophils Peripheral blood eosinophil chemotaxis in vitro allergic rhinitis and the healthy subjects (P < 0.05) (Figure 4). Furthermore, 7 h and 24 h after bronchial challenge, a sig- nificantly lower percentage of apoptotic peripheral blood eosinophils was recorded in the asthma patents’ group when compared with other groups and the baseline values (P < 0.05). p p Eotaxin at different concentrations (10, 100, and 1000 ng/mL) had an impact on peripheral blood eosino- phil chemotaxis in all the studied groups, but the highest concentration had the greatest effect. At the baseline, per- ipheral blood eosinophil chemotaxis after the stimulation with 1000 ng/mL of eotaxin was higher in the patients with allergic asthma compared with those with allergic rhinitis and the healthy subjects (P < 0.05). At 7 h and 24 h after bronchial challenge, eosinophil chemotaxis was significantly enhanced in the patients with allergic asthma compared with the baseline values, and it was greater than in the patients with allergic rhinitis and the healthy sub- jects (Figure 2). Meanwhile, bronchial allergen challenge had no significant effect on eosinophil chemotaxis in the peripheral blood of healthy subjects. ROS in peripheral blood eosinophils Before bronchial allergen challenge, spontaneous ROS production in peripheral blood eosinophils was signifi- cantly greater in the patients with allergic asthma com- pared than those with allergic rhinitis and the healthy subjects (P < 0.05). At 7 h and 24 h after bronchial chal- lenge, ROS generation was significantly greater in the pa- tients with allergic asthma compared with other groups and the baseline values (Figure 3). Bronchial allergen chal- lenge had no impact on ROS production in eosinophils isolated from the healthy subjects. Figure 2 Peripheral blood eosinophil chemotaxis (stimulated with 1000 ng/mL of eotaxin) in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Figure 2 Peripheral blood eosinophil chemotaxis (stimulated with 1000 ng/mL of eotaxin) in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. IL-5 Levels in induced sputum and serum There was a significant increase in the induced sputum IL-5 levels in the patients with allergic asthma and those with allergic rhinitis compared with the healthy subjects at 24 h before bronchial challenge (Figure 5A). At 7 h Figure 2 Peripheral blood eosinophil chemotaxis (stimulated with 1000 ng/mL of eotaxin) in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. ROS in peripheral blood eosinophils Characteristics Page 5 of 9 Page 5 of 9 Lavinskiene et al. Journal of Inflammation (2015) 12:25 Figure 1 Eosinophil count of patients with allergic asthma, allergic rhinitis and healthy subjects. A Eosinophil counts in peripheral blood 24 h before as well as 7 h and 24 h after bronchial challenge. B Eosinophil counts in the induced sputum 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Figure 1 Eosinophil count of patients with allergic asthma, allergic rhinitis and healthy subjects. A Eosinophil counts in peripheral blood 24 h before as well as 7 h and 24 h after bronchial challenge. B Eosinophil counts in the induced sputum 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Discussion h d h This study has demonstrated that peripheral blood eosino- phil chemotaxis, spontaneous ROS production, and eosino- phil apoptosis are altered in patients with allergic asthma during allergen-induced late-phase airway inflammation. The increased sputum eosinophil count and enhanced levels of IL-5 significantly correlated with the impaired functional activity of peripheral blood eosinophils. and 24 h after bronchial challenge, the induced sputum IL-5 levels increased significantly in the patients with al- lergic asthma and those with allergic rhinitis compared with the healthy subjects. Moreover, the sputum IL-5 levels at 7 h and 24 h after bronchial challenge were sig- nificantly higher in the patients with allergic asthma than those with allergic rhinitis. The same tendency was observed while analyzing serum IL-5 levels (Figure 5B). However, in patients with allergic rhinitis, there was no significant difference in the serum IL-5 levels comparing It is known that eosinophils are recruited to sites of in- flammation by released chemotactic agents. Eotaxin, a CC chemokine, is one of the strongest stimulator of eo- sinophil chemotaxis [24]. It also induces the release of various mediators from eosinophils and is known to play an integral role in the development of eosinophilic inflammation [25]. Therefore, in order to investigate chemotaxis in vitro, peripheral blood eosinophils were stimulated with different concentrations (10, 100, and 1000 ng/mL) of eotaxin. Our results showed that before bronchial allergen challenge, peripheral blood eosinophil chemotaxis stimulated with the highest eotaxin concen- tration was greatest in the patients with allergic asthma as compared with other groups. These data suggest that eosinophils in the peripheral blood of individuals with allergic asthma are already primed and more sensitive to a chemokine. Figure 4 The percentage of apoptotic peripheral blood eosinophils in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Apoptosis of peripheral blood eosinophils Before bronchial allergen challenge, the patients with allergic asthma had a significantly lower percentage of apoptotic peripheral blood eosinophils than those with Page 6 of 9 Lavinskiene et al. Journal of Inflammation (2015) 12:25 Figure 3 Production of reactive oxygen species in peripheral blood eosinophils of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10); MFI, mean fluorescence intensity. P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. the baseline values with those recorded 7 h after bronchial challenge. Bronchial challenge had no impact on the IL-5 levels in the sputum and serum of healthy subjects. Correlations Significant correlations were found only in the patients with allergic asthma at 24 h after bronchial allergen challenge. The migrated peripheral blood eosinophil count significantly correlated with the eosinophil count in the sputum (Rs = 0.89, P < 0.0001; Figure 6A). Moreover, there was a significant correlation between the percentage of apoptotic peripheral blood eosinophils and eosinophil count in sputum (Rs = −0.53, P =0.02; Figure 6B). The mi- grated peripheral blood eosinophil count significantly cor- related with sputum IL-5 levels (Rs = 0.68, P = 0.002; Figure 6C), and ROS production in peripheral blood eo- sinophils significantly correlated with the serum IL-5 levels (Rs = 0.71, P = 0.01; Figure 6D). Figure 3 Production of reactive oxygen species in peripheral blood eosinophils of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10); MFI, mean fluorescence intensity. P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Discussion h d h A IL-5 levels in the induced sputum 24 h before as well as 7 h and 24 h after bronchial allergen challenge. B IL-5 levels in serum 24 h before as well as 7 h and 24 h after bronchial allergen challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. levels of serum IL-5 and looked for a relationship with generated ROS in peripheral blood eosinophils. We found elevated serum IL-5 levels, especially 24 h after bronchial challenge, in the patients with allergic asthma compared with the baseline values and other groups. Moreover, a significant correlation was observed only 24 h after bronchial challenge and only in the patients with allergic asthma. The increased serum IL-5 levels correlated with the enhanced ROS production in periph- eral blood eosinophils (P < 0.05). These findings suggest that enhanced ROS generation can activate a number of redox-sensitive signaling cascades, stimulate production- interaction of proinflammatory cytokines, and promote inflammation [29]. activity of eosinophils, but response to eotaxin confirms the undeniable importance of this chemokine. More- over, at 24 h after bronchial challenge, the increased eo- sinophil count in the sputum significantly correlated with the increased migrated eosinophil count in the per- ipheral blood of asthmatic patients. That can reflect the hallmark characteristic of allergic asthma by infiltration of eosinophils to the airway. The inflammatory cells recruited to the asthmatic air- ways are exceptionally capable of producing ROS, result- ing in abnormal physiologic function of DNA, proteins, and lipids that clinically can augment bronchial hyperre- sponsiveness and inflammation [27]. However due to the possibility that eosinophils can be already activated in periphery, we examined spontaneous ROS production in peripheral blood eosinophils. As it has been reported that IL-5 can enhance eosino- phil migration by the upregulation of adhesion molecules on eosinophils [30], we analyzed sputum IL-5 levels and investigated the possible relationship to peripheral blood eosinophil chemotaxis. Thus, we determined a significant increase in sputum IL-5 levels in the patients with allergic asthma, and it correlated with the migrated peripheral blood eosinophil count in allergen-induced late-phase airway inflammation in asthma patients. Discussion h d h Experiments with in vivo injection of eotaxin into the skin of mice and rhesus monkeys showed local accumula- tion of eosinophils, and the kinetics of allergen-induced production of eotaxin is paralleled by eosinophil accumu- lation in a guinea-pig model of allergic airways [25,26]. Figure 4 The percentage of apoptotic peripheral blood eosinophils in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Figure 4 The percentage of apoptotic peripheral blood eosinophils in patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 h and 24 h after bronchial challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. We found that 7 h and 24 h after bronchial allergen challenge, eosinophil chemotaxis was significantly greater in the patients with allergic asthma than other groups and also compared with the baseline values. This shows that not only allergen challenge stimulates the functional Lavinskiene et al. Journal of Inflammation (2015) 12:25 Page 7 of 9 Figure 5 IL-5 levels of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects. A IL-5 levels in the induced sputum 24 h before as well as 7 h and 24 h after bronchial allergen challenge. B IL-5 levels in serum 24 h before as well as 7 h and 24 h after bronchial allergen challenge. Data are shown as median (range). AA indicates patients with allergic asthma (n = 18); AR, patients with allergic rhinitis (n = 14); HS, healthy subjects (n = 10). P < 0.05 compared with healthy subjects; #P < 0.05 compared with baseline values; †P < 0.05 compared with patients with allergic rhinitis. Figure 5 IL-5 levels of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects. Discussion h d h B Correlation between percentage of apoptotic peripheral blood eosinophils and eosinophil count in sputum. C Correlation between migrated peripheral blood eosinophil count and IL-5 level in sputum. D Correlation between ROS production in peripheral blood eosinophils and serum IL-5 levels. MFI indicates mean fluorescence intensity. Finally, we evaluated relationships between eosinophil counts in the sputum and eosinophil viability after bron- chi al allergen challenge, which could represent a link between eosinophilic influx to the airways possibly caused by delayed peripheral blood eosinophil apoptosis in allergen-induced late-phase airway inflammation. apoptotic peripheral blood eosinophils in the patients with allergic asthma was significantly lower compared with the patients with allergic rhinitis and the healthy subjects. Apoptosis of peripheral blood eosinophils in the patients with allergic asthma was more delayed 7 h and 24 h after bronchial challenge compared with the baseline and other groups. The same tendency was reported by Evans et al., who showed that in asthmatic patients demonstrating a late response, the survival of peripheral blood eosinophils is prolonged after the whole lung allergen challenge [32]. Moreover, Druilhe et al. and Vignola et al. demonstrated that asthmatic patients had greater numbers of eosinophils in the bronchial biopsy than healthy individuals [33,34]. All the above-mentioned results and findings of our study show that eosinophil survival through the inhibition of apoptosis can increase airway eosinophilia. apoptotic peripheral blood eosinophils in the patients with allergic asthma was significantly lower compared with the patients with allergic rhinitis and the healthy subjects. Apoptosis of peripheral blood eosinophils in the patients with allergic asthma was more delayed 7 h and 24 h after bronchial challenge compared with the baseline and other groups. The same tendency was reported by Evans et al., who showed that in asthmatic patients demonstrating a late response, the survival of peripheral blood eosinophils is prolonged after the whole lung allergen challenge [32]. Moreover, Druilhe et al. and Vignola et al. demonstrated that asthmatic patients had greater numbers of eosinophils in the bronchial biopsy than healthy individuals [33,34]. All the above-mentioned results and findings of our study show that eosinophil survival through the inhibition of apoptosis can increase airway eosinophilia. Significant correlations were observed only 24 h after bronchial challenge and only in the patients with allergic asthma. The lower percentage of peripheral blood eosino- phils was negatively correlated with the increased eosino- phil count in the sputum. Discussion h d h Our study showed that spontaneous ROS production in peripheral blood eosinophils was significantly greater in the patients with allergic asthma than those with al- lergic rhinitis and the healthy subjects. Moreover, at 7 h and 24 h after bronchial allergen challenge, ROS gener- ation in peripheral blood eosinophils was significantly greater in the patients with allergic asthma compared with the baseline values and other groups. The accumulation of eosinophils in the asthmatic lungs is a complex process, which involves their maturation in and release from the bone marrow, adhesion and transmigra- tion through the post-capillary endothelium, and then their chemotaxis to and activation/degranulation at inflamma- tory foci [9,31]. In the circulation and tissues, eosinophils are programmed to undergo apoptosis in the absence of viability-enhancing stimuli [31]. A defect in apoptosis might contribute to chronic tissue eosinophilia associated with asthma. Therefore, we aimed at evaluating peripheral blood eosinophil apoptosis during allergen-induced late-phase in- flammation. Before bronchial challenge, the percentage of The upregulation of ROS production by blood eosino- phils in allergic patients has been previously documented in several studies [14,28], and the results of these studies as well as our date suggest that eosinophils from allergic asthma patients may might be already activated in the per- ipheral blood stream before they infiltrate the tissues and the inhalation of aggravating compounds such as allergens can promote stronger activation of these cells. Moreover, it was demonstrated that a low concentra- tion of IL-5 enhanced chemokine-primed ROS produc- tion by eosinophils [14]. Consequently, we evaluated the Lavinskiene et al. Journal of Inflammation (2015) 12:25 Page 8 of 9 Figure 6 Correlations between peripheral blood eosinophil activity and eosinophil count as well as IL-5 levels in the induced sputum in the patients with allergic asthma 24 h after bronchial allergen challenge. A Correlation between migrated peripheral blood eosinophil count and eosinophil count in sputum. B Correlation between percentage of apoptotic peripheral blood eosinophils and eosinophil count in sputum. C Correlation between migrated peripheral blood eosinophil count and IL-5 level in sputum. D Correlation between ROS production in peripheral blood eosinophils and serum IL-5 levels. MFI indicates mean fluorescence intensity. Figure 6 Correlations between peripheral blood eosinophil activity and eosinophil count as well as IL-5 levels in the induced sputum in the patients with allergic asthma 24 h after bronchial allergen challenge. A Correlation between migrated peripheral blood eosinophil count and eosinophil count in sputum. 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Teran LM, Noso N, Carroll M, Davies DE, Holgate S, Schroder JM. Eosinophil recruitment following allergen challenge is associated with the release of the chemokine RANTES into asthmatic airways. J Immunol. 1996;157(4):1806–12. 13. Barnes PJ. Reactive oxygen species and airway inflammation. Free Radic Biol Med. 1990;9(3):235–43. 12. Teran LM, Noso N, Carroll M, Davies DE, Holgate S, Schroder JM. Discussion h d h It might be that diminished apoptotic potential represents a mechanism that promotes resolution of eosinophilic inflammation in asthma. In conclusion, this study has shown that allergic asthma patients demonstrates enhanced spontaneous ROS production, increased chemotaxis, and diminished Page 9 of 9 Page 9 of 9 Lavinskiene et al. Journal of Inflammation (2015) 12:25 apoptosis of peripheral blood eosinophils, and these al- terations are more pronounced during allergen-induced late-phase airway inflammation. of Health, National Heart, Lung and Blood Institute; 2002. Revised 2012. Available from: http://www.ginasthma.org/. of Health, National Heart, Lung and Blood Institute; 2002. Revised 2012. Available from: http://www.ginasthma.org/. of Health, National Heart, Lung and Blood Institute; 2002. Revised 2012. Available from: http://www.ginasthma.org/. 20. 20. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen) Allergy. 2008;63 Suppl 86:8–160. References Eosinophil recruitment following allergen challenge is associated with the release of the chemokine RANTES into asthmatic airways. J Immunol. 1996;157(4):1806–12. 13. Barnes PJ. Reactive oxygen species and airway inflammation. Free Radic Bio Med. 1990;9(3):235–43. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 14. 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Mechanisms of human eosinophil survival and apoptosis. Clin Exp Allergy. 1997;27(5):482–7. 17. Ilmarinen P, Kankaanranta H. Eosinophil apoptosis as a therapeutic target in allergic asthma. Basic Clin Pharmacol Toxicol. 2014;114(1):109–17. Upregulated response to chemokines in oxidative metabolism of eosinophils in asthma and allergic rhinitis. Eur Respir J. 2003;21(6):925–31. 15. Sedgwick JB, Geiger KM, Busse WW. Superoxide generation by hypodense eosinophils from patients with asthma. Am Rev Respir Dis. 1990;142(1):120–5. 15. Sedgwick JB, Geiger KM, Busse WW. Superoxide generation by hypodense eosinophils from patients with asthma. Am Rev Respir Dis. 1990;142(1):120–5. • Convenient online submission • Thorough peer review 16. Walsh GM. Mechanisms of human eosinophil survival and apoptosis. Clin Exp Allergy. 1997;27(5):482–7. Exp Allergy. 1997;27(5):482–7. 17. Ilmarinen P, Kankaanranta H. Eosinophil apoptosis as a therapeutic target in allergic asthma. 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https://openalex.org/W2599039856	https://www.scielo.br/j/ape/a/mR59TVqBVkhyzWzVBYbDKFr/?lang=pt&format=pdf	Portuguese	null	Qualidade do sono e cronotipo de estudantes de enfermagem	Acta Paulista de Enfermagem	2,016	cc-by	3,734	Qualidade do sono e cronotipo de estudantes de enfermagem Sleep quality and chronotype of nursing students Teresa Celia de Mattos Moraes dos Santos1 Milva Maria Figueiredo De Martino2,3 Jaqueline Girnos Sonati1,3 Ana Lucia De Faria1 Eliana Fátima de Almeida Nascimento1 Ana Lucia De Faria1 Eliana Fátima de Almeida Nascimento1 Resumo Objetivo: Verificar a qualidade do sono, o cronotipo e as características de saúde associadas à qualidade de Descritores Estudantes de enfermagem; Estudantes Descritores Estudantes de enfermagem; Estudantes de ciências da saúde; Sono; Ritmo circadiano/fisiologia Resumo Obj ti V if Objetivo: Verificar a qualidade do sono, o cronotipo e as características de saúde associadas à qualidade de sono de estudantes. Métodos: Estudo descritivo observacional e transversal com 204 estudantes do curso de graduação em enfermagem (faixa etária de 18 à 29 anos; 91,6% sexo feminino). Foi utilizado um questionário para caracterização sociodemográfica e de saúde. O questionário de Índice de Qualidade de Sono de Pittsburgh e o Questionário de Identificação de Indivíduos Matutinos e Vespertinos foram aplicados para a verificação da qualidade do sono e a identificação do cronotipo respectivamente. Foram analisadas a associação entre as variáveis de saúde e a qualidade do sono.i Keywords Students, nursing; students, health occupations; Sleep; Circadian rhythm/ physiology Resultados: A maioria dos estudantes foi identificada com preferência de cronotipo indiferente (56,37%) e com qualidade de sono ruim (84,31%). Houve associação entre ser estudante e trabalhar com os sintomas de má digestão, cefaleia, sonolência diurna e insônia. Submetido 23 de Setembro de 2016 Aceito 21 de Novembro de 2016 g Conclusão: Estudantes de enfermagem possuem qualidade de sono ruim e preferência de cronotipo indiferente; aqueles que, acumulam as funções estudo/trabalho, apresentam maior número se sintomas de má digestão, cefaleia, sonolência diurna e insônia. má digestão, cefaleia, sonolência diurna e insônia. Artigo Original Artigo Original Qualidade do sono e cronotipo de estudantes de enfermagem Sleep quality and chronotype of nursing students Teresa Celia de Mattos Moraes dos Santos1 Milva Maria Figueiredo De Martino2,3 Jaqueline Girnos Sonati1,3 Ana Lucia De Faria1 Eliana Fátima de Almeida Nascimento1 Abstract Obj ti T Objective: To verify the quality of sleep, chronotype, and health characteristics associated with the sleep quality of students. Methods: Descriptive, observational, and cross-sectional study with 204 undergraduate nursing students (age group 18-29 years, 91.6% female). A questionnaire was used for sociodemographic and health characterization. The Pittsburgh Sleep Quality Index Questionnaire and the Morningness-Eveningness Questionnaire were applied to verify the sleep quality and identify the chronotype, respectively. The association between health variables and sleep quality was analyzed.i Autor correspondente Jaqueline Girnos Sonati Av. Tiradentes, 500, 12030-180, Taubaté, SP, Brasil. j.girnos@gmail.com p q y y Results: The majority of students was identified with preference for the indifferent chronotype (56.37%) and poor sleep quality (84.31%). There was an association between being a student and working, with symptoms of poor digestion, headache, daytime sleepiness and insomnia. Conclusion: Nursing students have poor sleep quality and preference for the indifferent chronotype. Those who accumulate the study/work functions, present more symptoms of poor digestion, headache, daytime sleepiness and insomnia. 1Universidade de Taubaté, Taubaté, São Paulo, SP, Brasil. 2Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil. 3Universidade Estadual de Campinas, Campinas, São Paulo, SP, Brasil. Conflitos de interesse: não há conflitos de interesse a declarar. 658 Acta Paul Enferm. 2016; 29(6):658-63. Santos TC, Martino MM, Sonati JG, Faria AL, Nascimento EF Introdução os indiferentes adaptam-se com maior facilidade aos horários.(7) Fatores importantes têm atuado sobre os processos regulatórios do sono-vigília nos seres humanos, como sexo, idade, cronotipo, duração do sono habitual e va- riações genéticas.(1) O ciclo claro/escuro é considerado o mais importante zeitgeber dos ritmos de mamíferos, porém em humanos, com a descoberta da luz elétrica, os padrões de sincronização foram alterados. A expo- sição à luz artificial durante a fase escura, nos casos de situações de trabalho e estudo noturno, viagens trans- meridianas e costumes como televisão e internet, está associada à dessincronização dos ritmos circadianos.(2) Assim, torna-se necessário entender melhor os ciclos biológicos diários dos estudantes, já que eles desenvolvem comportamentos de privação de sono, principalmente quando acumulam as tarefas de es- tudante com a do trabalho, para propor um plane- jamento no que diz respeito ao aproveitamento do tempo destinado aos estudos e despertar a atenção para o cuidado com a saúde. Este estudo teve com objetivo identificar o cro- notipo, a qualidade do sono e a presença de sinto- mas relacionados com a qualidade do sono do estu- dante de enfermagem que estuda e trabalha. Essa dessincronização é mantida ao longo da vida pelo perfil do ritmo do homem contemporâ- neo, que acumula tarefas, como trabalho, estudo e lazer noturno, podendo acarretar na dificuldade em adormecer e na incapacidade para acordar de ma- nhã, refletindo na qualidade dos estudos e do tra- balho. Quando se é permitido escolher os horários preferenciais naturais, o sono é geralmente de boa qualidade e segue o curso natural. Ao tentar cumprir as obrigações escolares e profissionais, surge o débito de sono, resultando em privação crônica parcial do sono e sonolência diurna excessiva.(3) Métodos 659 Qualidade do sono e cronotipo de estudantes de enfermagem Tabela 1. Distribuição dos estudantes de enfermagem quanto às características sociodemográficas derando as faixas de zero a 5 como qualidade de sono boa; de 5 a 10, como ruim; e maior que 10, com presença de distúrbio do sono.(8) Utilizou-se a versão do instrumento validado no Brasil cuja con- sistência interna foi de 0,82 (alfa de Cronbach).(8) Tabela 1. Distribuição dos estudantes de enfermagem quanto às características sociodemográficas Variáveis Estudar n=64 Estudar e trabalhar n=140 Total n=204 n(%) n(%) n(%) Sexo Feminino 61(95,31) 126(90,00) 187(91,67) Masculino 3(4,69) 14(10,00) 17(8,33) Faixa etária, anos 18-29 56(27,45) 105(51,47) 161(78,92) 30-39 4(1,96) 28(13,73) 32(15,69) 40-49 3(1,47) 7(3,43) 10(4,90) 50 ou mais 1(0,49) - 1(0,49) Estado civil Solteiro 55(26,96) 100(49,02) 155(75,98) Casado 7(3,43) 34(16,67) 41(20,10) Divorciado 2(0,98) 6(2,94) 8(3,92) Ter filhos Sim 9(4,41) 32(15,69) 41(20,10) Não 55(26,96) 108(52,94) 163(79,90) Turno escolar Manhã 53(25,98) 100(49,02) 153(75,00) Noite 11(5,39) 40(19,61) 51(25,00) Turno de trabalho Diurno* - 71(50,71) 71(50,71) Noturno† - 48(34,29) 48(34,29) Vespertino/noturno‡ - 21(15,00) 21(15,00) Categoria profissional Enfermagem - 55(39,29) 55(39,29) Estágio renumerado - 18(12,86) 18(12,86) Ensino - 2(1,43) 2(1,430) Outras atividades - 18(12,86) 18(12,86) Não informaram - 47(33,57) 47(33,57 Das 7h às 19h, das 6h às 18h, das 12h às 18h, das 13h às 19h ; †das 19h às 7h, das 18h às 6h; ‡das 17h às 22h A preferência do cronotipo foi verificada pelo Questionário de Identificação de Indivíduos Matu- tinos e Vespertinos. Esse questionário é composto de questões a respeito de situações habituais da vida diária, e o indivíduo deve registrar seus horários pre- ferenciais para essas situações, partindo-se do pres- suposto de que há total disponibilidade de tempo para escolha. O resultado é um valor numérico que varia entre 16 e 86 pontos. A classificação indica a preferência dentre cinco cronotipos: vespertino ex- tremo/definitivamente vespertino (16 a 30 pontos), moderadamente vespertino (31 a 41 pontos), indi- ferentes/nem vespertinos e nem matutinos (42 a 58 pontos), moderadamente matutino (59 a 69 pon- tos) e matutino extremo/definitivamente matutino (70 a 86 pontos).(9) Os dados foram analisados no programa Statis- tical Analysis System (SAS), versão 9,2, com auxílio de profissional estatístico. Foi utilizada estatística descritiva (medidas de tendência central e disper- são, frequências e proporções). Para estudar possí- veis associações entre as variáveis, foi aplicado o tes- te não paramétrico de qui quadrado para amostras independentes. Métodos O valor de p foi definido em 0,05 para que os resultados das análises fossem conside- rados significantes. A análise da qualidade do sono demonstrou que a maioria dos estudantes de enfermagem pesquisa- dos estava com qualidade de sono ruim, tanto aque- les que estudavam (24,51%) quanto para aqueles que estudavam e trabalhavam (59,80%). i O estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade de Taubaté, CEP/UNI- TAU: nº014/12. A percepção individual, quanto ao cronotipo, apontou que a maioria dos estudantes era indife- rente (56,38%), independente de terem ou não ati- vidade laboral. Já quanto ao período de estudo e ao turno trabalhado, verificou-se predomínio dos indi- ferentes no período da manhã (42,16%) e no turno diurno (27,86%) (Tabela 2). Métodos Trata-se de uma pesquisa descritiva, observacional de corte transversal, realizada em uma universida- de do Vale do Paraíba, no interior do estado de São Paulo, entre agosto de 2012 e junho de 2013. Participaram 204 estudantes voluntários do pri- meiro ao quinto ano do curso de graduação em enfermagem, de ambos os sexos, com idade supe- rior a 18 anos. A amostra constou de todos os alu- nos matriculados nos períodos matutino (7h30m às 11h10m) e noturno (19h00m às 22h40m), considerando toda a população de estudantes de enfermagem da universidade. O recrutamento e o preenchimento dos questionários foram efetuados durante o período de aula e, para análise dos da- dos, os estudantes foram divididos em dois grupo: os estudantes que trabalham e estudam e os estu- dantes que somente estudam. É importante salientar a existência das diferenças individuais que podem ser observadas de acordo com os aspectos cronobiológicos, pois são consideradas a pre- ferência de horários para dormir e acordar e as fases de maior disposição física e cognitiva, o que permite uma classificação de acordo com a preferência de cronotipo, pois respeita a percepção individual entre as relações de fase distintas da expressão dos ritmos circadianos e os sincronizadores externos em seres humanos.(4,5) A cronobiologia permite conhecer as caracte- rísticas individuais que compõem um fenótipo ba- seado no comportamento, assegurando, assim, que indivíduos sejam identificados dentre cinco pre- ferências de cronotipo, denominados matutinos e vespertinos extremos ou moderados, e indiferentes ou intermediários.(6) Os indivíduos matutinos são aqueles que preferem dormir cedo, em torno das 21h ou 22h, e acordar cedo, em torno das 6h, sem interferências em seu desempenho físico e mental. Os vespertinos preferem dormir após as 22h e sen- tem mais disposição à tarde e no início da noite. Já As características sociodemográficas e de saúde foram obtidas por meio de questionário com ques- tões abertas e fechadas, para sexo, idade, estado civil, ter filhos, possuir outras atividades, tipo e horário da atividade, queixas com relação ao estado de saúde, ho- rário de trabalho e prejuízo com a saúde, e horário de estudo e consideradas como variáveis independentes. A qualidade do sono foi verificada por meio do instrumento de Índice de Qualidade de Sono de Pittsburgh, que quantifica a qualidade do sono por uma escala que varia de zero a 21 pontos, consi- Acta Paul Enferm. 2016; 29(6):658-63. Acta Paul Enferm. 2016; 29(6):658-63. Resultados Os resultados sociodemográficos mostraram pre- domínio do sexo feminino (91,67%) com idade média de 24,97(±6,82), solteiros (75,98%), sem fi- lhos (79,90%), alocados no turno escolar matutino (75%), terem atividade laboral (68,62%) na área de enfermagem (39,39%) e essa atividade se concen- trar nos turnos diurno (50,71%) e noturno (34,29) (Tabela 1). Ser estudante e trabalhar implicou em maior frequência de sintomas de má digestão (p=0,0016), cefaleia (p=0,0357), sonolência na aula (p=0,0395) e insônia (p=0,0369) (Tabela 3). 660 Acta Paul Enferm. 2016; 29(6):658-63. Santos TC, Martino MM, Sonati JG, Faria AL, Nascimento EF Tabela 2. Distribuição dos estudantes de enfermagem quanto o cronotipo Cronotipo Total (n=204) Estudar e trabalhar (n=140) Manhã Noite Total Diurno Noturno Vespertino/noturno Total n(%) n(%) n(%) n(%) n(%) n(%) n(%) Definitivamente matutino 5(2,45) - 5(2,45) 2(1,43) 2(1,43) - 4(2,86) Definitivamente vespertino 5(2,45) 3(1,47) 8(3,92) 2(1,43) 2(1,43) 1(0,71) 5(3,57) Moderadamente matutino 32(15,69) 7(3,43) 39(19,12) 16(11,43) 10(7,14) 3(2,14) 29(20,71) Moderadamente vespertino 25(12,25) 12(5,88) 37(18,13) 12(8,57) 11(7,86) 3(2,14) 26(18,57) Indiferente 86(42,16) 29(14,22) 115(56,38) 39(27,86) 23(16,43) 14(10,00) 76(54,29) Tabela 2. Distribuição dos estudantes de enfermagem quanto o cronotipo Tabela 3. Associação entre as variáveis de sintomas relatados de má digestão, cefaleia, sonolência e insônia e o período escolar e turnos de trabalho de estudantes de enfermagem Sinais e sintomas Turno escolar (n=204) p-value Turno de trabalho (n=140) p-value Manhã Noite Diurno Noturno Vespertino/noturno n(%) n(%) n(%) n(%) n(%) Má digestão Sim 37(80,43) 9(19,57) 0,3334 9(25,00) 19(52,78) 8(22,22) 0,0016* Não 116(73,42) 42(26,58) 62(59,62) 29(27,88) 13(12,50) Cefaleia Sim 75(78,95) 20(21,05) 0,2242* 30(46,88) 19(29,69) 15(23,44) 0,0357* Não 78(71,56) 31(28,44) 41(53,95) 29(38,16) 6(7,89) Sonolência na aula Sim 80(76,92) 24(23,08) 0,5177* 29(40,28) 30(41,67) 13(18,06) 0,0395* Não 73(73,00) 27(27,00) 42(61,76) 18(26,47) 8(11,76) Insônia Sim 25(71,43) 10(28,57) 0,5919* 8(29,63) 12(44,44) 7(25,93) 0,0369* Não 128(75,74) 41(24,26) 63(55,75) 36(31,86) 14(12,39) p-valor obtido por meio do teste qui quadrado. Tabela 3. Associação entre as variáveis de sintomas relatados de má digestão, cefaleia, sonolência e insônia e o período escolar e turnos de trabalho de estudantes de enfermagem Sinais e sintomas Turno escolar (n=204) p-value Turno de trabalho (n=140) p-value Manhã Noite Diurno Noturno Vespertino/noturno Tabela 3. Associação entre as variáveis de sintomas relatados de má digestão, cefaleia, sonolência e insônia e o período escolar e turnos de trabalho de estudantes de enfermagem as variáveis de sintomas relatados de má digestão, cefaleia, sonolência e insônia e o período escolar e ntes de enfermagem p-valor obtido por meio do teste qui quadrado. Discussão A maioria dos estudantes estava inserida no período da manhã, e com turno de trabalho concentrado no período diurno e noturno. Essa é uma característica do profissional de enfermagem, que prioriza o trabalho no- turno em detrimento de maior ganho salarial, já que os pesquisados também eram maioria no trabalho na área de enfermagem. A dupla jornada (estudo-trabalho), sobretudo para os turnos noturnos, leva a dificuldades para dormir durante o dia, comprometendo a saúde, as relações sociais como família e amigos,(11) repercutindo também na vida acadêmica, pois a permanência na gra- duação depende da organização da vida laboral que é sempre priorizada.(12) O presente estudo apresenta limitações caracterís- ticas de estudo transversal não avaliando causa e efeito, mas indicando preocupação quanto o sono e saúde da população estudada. Os resultados desta pesquisa, aliados com os dados encontrados na literatura, sugerem que os estudantes de graduação em enfermagem são do sexo feminino, com predomínio de jovens na faixa etária de 18 a 29 anos, seguidos por indiví- duos na faixa etária de 30 a 39 anos, e com me- nor porcentual para a idade acima dos 40 anos. Essas características são também encontradas em estudo sobre qualidade de vida de estudantes in- gressantes no curso de enfermagem da Universi- dade Federal Fluminense.(10) Já quanto ao estado civil, os resultados da presente pesquisa foram divergentes, pois foram verificados alguns estu- dantes casados e com filhos, o que por ter sido encontrado por incluir os estudantes de todos os anos do curso e não somente os ingressantes. A maioria dos estudantes apresentou qualidade de sono ruim. Esses dados estão de acordo com a literatura, corroborando estudo realizado com 701 alunos da Universidade Federal do Ceará, o qual evidenciou que 95,3% dos estudantes estavam com má qualidade do sono(13) e com estudo realizado na Universidade Federal de Pernambuco com 173 es- tudantes (92 dos cursos da área de exatas, matemá- tica, física e computação, e 81 da área de biológicas Acta Paul Enferm. 2016; 29(6):658-63. Discussão 661 Qualidade do sono e cronotipo de estudantes de enfermagem e educação física), que verificou a má qualidade do sono e sonolência diurna excessiva em estudantes da área de saúde e exatas.(14) A concordância entre a preferência de crono- tipo e o período de trabalho e estudo também é importante na determinação do desempenho escolar, laboral e na melhor qualidade de vida.(1) O perfil cronobiológico pode minimizar ou po- tencializar os efeitos negativos das alterações do ciclo vigília-sono. Com relação aos estudantes que também traba- lham, os dados quanto à sonolência estão de acordo com os apresentados na literatura, corroborando o estudo realizado em uma faculdade particular do in- terior de São Paulo e envolvendo estudantes de en- fermagem que trabalhavam no turno noturno. Esse estudo demonstrou que, de acordo com a escala de Sonolência de Epworth, os sujeitos apresentaram escores de sonolência que variaram de 7,2 a 15,9, com média de 11,4, caracterizando prevalência de sonolência diurna excessiva. O autor conclui que, por conta dos estudos, os estudantes aumentam suas horas de vigília, resultando em alta incidência de sonolência diurna.(15) A preferência de cronotipo encontrada na pes- quisa foi do tipo indiferente (nem vespertinos e nem matutinos). Esse perfil foi semelhante ao en- contrado em estudo realizado com universitários do curso de ciências biológicas da Universidade Metodista de Piracicaba, no interior do Estado de São Paulo. Os dados indicaram que os tipos indiferentes e os moderadamente vespertinos são sete vezes maiores do que os matutinos típicos, 1,4 vez maior que os moderadamente matutinos e 3,5 vezes maiores que os vespertinos típicos.(6) Essas características mostram uma adaptação ao cronotipo indiferente aos ritmos sociais. Portan- to, entender as questões que envolvem o cansaço, sonolência excessiva e duração, e qualidade do sono de estudantes é de grande interesse, uma vez que esses fatores estão associados a um menor desempenho escolar. Discussão O impacto negativo da dupla jornada, trabalho e estudo, no sono de jovens estudantes brasileiros, da ci- dade de São Paulo, foi confirmada comparando o dé- bito de sono antes dos estudantes assumirem a dupla jornada e após estarem comprometidos com o estudo e o trabalho, mostrando a existência de diferença sig- nificante entre os valores médios do tempo de sono.(16) i Esses achados somados aos resultados encontra- dos nessa pesquisa é preocupante, já que o sono está associado a diferentes modos de processamento da memória, favorecendo sua consolidação e a recupe- ração de estímulos que são requeridos no momento da vigília,(17) podendo, portanto, interferir no pro- cesso de consolidação do conhecimento dos estu- dantes e na qualidade do profissional formado. Conclusão O estudo encontrou preferência de cronotipo pre- dominantemente indiferente com qualidade de sono ruim e associação entre insônia, cefaleia, má digestão e sonolência na aula para os estudantes que trabalhavam e estudavam. Essas conclusões mos- tram a importância de se investir no entendimento das alterações que podem ocorrer entre os ritmos biológicos e os ciclos ambientais, principalmente em relação às adequações nos horários do turno es- colar e do trabalho em turnos. q pi As consequências da dupla jornada (trabalho-es- tudo) para com a saúde, se confirmaram na presente pesquisa com os achados de associações entre as va- riáveis sonolência em sala de aula, insônia, cefaleia e má digestão e a presença da dupla jornada. O tra- balho desenvolvido em turno, em especial aqueles que proporcionam maior débito de sono, têm sido associados com fadiga e sonolência, condições não esperadas para indivíduos que desempenham ativi- dades que exigem concentração e atenção.(18) Esses achados indicam a necessidade de mudança no es- tilo de vida, que promovam hábitos saudáveis en- volvendo melhor qualidade de sono, alimentação e atividade física.(19) Acta Paul Enferm. 2016; 29(6):658-63. Colaborações Colaborações Santos TCMM, De Martino MMF, Sonati JG, De Faria AL, Nascimento EFA declaram que contribuí- ram com a redação do artigo, revisão crítica rele- vante do conteúdo intelectual e aprovação final da versão a ser publicada. Santos TCMM, De Martino MMF colaboraram nas etapas de concepção do es- tudo, análise, interpretação dos dados, redação do artigo, revisão crítica relevante do conteúdo intelec- tual e aprovação final da versão a ser publicada. 9. Benedito Silva AA, Menna-Barreto L, Marques N, Tenreiro S. A self- assessment questionnaire for the determination of morningness- eveningness types in Brazil. In: Hayes DK, Pauly JE, Reiter RJ, editors. Cronobiology: its role in clinical medicine, general biology and agriculture. Part B. New York: Wiley-Liss; 1990. p.89-98. 10. Almeida PF, Espírito Santo FH. Qualidade de vida: um estudo com ingressantes do curso de graduação em enfermagem e licenciatura. Rev Pesq Cuid Fundam. 2012; 4(1):2647-53. 11. Korompeli A, Muurlink O, Tzavara C, Velonakis E, Lemonidou C, Sourtzi P. Influence of Shiftwork on greek nursing personnel. Saf Health Work. 2014; 5(2):73-9. 12. Maier SRO, Mattos M. O trabalhar e o estudar no contexto universitário: uma abordagem com trabalhadores-estudantes. Rev Saúde (Santa Maria). 2016; 42(1):179-85. Agradecimentos Agradecemos a Universidade de Taubaté por permitir o desenvolvimento do projeto, espe- cialmente a diretora do Departamento de Enfer- magem e Nutrição Professora Ms. Maria Angela Petrini. Acta Paul Enferm. 2016; 29(6):658-63. 662 Santos TC, Martino MM, Sonati JG, Faria AL, Nascimento EF 8. Bertolazi AN, Fagondes SC, Hoff LS, Dartora EG, Miozzo IC, Barba ME, et al. Validation of the Brazilian portuguese version of the Pittsburg Sleep Quality Index. Sleep Med. 2011; 12(1):70-5. Referências 13. Araújo MF, Lima AC, Alencar AM, Araújo TM, Fragoso LV, Damasceno MM. Avaliação da qualidade do sono de estudantes universitários de Fortaleza-CE. Texto Contexto Enferm. 2013; 22(2):352-60. 1. Maire M, Reichert CF, Schmidt C. Sleep-wake rhythms and cognition. J Cogn Behav Psychother. 2013; 13(1a):133-70. 14. Carvalho TM, Silva Jr II, Siqueira PP, Almeida JO, Soares AF, Lima AM. Qualidade do sono e sonolência diurna entre estudantes universitários de diferentes áreas. Rev Neurociênc. 2013; 21(3):383-7. 2. Pereira E, Anacleto TS, Louzada FM. Interação entre sincronizadores fóticos e sociais: repercussões para a saúde humana. Rev Biol. 2012; 9(3):68-73. 15. Ferreira LR, De Martino MM. [Sleep patterns and fatigue of nursing students who work]. Rev Esc Enferm USP. 2012; 46(5):1176-81. Portuguese. 3. Zhu L, Zee PC. Circadian rhythm sleep disorders. Neurologic Clin. 2012; 30(4):1167-91. 16. Fischer FM, Wey D, Valente D, Luz AA, Pinheiro F, Fonseca BC, Silva-Costa A, Moreno CR, Menna-Barreto L, Teixeira LR. Sleep patterns and sleepiness among Young students: A longitudinal study before and after admission as trainees and apprentices. Chronobiol Int. 2015; 32(4): 478-85. 4. Horne JA, Ostberg O. A self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms. Int J Chronobiol. 1976; 4(2):97-110. 5. Duarte LL, Menna-Barreto L, Miguel MA, Louzada F, Araújo J, Alam M, et al. Chronotype ontogeny related to gender. Braz J Med Biol Res. 2014; 47(4):316-20. 17. Rasch B, Born J. About sleep’s role in memory. Physiol Rev. 2013; 93(2):681-766. 18. Sonati JG, De Martino M, Vilarta R, Maciel E, Moreira E, Sanchez F, De Martino G, Sonati R. Quality of life, health, and sleep of air traffic controllers with different shift systems. Aerosp Med Hum Perform. 2015; 86(10):895-900. 6. Duarte M, Silva CA. Identificação do cronotipo e perfil cronobiológico de uma população de acadêmicos de Ciências Biológicas da Unimep. Saúde Rev. 2012; 12(31):53-60. 7. Andreoli CP, De Martino MM. Academic performance of night-shift students and its relationship with the sleep-wake cycle. Sleep Sci. 2012; 5(2):45-8. 19. Sonati JG, De Martino MMF, Vilarta R, Maciel ES, Sonati RJF, Paduan PC. Quality of life, sleep, and health of air traffic controllers with rapid counteclockwise shift rotation. Workplace Health Saf. 2016; 64(8): 377-84. 663 Acta Paul Enferm. 2016; 29(6):658-63.
https://openalex.org/W4212878976	https://www.swsc-journal.org/10.1051/swsc/2022002/pdf	English	null	GEANT4 simulation study of the response of a miniature radiation detector in Galactic Cosmic Rays and inside a spacecraft.	Journal of space weather and space climate	2,022	cc-by	6,908	Received 19 May 2021 / Accepted 13 February 2022 Abstract – The Miniaturized Detector for Application in Space (MIDAS) is a compact device with dimensions 5 5 1 cm3 that combines position-sensitive Si detectors and a fast neutrons spectrometer. MIDAS is developed with the purpose of acting as a linear energy transfer (LET) spectrometer for the charged particles and measuring dose and dose equivalent from both charged particles and neutrons. It is based on fully depleted monolithic active Si pixel sensors for the charged track and energy deposition measurements, while a plastic scintillator read out by a silicon photomultiplier is used to determine energy depositions from fast neutrons. A simulation study of the detector response in galactic cosmic ray (GCR) radiation ﬁelds with the aid of GEANT4 has been performed. Energy depositions and hit pixel addresses have been used to reconstruct tracks and calculate LET spectra. A method to calculate LET1 in water from the measured LET has been elaborated. The dose rate in water and dose equivalent rate has been calculated. The energy and particle composition of the radiation ﬁeld produced by the interaction of GCR with the Al walls of a spacecraft model has been determined, and the response of MIDAS in this radiation ﬁeld has been investigated. Keywords: GEANT4 simulations / space dosimetry / depleted monolithic active pixel sensors It is well known that the absorbed dose of radiation is mea- sured in units of Gray (1 Gy = 1 J/kg). As not all sources of radiation have the same biological effectiveness, the dose equiv- alent measured in units of Sieverts (Sv) takes this into account. The dose equivalent (in Sv) is equal to the dose (in gray) times the quality factor (Q), where Q is a function of the linear energy transfer (LET) – the rate of energy loss of a particle in water measured in keV lm1. GEANT4 simulation study of the response of a miniature radiation detector in Galactic Cosmic Rays and inside a spacecraft Konstantinos Karafasoulis1,2 , Christos Papadimitropoulos2 , Constantinos Potiriadis2,3, and Charalambos Pan Lambropoulos2,* 1 Hellenic Army Academy, Vari, Attiki 16673, Greece 2 1 Hellenic Army Academy, Vari, Attiki 16673, Greece Hellenic Army Academy, Vari, Attiki 16673, Greece 2 Department of Aerospace Science and Technology, National and Kapodistrian University of Athens, Psahna-Evia 34400, Greece 3 Greek Atomic Energy Commission, Agia Paraskevi, Attiki 15310, Greece e e c y cade y, Va , tt 6673, G eece 2 Department of Aerospace Science and Technology, National and Kapodistrian University of Athens, Psahna-Evia 34400, Greece 3 Greek Atomic Energy Commission, Agia Paraskevi, Attiki 15310, Greece 2 Department of Aerospace Science and Technology, National and Kapodistrian University of Athens, Psahn 3 Greek Atomic Energy Commission, Agia Paraskevi, Attiki 15310, Greece *Corresponding author: lambrop@uoa.gr This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Topical Issue - Space Weather Instrumentation Topical Issue - Space Weather Instrumentation OPEN ACCESS J. Space Weather Space Clim. 2022, 12, 8 J. Space Weather Space Clim. 2022, 12, 8 K. Karafasoulis et al., Published by EDP Sciences 2022 Available online at: www.swsc-journal.org Available online at: www.swsc-journal.org Available online at: www.swsc-journal.org TECHNICAL ARTICLE OPEN ACCESS p p K. Karafasoulis et al., Published by EDP Sciences 2022 J. Space Weather Space Clim. 2022, 12, 8 1 Hellenic Army Academy, Vari, Attiki 16673, Greece . a a asou s et a ., ub s ed by Sc e ces 0 https://doi.org/10.1051/swsc/2022002 1 Introduction We designed in-pixel electronics to handle a wide dynamic range of energy depositions in Si voxels with dimensions 106 106 50 lm3 from less than the most prob- able value of energy loss of minimum ionizing protons up to more than 2.33 MeV lm1 (10 MeV cm2 mg1 in Si). MIDAS is a cube whose 5 facets house 2 layers of DMAPS, while the one facet is covered by a silicon photomultiplier (SiPM) and one layer of DMAPS. The SiPM reads the light output of a plas- tic scintillator, which occupies the interior volume of the cube with dimensions 7 7 7 mm3. The ﬁve facets of the scintil- lator are covered by a Ti box with 1 mm thickness which absorbs the recoil protons with kinetic energy up to 18 MeV. The device concept, ﬁrst prototype iteration, initial measure- ments, and simulations have been described in Lambropoulos et al. (2019). MIDAS is at technology readiness level 4. with 51 lm thickness. The parameters were selected purposely to compare with the values reported in Table VIII of Bichsel (1988) for protons with bc = 2.1 (momentum 2 GeV/c, kinetic energy 1244.1 MeV) and taken from Bak et al. (1987). The peak of the distribution of the deposited energy was found at 13.82 keV, and the Full Width at Half Maximum (FWHM) is 7.2 keV. The corresponding experimental values are 12.64 keV for the distribution peak of the deposited energy and 7.446 keV for the FWHM. With the aid of the OMERE freeware (OMERE), the differ- ential ﬂux distributions of galactic cosmic rays (GCR) according to the ISO 15390 model for the solar minimum 1996.4 between the end of Cycle 22 and the beginning of Cycle 23 were obtained and used as energy distributions of a spherical source with a radius of 20 cm. The energy range is from 0.1 MeV/n up to 20 GeV/n, and it is partitioned in 835 logarithmic bins. The device was placed at the center of this source, which emitted par- ticles with equal probability from all points of its interior surface, with cosh and u uniformly distributed in the interval [1, 1] and [0, 2p] respectively, where h is the polar angle with respect to the radius of the sphere from the point of emission and u is the azi- muthal angle in the plane perpendicular to this radius. 1 Introduction Equal numbers of particles (107) were generated for each element with Z = 1 up to Z = 26. The distribution of a calculated quantity resulting from the Monte Carlo experiment for one element was scaled with the integral over the energy of the element’s ﬂux with purpose to assign to this distribution the correct weight in the weighted sum for all particles. This weighted sum gives the ﬁnal distribution of the quantity due to the GCR spectrum. The geometry of the device, the visualization of the tracks of the particles produced due to the passage of a Fe56 ion with 10 GeV kinetic energy, and the die photo of the ﬁrst version of the Si active pixel sensors can be seen in Figure 1. ( ) gy In the present paper, based on simulation experiments per- formed with the aid of the GEANT4 (GEometry ANd Tracking) package, we present a detailed study of the following subjects: (a) The reconstruction of the charged particle tracks using the energy deposition patterns on the pixel layers. (b) The calcula- tion of the measured LET distribution. (c) The correction of the measured LET for the escaped energy and the determination of LET1 in water. (d) The calculation of the dose and dose equiv- alent rate using LET1 in water. (e) The response of MIDAS inside a spacecraft model, where the GCR radiation ﬁeld is modiﬁed due to the interaction with the Al walls. Although the purpose of the work presented is to elucidate some of the capabilities of this novel device with the aid of Monte Carlo tools, the obtained results agree with some ﬁeld measurements and with simulation results reported by other groups. The analysis presented here has been performed with the following assumptions: (a) the depleted monolithic active pixel sensors have 50 lm thickness, and all the energy deposited to a voxel with dimensions 106 106 50 lm3 is registered. The wafers of the ﬁnal version of the MIDAS pixel detectors will be processed to become 50 lm thick. (b) A layer of silicon sensors is mounted below the SiPM and at the bottom layer of the asso- ciated electronics board. This means that all the plastic scintilla- tor cube facets are covered by at least one silicon layer, something which was not foreseen in the initial design of the device, as explained in Lambropoulos et al. (2019). 1 Introduction (c) In the calculations, only energy depositions above 20 keV are used as a conservative estimate of the pixel readout electronics sensitivity. 2 The simulation environment, geometry, and materials The Monte Carlo simulations were performed using the GEANT4 toolkit version 4.10.06.p02 (Agostinelli et al., 2003; Allison et al., 2006, 2016). The QGSP_BERT_HP physics list has been used. The acronym stands for the “Quark gluon string model,” which allows to simulate reactions of high energy hadrons with nuclei and to simulate high energy electro- and gamma-nuclear reactions; the “Bertini cascade model”, which treats nuclear reactions initiated by long-lived hadrons and gam- mas with energies between 0 and 10 GeV, and the “High pre- cision neutron model”, which uses cross-section for neutrons with energies up to 20 MeV from the ENDF/B-VI evaluated data library. It includes the G4EmStandardPhysics list, and the production threshold for gammas, electrons, and positrons is 1 mm. For space applications, the correct treatment of low-energy protons is important. For this reason, the default GEANT4 model (Lassila-Perini & Urbán, 1995) for the calcu- lation of energy depositions by charged particles was checked: in a simulation experiment, protons with 1244.1 MeV kinetic energy were impinging vertically on a fully depleted silicon slab 1 Introduction Coarsely segmented Si diode detectors are used in almost all the instruments measuring the mixed radiation ﬁelds of the space environment (Spence et al., 2010; Hassler et al., 2012; Rodríguez-Pacheco et al., 2020). For almost a decade, hybrid Si pixel detectors have started to penetrate the ﬁeld of space dosimetry with starting event the installation of 5 Timepix units onboard the International Space Station in 2012 (Pinsky et al., 2014). Instruments using Si pixel detectors for monitoring radia- tion in the space environment can achieve orders of magnitude reduction in mass, size, and power consumption. Wide ﬁelds of view without compromising the geometric resolution can be achieved, something that cannot be done with coarsely seg- mented Si diodes in telescopic conﬁgurations. Miniaturized telescopes based on partially depleted active Si pixel sensors using the Silicon on Insulator technology with heritage from X-ray imaging have been proposed and are developed (Vrba et al., 2018). l MIDAS is developed in response to the European Space Agency (ESA) request for a device whose size, power consump- tion, and radiation data output would increase the level of space- ﬂight crew autonomy regarding operational decisions related to radiation hazards. ESA required a device sensitive to both charged particles and neutrons to enable measurement of dose, dose rate, energy deposition, LET, and calculation of dose equiv- alent. For neutrons, the desired energy ranges from 100 keV up to 200 MeV. For charged particles, the capability to measure LET values of at least 10 MeV cm2 mg1 in Si was also re- quired. The mass limit of the device was set at 50 g, with dimensions up to 5 5 1 cm3. Power consumption should allow 30 days of autonomous operation. For measuring the K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 tracks and the energy depositions from charged particles, we proposed to use MIDAS fully depleted monolithic active pixel sensors (DMAPS). In the last decade, DMAPS have been stud- ied and introduced in high-energy physics experiments for mea- suring minimum ionizing particles (Peric, 2007). Fully depleted Si sensors achieve fast charge collection and are less prone to radiation damage. 3 Track reconstruction of charged particles The correct estimation of the track direction is necessary for the calculation of the distance traveled by the particle in a Si layer and, consequently, of the measured LET, which can be deﬁned as, LETmeas ¼ Edep=L; ð1Þ ð1Þ where Edep is the deposited energy in the Si sensor and L is the track length in it. The deposited energy and the track length are quantities that can be found using either Monte Carlo or real data. Page 2 of 8 Page 2 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 Fig. 1. (a) The geometry of the sensitive cube: two layers of Si pixel detectors are placed on each facet of a Ti box, which contains a plastic scintillator. At the bottom, a silicon photomultiplier in touch with the plastic scintillator and beneath them one layer of Si. (b) The die of the Si active pixel sensor. (c) Visualization of the tracks of secondary particles produced when a 10 GeV 56Fe ion hits the device. Gray: the 56Fe track; red: photon tracks; green: neutron tracks; blue: proton tracks. Fig. 1. (a) The geometry of the sensitive cube: two layers of Si pixel detectors are placed on each facet of a Ti box, which contains a plastic scintillator. At the bottom, a silicon photomultiplier in touch with the plastic scintillator and beneath them one layer of Si. (b) The die of the Si active pixel sensor. (c) Visualization of the tracks of secondary particles produced when a 10 GeV 56Fe ion hits the device. Gray: the 56Fe track; red: photon tracks; green: neutron tracks; blue: proton tracks. The track direction is estimated with the following steps: The track direction is estimated with the following steps: not from the primary track. Usually, the energy deposi- tions due to delta rays are much lower than the ones pro- duced by the primary ion. We found that by excluding from the 3D line ﬁt clusters with energy less than 7% of the total energy of the four candidates, the distribution of the difference angle between the real and the recon- structed direction of the primary track becomes narrower. (a) The pixels of a Si sensor with registered hits are grouped into clusters, provided they have a common edge or ver- tex, and the deposited energies in the voxels deﬁned by these pixels are summed. The cluster having the highest deposited energy is selected. (b) As every Si layer contains 4 sensors, the cluster or iso- lated pixel with the highest deposited energy among all the 4 sensors is selected as a candidate for the 3D line ﬁt that will provide the estimation of the track direction. For this cluster, the energy weighted barycenter is calculated. (e) A 3D line ﬁt is performed to the barycenter points of the clusters that have passed the cut imposed in step (d), pro- vided they are more than two. If two clusters have remained, then the line that connects their barycenter points is calculated. (f) When a 3D line ﬁt is performed, the sum of the squares of the residuals of the ﬁt is computed. If the sum is above 0.01 mm2, the least energetic cluster is excluded, and the line is estimated again using the remaining clusters. (f) When a 3D line ﬁt is performed, the sum of the squares of the residuals of the ﬁt is computed. If the sum is above 0.01 mm2, the least energetic cluster is excluded, and the line is estimated again using the remaining clusters. (c) If energy has been deposited in more than four Si layers, there will be more than four candidate energy clusters. In this case, the barycenter points of the four most energetic ones are used as candidate points to which a 3-D line is ﬁtted. (g) The direction cosines of a unit vector along the estimated track line are calculated. (g) The direction cosines of a unit vector along the estimated track line are calculated. 4 LET estimation The measured LET is calculated using (1) for each Si layer traversed by the ﬁtted track line, and the average value for these layers is used to construct the distribution of LETmeas. As this distribution has been calculated separately for each sample of primary ions, it is scaled with the ion’s relative contribution to the GCR ﬂux spectrum, and the weighted sum for all of them is the ﬁnal measured LETmeas(Si). The result is presented in Figure 3 together with LETH2O deduced from LETmeas with the procedure described below. Fig. 4. Scatter plot of LETH2O as calculated with G4EMCalculator and of LETmeas for all ions. A reduced number of points for each sample is included to improve visibility. The ﬁtted line is computed using the full samples. For each sample of ion species, the Monte Carlo truth infor- mation is used to calculate the LET in water (LETH2O) using the Geant4 G4EMCalculator for every reconstructed track. the wrong inclusion of secondary or, sometimes, fake tracks as primaries. The reconstruction efﬁciency can be deﬁned as the number of simulated events whose reconstructed tracks contribute to the measured LET spectra over the events handled by the Geant4 G4EMCalculator. Consequently, correction factors Cj, rec as a function of LETH2O are calculated as the inverse of the reconstruction efﬁciency. They are given by the following expression: LETmeas and LETH2O for all ion species are inserted in the scatter plot presented in Figure 4. The best ﬁt line to this diagram is found to be, ð2Þ log10ðLETH2OÞ ¼ p0 þ p1 log10ðLETmeasÞ ð2Þ 10ð H2OÞ 0 1 10ð measÞ ð Þ where p0 = 0.25797 ± (0.00085) and p1 = 0.98514 ± (0.00038). With the aid of this equation, the obtained LETmeas values are converted to LETH2O values. This result is very close to that by Benton et al. (2010) obtained from range/ energy relations for ions ranging in charge from 1 to 26 over an energy interval of 0.8–2000 MeV amu1. where p0 = 0.25797 ± (0.00085) and p1 = 0.98514 ± (0.00038). With the aid of this equation, the obtained LETmeas values are converted to LETH2O values. This result is very close to that by Benton et al. (2010) obtained from range/ energy relations for ions ranging in charge from 1 to 26 over an energy interval of 0.8–2000 MeV amu1. The track direction is estimated with the following steps: (d) Since heavy ions produce secondary particles in interac- tions with either the active (silicon and plastic scintillator) or the non-active materials (titanium box, enclosure, substrate PCBs) of the device, it is possible that the Si sensors register hits due to the secondary radiation and (d) Since heavy ions produce secondary particles in interac- tions with either the active (silicon and plastic scintillator) or the non-active materials (titanium box, enclosure, substrate PCBs) of the device, it is possible that the Si sensors register hits due to the secondary radiation and Using the described algorithm, a track direction is calculated for every event, and this is considered as the direction to be used for the determination of LET. However, it is probable that a primary ion will interact with the non-active parts of the device, Page 3 of 8 Page 3 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 Fig. 3. LETmeas(Si) resulting from the addition of the weighted distributions from all the ions with Z = 1 to Z = 26 using as weights their relative contribution to the GCR ﬂux spectrum (blue) and LETH2O computed using the method described in the text. The bin width is 1 keV lm1. Fig. 2. Distribution of the angle between the real (known from MC) and reconstructed track. Fig. 3. LETmeas(Si) resulting from the addition of the weighted distributions from all the ions with Z = 1 to Z = 26 using as weights their relative contribution to the GCR ﬂux spectrum (blue) and LETH2O computed using the method described in the text. The bin width is 1 keV lm1. Fig. 3. LETmeas(Si) resulting from the addition of the weighted distributions from all the ions with Z = 1 to Z = 26 using as weights their relative contribution to the GCR ﬂux spectrum (blue) and LETH2O computed using the method described in the text. The bin width is 1 keV lm1. Fig. 2. Distribution of the angle between the real (known from MC) and reconstructed track. Fig. 2. Distribution of the angle between the real (known from MC) and reconstructed track. Fig. 3. LETmeas(Si) resulting from the addition of the weighted distributions from all the ions with Z = 1 to Z = 26 using as weights their relative contribution to the GCR ﬂux spectrum (blue) and LETH2O computed using the method described in the text. The track direction is estimated with the following steps: The bin width is 1 keV lm1. Fig. 2. Distribution of the angle between the real (known from MC) and reconstructed track. and the Si sensors will register only the secondary tracks and not the primary one. As in reality, it will not be possible to discriminate against such an event, we do not exclude the false primary tracks from our analysis. This practically means that a highly energetic delta ray may be treated as if it were a proton. In Figure 2 is presented the distribution of the angle between the real and the reconstructed track for a sample of 56Fe events with the GCR energy distribution. One can see that although the distribution has peaks at 0 rad and p rad, there is a contin- uum of events between these two angles because the recon- structed track assigned to a primary particle is the track of a secondary delta ray. Fig. 4. Scatter plot of LETH2O as calculated with G4EMCalculator and of LETmeas for all ions. A reduced number of points for each sample is included to improve visibility. The ﬁtted line is computed using the full samples. 5 Dose and dose equivalent calculation We have found a dose equivalent rate in water for the GCR spectrum H = 2.32 mSv day1 when summing up to 400 keV lm1 with an average Quality factor <Q> = 4.58 and H = 2.57 mSv day1 with <Q> = 4.89, when summing up to 1000 keV lm1. As the purpose of this study is to provide estimates of the dose and dose rate indications of the MIDAS device in the GCR ﬁeld, a computation of count rate is necessary. For this reason, the effective cross-section of the device has been determined with the following procedure: In the interior of the spherical source used to generate 107 primary particles with their directions distributed uniformly, a series of concentric spheres were placed. In these spheres, the primary particles do not undergo any interaction; they are used only for counting the primary particles entering their interior. The area of the great circles of the concentric spheres as a function of the number of the particles entering them shows a linear behavior thus a linear equation relating the area of the great circle to the number of entering particles was obtained. The number of particles enter- ing the MIDAS sensitive volume without surrounding enclosure was counted with the aid of a separate Monte Carlo experiment. It was found that the number of particles entering MIDAS is the same as the number of those entering a sphere with 0.422 cm radius and a great circle with 0.5595 cm2 area, which is the effective cross-section, Aeff, of MIDAS. The time corresponding to the 107 particles generated was deduced using the relation, p l In the correction calculation of the inefﬁciency of MIDAS in reconstructing the LET spectrum, the model of the radiation environment is used. As in reality, the radiation environment is not known beforehand, we tried to estimate the effect on the dose calculation using the measured LET distribution for the 1996.4 solar minimum corrected with Cj,rec evaluated for solar maximum. The resulting dose rate in water is 0.460397 mGy day1, the dose equivalent rate in water is 1.99588 mSv day1 and <Q> = 4.33513. See the Equation (5) bottom of the page and the relation, H ¼ 1 qH2O X N i¼1 LETi QðLETiÞ N i ð6Þ ð6Þ ð6Þ 4 LET estimation Cj; rec LETH2O ð Þ ¼ P ions W ion ^nj; ion LETH2O ð Þ P ions W ion nj;ion LETH2O ð Þ where the sum is over all the ions contributing to the simula- tion, Wion is the relative contribution of the ion in the GCR spectrum, ^nj; ion LETH2O ð Þ is the content of the bin j of the gy The measured LET spectrum should be corrected both for inefﬁciencies in the estimation of the primary tracks and for Page 4 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 LET distribution constructed using the Geant4 G4EM Calculator to ﬁnd the values of LET in water from all the primary particles entering the silicon volumes, while nj;ion LETH2O ð Þ is the content of bin j of the measured LET distribution, calculated from the reconstructed tracks and converted using equation (2). Cj; rec LETH2O ð Þ have been calcu- lated from samples of events different from those used to extract the measured LET distribution to which they have been applied. of bin i expressed in joules cm1 and qH2O) is the water density in kg cm3. The LET bin width has been set to 1 keV lm1 and the sum was performed for LET values up to 400 keV lm1 and for LET values up to 1000 keV lm1. We have found an absorbed dose rate in water D = 0.506 mGy day1 when summing up to 400 keV lm1 and D = 0.526 mGy day1 when summing up to 1000 keV lm1 for the GCR spectrum. For the dose equivalent calculation, we have used the (ICRP, 1991) quality factors Q: Consequently, the LETH2O distribution presented in Figure 3 and the dose estimation described in the following section have been deduced using the content nj(LETmeas) of each bin j of the measured LET distribution (either from a real or a Monte Carlo sample) converted using equation (2) to nj LETH2O ð Þ and multiplied with Cj; rec LETH2O ð Þ. See the Equation (5) bottom of the page 6 Radiation ﬁeld inside the spacecraft The radiation ﬁeld inside a spacecraft is modiﬁed due to the partial absorption of the primary particles coming from GCR and SEP (solar energetic particles) events in the walls and the reactions of these particles with the spacecraft materials. There- fore, the response of MIDAS in a radiation ﬁeld representative of what will be encountered inside a spacecraft has been studied. time ¼ N incident Aeff Utot ; ð3Þ ð3Þ where Nincident is the number of particles entering MIDAS and Utot is the total integral ﬂux of all ions with Z = 1 up to Z = 26 provided by the OMERE freeware. The ﬁeld distributions were constructed using a spacecraft model placed inside a spherical source with a radius of 1 m. The spacecraft was bombarded with particles having the same differential ﬂux spectrum as in the calculations presented in the previous sections. The simpliﬁed spacecraft model is a cylinder whose both inner radius and height are 80 cm. The material of its walls consists of 7.407 cm (20 g/cm2) thick Al. Its interior is ﬁlled with air. A probing sphere with a 20 cm radius is placed at the cylinder’s center to record the incoming The LETH2O spectrum is then normalized in order to be expressed as particles cm2 s1 and the dose rate is calculated as, D ¼ 1 qH2O X N i¼1 LETi N i; ð4Þ ð4Þ where Ni is the content of bin i of the LETH2O spectrum expressed in particles cm2 s1, LETi is the LET at the center Q L ð Þ ¼ 1 L < 10 keV lm1 0:32 L 2:210 keV lm1 10 keV lm1 L 100 keV lm1 300= ﬃﬃﬃ L p L > 100 lm1 ; 8 > > < > > : ð5Þ Q L ð Þ ¼ 1 L < 10 keV lm1 0:32 L 2:210 keV lm1 10 keV lm1 L 100 keV lm1 300= ﬃﬃﬃ L p L > 100 lm1 ; 8 > > < > > : ð5Þ ð5Þ Page 5 of 8 Page 5 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 (a) (b) (c) (d) Fig. 5. 6 Radiation ﬁeld inside the spacecraft Figure 5d shows the energy spectra of the 4 particles which are not present in the GCR external ﬁeld, but they have the highest contribution in ﬂux. Fig. 6. LETH2O spectrum for the ﬁeld inside the spacecraft (red) and LETexp(Si) (blue). Both are calculated using the energy depositions in the pixel detectors of MIDAS. The resulting energy spectra were used as inputs for the def- inition of the spherical sources surrounding MIDAS. Equal numbers of particles (107) were generated for each species. Again, the distribution of a calculated quantity resulting from the Monte Carlo experiment for one particle species was scaled with its relative contribution, and the sum for all species was derived. 6 Radiation ﬁeld inside the spacecraft (a) For the particles with Z = 1 to Z = 26 the ratio of their contribution in the integrated ﬂux of the spacecraft internal ﬁeld over their contribution in the integrated ﬂux of the GCR ﬁeld is shown. (b) The relative contribution of the particles in the integrated ﬂux of the spacecraft’s internal radiation ﬁeld is presented. (c) The relative contribution to dose in water of the particles of the spacecraft internal radiation ﬁeld. (d) Energy spectra for gammas, neutrons, electrons, and positrons, which are the most abundant species in the internal ﬁeld. (a) (b) (a) (b) (c) d) (d) (c) Fig. 5. (a) For the particles with Z = 1 to Z = 26 the ratio of their contribution in the integrated ﬂux of the spacecraft internal ﬁeld over their contribution in the integrated ﬂux of the GCR ﬁeld is shown. (b) The relative contribution of the particles in the integrated ﬂux of the spacecraft’s internal radiation ﬁeld is presented. (c) The relative contribution to dose in water of the particles of the spacecraft internal radiation ﬁeld. (d) Energy spectra for gammas, neutrons, electrons, and positrons, which are the most abundant species in the internal ﬁeld. Fig. 6. LETH2O spectrum for the ﬁeld inside the spacecraft (red) and LETexp(Si) (blue). Both are calculated using the energy depositions in the pixel detectors of MIDAS. particles and their energies. The particles considered for the construction of the internal ﬁeld distributions to be used are nu- clei with Z = 1 to Z = 26 plus 12 non-existing in the primary ﬁeld species produced from the interactions of the GCR with Al and air. The additional 12 particles are neutrons, gammas, e/e+, l/l+, Κ/Κ+, p/p+, deuterons and tritons. For each particle, its relative contribution to the internal ﬁeld was calcu- lated. In Figure 5a are shown the ratios of the ﬂux relative con- tributions of the charged particles present in both the GCR and the spacecraft ﬁeld. The error bars represent statistical ﬂuctua- tions of the Monte Carlo samples used. In Figures 5b and 5c are presented the relative contributions in the ﬂux and in the dose in water, respectively, of all the particles of the spacecraft ﬁeld. Dose in water has been calculated with the aid of the G4EMCalculator. 7 Estimation of LET and dose inside the spacecraft The measured LET was estimated using (1) and converted to LET in water with the procedure described in the previous Page 6 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 Table 1. Dose rates and quality factors from the MIDAS simulated response in 1996.4 solar minimum calculated using correction coefﬁcients resulting from solar minimum and solar maximum GCR ﬁelds. They are compared with the measurements on the lunar surface, which are multiplied by 2. The 3rd column reports simulation results obtained using the Badwar O’Neil model and completely different methodologies than the one reported. MIDAS simulation result for GCR ﬁeld at 1996.4 solar minimum Measurement on the lunar surface (Zhang et al., 2020) Dose rate in water (LET limit 400 keV lm1) 0.460–0.506 mGy day1 0.318 ± 0.034 mGy day1 2 (0.636 ± 0.068) Simulation result by Banjac et al. (2019): 0.6 mGy day1 Dose equivalent rate in water (LET limit 400 keV lm1) 2.32–1.99 mSv day1 1.37 ± 0.25 mSv day1 2 (2.74 ± 0.5) <Q> (LET limit 400 keV lm1) 4.58–4.33 4.3 ± 0.7 <Q> under 20 g cm2 of Al shield 2.55 (LET limit 400 keV lm1) 2.61 (LET limit 1000 keV lm1) Simulation result by Zeitlin et al. (2019): 2.3–2.4 paragraphs. It is shown in Figure 6. With a purpose to check that equation (2) can be used for the internal ﬁeld too, the ﬁt was repeated for the corresponding LETmeas and LETH2O and the result was almost identical. The count rate calculation for the internal ﬁeld has been performed. For each GCR particle species, 25 106 events were generated, and the number of particles, either primary or secondary, entering the probing sphere was counted, weighted with the relative contribution in the GCR ﬂux of the parent species, and summed. The ratio of this number to 25 106 is the estimate of the coefﬁcient, which relates any number of particles generated from a spherical source with a radius of 20 cm and with the composition and energy distribution of the internal ﬁeld to the number of the primary GCR particles sample from which it originates. This coefﬁcient is used to retrieve an effective Nincident in (3). and simulations reported for instruments that have been ﬂown and have an order of magnitude higher dimensions mass and power consumption. 7 Estimation of LET and dose inside the spacecraft This fact provides evidence that the methodology presented is valid, and on the other hand, it accentuates the potential of this miniaturized LET spectrometer. p The dose equivalent rate on the Lunar surface reported by the LND experiment (Zhang et al., 2020) for charged particles is 1.37 mSv day1 and <Q> is 4.3. Dose equivalent due to GCR charged particles on the lunar surface must be multiplied by 2 when compared to the dose equivalent in interplanetary space. The quality factor found by us for the ﬁeld inside the spacecraft is almost the same as the quality factor for the same shielding thickness (20 g cm2) calculated using the PHITS Monte Carlo code and presented in Figures B1 and B2 (Zeitlin et al., 2019). Fig. 7 of (Banjac et al., 2019) reports comparable values for dose rates in a water slab for the u = 420 MV param- eter of the BON model used to model the GCR ﬂux of the 1996.4 solar minimum. The resulting absorbed dose rate in water is D = 0.25 mGy day1, for integration up to 1000 keV lm1 and D = 0.2486 mGy day1 for integration up to 400 keV lm1. The corresponding dose equivalent rates and quality factors are H = 0.654 mSv day1, <Q> = 2.61 for integration up to 1000 keV lm1 and H = 0.634 mSv day1, <Q> = 2.55 for integration up to 400 keV lm1. In Table 1 are collected the results of the calculations presented in this paper and the results from measurements and simulations with which they are compared. The contribution of neutrons or other particles present in the radiation ﬁeld inside a spacecraft has not been considered in the calculations and the results of other works used for the comparisons. References for real-time determination of the identity and energy of particles in space. Space Weather 18: e2019SW002344. https://doi.org/ 10.1029/2019SW002344. Agostinelli S, Allison J, Amako K, Apostolakis J, Araujo H, et al. 2003. GEANT4 – A simulation toolkit. Nucl Instrum Methods Phys Res Sect A 506(3): 250–303. https://doi.org/10.1016/S0168- 9002(03)01368-8. Lassila-Perini K, Urbán L. 1995. Energy loss in thin layers in GEANT. Nucl Instrum Methods Phys Res Sect A 362: 416–422. https://doi.org/10.1016/0168-9002(95)00344-4. Peric I. 2007. A novel monolithic pixelated particle detector implemented in high-voltage CMOS technology. Nucl Instrum Methods Phys Res A 582: 876–885. https://doi.org/10.1016/j. nima.2007.07.115. Allison J, Amako K, Apostolakis J, Araujo H, Arce Dubois P, et al. 2006. Geant4 developments and applications. IEEE Trans Nucl Sci 53(2006): 270. https://doi.org/10.1109/TNS.2006.869826. Allison J, Amako K, Apostolakis J, Arce P, Asai M, Aso T, et al. 2016. Recent developments in GEANT4. Nucl Instrum Meth A Pinsky L, Hoang SM, Idarraga-Munoz J, Kruppa M, Stofﬂe N, et al. 2014. Summary of the ﬁrst year of medipix-based space radiation monitors on the ISS. In: Yellowstone Conference Center in Big Sky, Montana US, March 7–14, 2015, pp. 1–8. https://doi.org/ 10.1109/AERO.2014.6836502. 835: 186–225. https://doi.org/10.1016/j.nima.2016.06.125. Bak JF, Burenkov A, Petersen JBB, Uggerhoj E, Moller SP, Siffert P. 1987. Large departures from Landau distributions for high energy particles traversing thin Si and Ge targets. Nucl Phys B 288: 681. https://doi.org/10.1016/0550-3213(87)90234-3. Rodríguez-Pacheco J, Wimmer-Schweingruber RF, Mason GM, Ho GC, Sánchez-Prieto S, et al. 2020. The energetic particle detector. Energetic particle instrument suite for the solar orbiter mission. Banjac S, Berger L, Burmeister S, Guo J, Heber B, et al. 2019. Galactic cosmic ray induced absorbed dose rate in deep space – accounting for detector size, shape, material, as well as for the solar modulation. J Space Weather Space Clim 9: A14. https://doi. org/10.1051/swsc/2019014. A&A 642: A7. https://doi.org/10.1051/0004-6361/201935287. Spence HE, Case AW, Golightly MJ, Heine T, Larsen BA, et al. 2010. CRaTER: The cosmic ray telescope for the effects of radiation experiment on the lunar reconnaissance orbiter mission. Space Sci Rev 150: 243–284. https://doi.org/10.1007/s11214-009- 9584-8. Benton ER, Benton EV, Frank AL. 2010. Conversion between different forms of LET. Radiat Meas 45(8): 957–959. https://doi. org/10.1016/j.radmeas.2010.05.008. Zhang S, Wimmer-Schweingruber RF, Yu J, Wang C, Zou Y, et al. 2020. First measurements of the radiation dose on the lunar surface. Sci Adv 6(39). https://doi.org/10.1126/sciadv.aaz1334. Bichsel H. 1988. Straggling in thin silicon detectors. Rev Mod Phys 60(3): 663. https://doi.org/10.1103/RevModPhys.60.663. 8 Discussion The calculations have been based on clusters of energy depositions in Si voxels with lateral dimensions equal to the pixel pitch and 50 lm thickness. This assumption considers the spread of energy deposition due to the delta rays, but it does not account for the pixel detector nonidealities such as the charge diffusion and the ﬂuctuations of gain or other systematic and random effects. These effects can be included in the simu- lation when a working prototype of the Silicon pixels detectors will be experimentally characterized. The work presented concerns the development of the methodology to extract LET spectra, dose, and dose equivalent from the energy depositions by charged particles in a novel device based on Si pixel detectors. The radiation ﬁelds used in the simulation represent what could be encountered in ﬂight outside the geomagnetic ﬁeld without shielding and inside a spacecraft. Obviously, the conditions in a real ﬂight will vary, but the qualitative characteristics of the ﬁelds will not be different from those used in this study. The particles and their energies encountered in a solar energetic particle (SEP) event are a subset of those included in the study. In the case of SEP events, the high rate may affect the response of an instrument. Pixel detectors are advantageous in coping with high rates because of the independent signal processing within each pixel. Acknowledgements. This work has been funded by the European Space Agency Contract 4000119598/17/NL/LF for the development of a highly miniaturized ASIC radiation detector. Also, it has been supported by computational time granted from the Greek National Infrastructures for Research and Technology S.A. (GRNET) in the National HPC facility – ARIS. The editor thanks Jingnan Guo and two anonymous reviewers for their assistance in evaluating this paper. The end results obtained from this analysis of the simulated response of MIDAS are in fair agreement with measurements Page 7 of 8 Page 7 of 8 K. Karafasoulis et al.: J. Space Weather Space Clim. 2022, 12, 8 Cite this article as: Karafasoulis K, Papadimitropoulos C, Potiriadis C & Lambropoulos CP 2022. GEANT4 simulation study of the response of a miniature radiation detector in Galactic Cosmic Rays and inside a spacecraft. J. Space Weather Space Clim. 12, 8. https://doi. org/10.1051/swsc/2022002. References Hassler DM, Zeitlin C, Wimmer-Schweingruber RF, Böttcher S, Martin C, et al. 2012. The radiation assessment detector (RAD) Investigation. Space Sci Rev 170: 503–558. https://doi.org/ 10.1007/s11214-012-9913-1. Zeitlin C, Narici L, Rios RR, Rizzo A, Stofﬂe N, et al. 2019. Comparisons of high-linear energy transfer spectra on the ISS and in deep space. Space Weather 18: e2019SW002344. https://doi. org/10.1029/2018SW002103. ICRP. 1991. 1990 Recommendations of the International Commission on Radiological Protection. ICRP Publication 60. Ann ICRP 21(1–3). ICRP. 1991. 1990 Recommendations of the International Commission on Radiological Protection. ICRP Publication 60. Ann ICRP 21(1–3). Lambropoulos CP, Potiriadis C, Theodoratos G, Kazas I, Papadimitropoulos C, et al. 2019. MIDAS: A miniature device Vrba V, Benka T, Fojtik J, Havranek M, Janoska Z, et al. 2018. The SpacePix-D radiation monitor technology demonstrator. JINST 13: C12017. https://doi.org/10.1088/1748-0221/13/12/C12017. Lambropoulos CP, Potiriadis C, Theodoratos G, Kazas I, Papadimitropoulos C, et al. 2019. MIDAS: A miniature device Page 8 of 8
https://openalex.org/W1997845502	https://hal.science/hal-00856210/document	English	null	A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots	PloS one	2,011	cc-by	11,229	A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5’->3’ exoribonuclease (XRN) activities in Arabidopsis thaliana roots Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, Gonzalo M Estavillo, Hélène Javot, Serge Chiarenza, Allison C Mallory, Alexis Maizel, et al. Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, Gonzalo M Estavillo, Hélène Javot, Serge Chiarenza, Allison C Mallory, Alexis Maizel, et al. To cite this version: Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, et al.. A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5’->3’ exoribonuclease (XRN) activities in Arabidopsis thaliana roots. PLoS ONE, 2011, 6 (2), pp.e16724. 10.1371/journal.pone.0016724. hal-00856210 Distributed under a Creative Commons Attribution 4.0 International License Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an ANR-GENOPLANT grant (RIBOROOT-ANR06 GPLA 011) and the CEA agency. Array hybridizations have been partly supported by RNG (Re´seau National des Ge´nopoles, Evry, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: elena.marin@cea.fr . These authors contributed equally to this work. . These authors contributed equally to this work. ¤ Current address: UMR BGPI, Campus International de Baillarguet, TA A54/K, Montpellier, France ¤ Current address: UMR BGPI, Campus International de Baillarguet, TA A54/K, Montpellier, France HAL Id: hal-00856210 https://hal.science/hal-00856210v1 Submitted on 5 Oct 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Judith Hirsch1¤, Julie Misson1., Peter A. Crisp2., Pascale David1., Vincent Bayle1., Gonzalo M. Estavillo2, He´le`ne Javot1, Serge Chiarenza1, Allison C. Mallory3, Alexis Maizel4, Marie Declerck5, Barry J. Pogson2, Herve´ Vaucheret3, Martin Crespi5, Thierry Desnos1, Marie-Christine Thibaud1, Laurent Nussaume1, Elena Marin1* 1 CEA, DSV IBEB, Laboratoire de Biologie du De´veloppement des Plantes, UMR 6191 CNRS, CEA, Aix-Marseille II, Saint-Paul-lez-Durance, France, 2 ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia, 3 Institut Jean-Pierre Bourgin, UMR 1318, INRA, Versailles, France, 4 Department of Stem Cell Biology, University of Heidelberg, Heidelberg, Germany, 5 Institut des Sciences du Ve´ge´tal, CNRS, Gif sur Yvette, France Abstract Background: Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 39,(29),59-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. Principal Findings: A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 39-polyadenosine 59-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. Conclusions/Significance: Our results indicate that the 39,(29),59-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs. Citation: Hirsch J, Misson J, Crisp PA, David P, Bayle V, et al. (2011) A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 59-.39 Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots. PLoS ONE 6(2): e16724. doi:10.1371/journal.pone.0016724 Editor: Edward Newbigin, University of Melbourne, Australia Received October 18, 2010; Accepted December 22, 2010; Published February 3, 2011 Copyright: 2011 Hirsch et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Hirsch et al. PLoS ONE \| www.plosone.org Citation: Hirsch J, Misson J, Crisp PA, David P, Bayle V, et al. (2011) A Novel fry1 Allele Reveals the Existence of a Mutant Phen Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots. PLoS ONE 6(2): e16724. doi:10.1371/journal.pone.0016724 February 2011 \| Volume 6 \| Issue 2 \| e16724 A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 59-.39 Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots Judith Hirsch1¤, Julie Misson1., Peter A. Crisp2., Pascale David1., Vincent Bayle1., Gonzalo M. Estavillo2, He´le`ne Javot1, Serge Chiarenza1, Allison C. Mallory3, Alexis Maizel4, Marie Declerck5, Barry J. Pogson2, Herve´ Vaucheret3, Martin Crespi5, Thierry Desnos1, Marie-Christine Thibaud1, Laurent Nussaume1, Elena Marin1* Introduction regulator of both ABA-independent and ABA-dependent stress response pathway ([2], Estavillo and Pogson, personal communi- cation) and is involved in leaf venation patterning [3]. Indepen- dent screens also identified fry1 alleles affecting the regulation of photo-morphogenic processes, including hypocotyl elongation and flowering time [4] and lateral root initiation [5]. In the last ten years, a variety of independent genetic screens have identified defects in the enzyme FIERY1/SAL1 (FRY1). The first fry1 mutants were identified in a genetic screen based on the deregulation of an ABA reporter gene [1]. FRY1 was described as a repressor of ABA-mediated stress signal transduction, as the corresponding mutant presented an increased sensitivity to cold, salt and drought stresses [1]. FRY1 seems to act as a negative Such a diversity of phenotypes could be explained by the complexity of FRY1 activity. FRY1 was originally identified as a bifunctional enzyme presenting both an inositol polyphosphate 1- PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 1 February 2011 \| Volume 6 \| Issue 2 \| e16724 FRY1 Activity Unlinked to XRNs Figure 1. Phenotype of the fry1-7 mutant. (A-B) Root cross section of a ten day-old PHT1;4:GUS parental line (A) and the PHT1;4:GUS/fry1-7 mutant (B) after GUS staining. Scale bars, 30 mm. (C) Ten day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, note the reduced root system in the mutant. Scale bar, 10 mm. doi:10.1371/journal.pone.0016724.g001 phosphatase activity that hydrolyses inositol 1,4,5-trisphosphate (IP3) in vitro, complementing a salt sensitive yeast strain [6], and a highly specific 39,(29),59-bisphosphate nucleotide phosphatase activity converting PAP (39-polyadenosine 59-phosphate) into AMP and phosphate (Pi) [6,7]. This latter activity was predicted to negatively impact the amount of PAP available in the cell. Indeed, in a separate paper that focuses on chloroplast to nuclear signaling in leaves, several authors from the current study shown that PAP content and not inositol phosphates are regulated by FRY1/SAL1 (Estavillo and Pogson, personal communication). In vitro, PAP suppresses the activity of the yeast 59-.39 exoribonu- cleases Rat1 and Xrn1 [8]. Thus, the accumulation of PAP in loss- of-function fry1 mutants could inhibit the three Rat1 Arabidopsis orthologs XRN2, XRN3 and XRN4, which are all RNA silencing-suppressors [9]. Indeed, some phenotypes of xrn3, xrn2 xrn3 and xrn4 mutants mimic some fry1 traits such as an altered leaf shape, hypocotyl length and reduction of lateral root initiation [4,5,9]. Map-based cloning identifies a new allele of fry1 The mutation was mapped on chromosome 5 between microsatellite markers 5.74 and 5.80, which define an approxi- mately 110 kb interval containing 29 genes. A transcriptomic analysis showed that a transcript corresponding to At5g63980 (FRY1/HOS2/SAL1) was down-regulated in the mutant to 30% of the level detected in the PHT1;4:GUS parental line. Sequencing of the corresponding locus in the mutant line revealed a point mutation (G to A exchange) at nucleotide position 559 in the donor site of the second intron of the FRY1 genomic sequence (Fig. 2A). This mutation altered the splicing of FRY1 transcripts, as confirmed by the cloning and sequencing of four FRY1 splice Results and Discussion Identification of a mutant deregulating PHT1;4::GUS expression and root development Introduction Nevertheless, the contribution of the roots to the reported alx8 and fry1-1 drought tolerance [2] and the role of XRNs in root morphology and drought tolerance have not been analyzed. Using a reporter gene strategy to identify mutations deregulat- ing the expression of the high affinity phosphate transporter PHT1;4 [10], we identified a novel allele of fry1. In addition to root deregulation of the gene reporter, the mutant exhibited strong root architecture defects and a drought resistance phenotype. Through physiological approaches, grafting experiments and mutant analysis, we show that FRY1 plays a role in long distance signaling to roots through its proposed impact on XRN activities in leaves. In contrast, we reveal a new role for FRY1 in the local regulation of phosphate starvation response genes likely linked to a local depletion of Pi in the root stele. Figure 1. Phenotype of the fry1-7 mutant. (A-B) Root cross section of a ten day-old PHT1;4:GUS parental line (A) and the PHT1;4:GUS/fry1-7 mutant (B) after GUS staining. Scale bars, 30 mm. (C) Ten day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, note the reduced root system in the mutant. Scale bar, 10 mm. doi:10.1371/journal.pone.0016724.g001 Identification of a mutant deregulating PHT1;4::GUS expression and root development variants in the mutant (Fig. 2B). All splice variants encoded truncated forms of the FRY1 protein, suggesting that this mutant, referred to as fry1-7, is a loss-of-function allele. Expression of the FRY1 cDNA under the control of the 35S promoter in the fry1-7 line complemented the root phenotype (Fig. S1A), confirming that the mutation in fry1 was responsible for the root defect. Importantly, the expression of the GUS reporter was also com- plemented in PHT1;4:GUS/fry1-7/35S::FRY1 lines (data not shown) and was indistinguishable from the original PHT1;4:GUS line. An allelism test between fry1-7 and the T-DNA insertion allele fry1-6 (Fig. 2A; [9]) further confirmed that FRY1 was the causal gene (data not shown). When driven by the promoter of the high affinity phosphate transporter gene PHT1;4, the GUS reporter gene is induced by phosphate starvation and primarily expressed in the Arabidopsis root. We screened seedlings for the deregulation of this root- expressed reporter gene, in an EMS-derived population of a transgenic line. In our screening conditions (i.e. on phosphate-rich medium) the expression of this reporter marker was not detectable in roots of the parental line [10]. Ten day-old seedlings from each of the 1400 M2 families were stained and roots were screened for seedlings with detectable GUS expression [11]. We identified a recessive mutant (fry1-7, see below) that constitutively expressed the GUS reporter gene in the central cylinder and the pericycle of the root and in primary root meristems (Fig. 1A, B). This mutant also displayed shorter primary and lateral roots (Fig. 1C). Together with the root developmental defects, fry1-7 mutants displayed the aerial growth and developmental defects previously described for other fry1 alleles, including fry1-6 [2,4,9]. Young rosette leaves were crinkly and presented rounded leaf margins and shorter petioles (Fig. S1B), whereas older leaves were serrated. In addition, when transferred to soil, the mutant was more tolerant to drought stress than the wild type control (see below) and displayed a general delay in growth (Fig. S1C) and flowering time (data not shown). PLoS ONE \| www.plosone.org Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect PHT1;4 expression level in this line by qRT-PCR. In both leaves and roots, we observed an increase in PHT1;4 transcript levels in the fry1-7 single mutant as compared to the Ws control (Fig. 3A). The induction of PHT1;4 was also detected in the fry1-6 allele (Fig. S3), which confirms that the expression of the PHT1;4:GUS transgene in the PHT1;4:GUS/fry1-7 mutant reflects the activation of the endogenous PHT1;4 gene. Thus, in high phosphate conditions, fry1 mutants show a constitutive induction of PHT1;4 in the central cylinder of the root. Alteration of fry1 root architecture has been recently reported [5], but the description of the root phenotype was limited to lateral root initiation. Our analysis indicated that the root system of the fry1-7 mutant is reduced compared to the parental control line both at the primary root (PR) and the lateral root (LR) levels. Seven days post germination (dpg), the fry1-7 mutant primary root was 37% shorter than its parental line, and the fry1-6 primary root was 32% shorter than the Col PR (Fig. 4A). Quantification of PR growth rate during in vitro development in both the fry1-7 and the fry1-6 mutant alleles revealed a statistically significant difference in growth rate when compared with controls (determined by Student’s t test, P,0.01), which likely explains the growth delay observed in the mutant (Fig. 4B). We tested whether the fry1 mutation stimulates the expression of other genes related to PHT1-4. This phosphate transporter belongs to a multigenic family (the PHT1 gene family) that exhibits a tight co-regulation (in particular during Pi deficiency [12]). We found that PHT1;1 and PHT1;2 (revealed by a common pair of primers), PHT1;7 and PHT1;8 transcripts were also induced in fry1-7 (Fig. 3A, B) as compared to the wild type control. In order to assay if genes modulated by Pi starvation distinct from PHT1 family could also be affected by fry1 mutation, we tested two other markers associated with Pi deficiency in the stele: Pho1H1 [13] and the At1G73010 phosphatase (Fig. S2). Both genes were found significantly induced in roots of the fry1 background (Fig. 3C and Fig. S4C, D). Analyses revealed an absence of obvious alterations in Pi content, uptake or transport capacity of the fry1 mutant (data not shown). Nevertheless, the levels of gene induction measured here by qRT-PCR are substantially lower than those observed during phosphate starvation [12,14]. fry1 stimulates the transcription of several genes induced by Pi starvation in the stele To test whether the GUS expression in PHT1;4:GUS/fry1 was due to the upregulation of the endogenous PHT1,4 gene or specific to the T-DNA reporter construct inserted in PHT1;4, we generated a fry1-7 line devoid of any T-DNA insertion by performing a series of back-crosses. We then measured the PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 2 FRY1 Activity Unlinked to XRNs Figure 2. Schematic of the mutant fry1 alleles. (A) FRY1 gene structure and position of fry1 mutations. White boxes represent the exons, the horizontal lines represent the introns and the UTRs. In the first exon (e1) the grey box corresponds to the plastid transit peptide (54 amino acids long) predicted in the TAIR database. Positions of the T-DNA insertion in the fry1-3 and fry1-6 mutants alleles are indicated by triangles. The nature of the untagged fry1 alleles is indicated: Lines indicate point mutations, numbers show the position of amino acids, asterisks indicate stop codons. (B) CDS of the FRY1 locus and structure of different splice variants identified in the fry1-7 mutant. In one of the splice variants, the second intron (i2) has been conserved due to the point mutation in fry1-7. The 3rd exon is marked (e3) to clarify the interpretation of the figure. The protein length indicated for each splice variant includes the 54 amino acids of the transit peptide. Asterisks indicate stop codons. doi:10.1371/journal.pone.0016724.g002 Figure 2. Schematic of the mutant fry1 alleles. (A) FRY1 gene structure and position of fry1 mutations. White boxes represent the exons, the horizontal lines represent the introns and the UTRs. In the first exon (e1) the grey box corresponds to the plastid transit peptide (54 amino acids long) predicted in the TAIR database. Positions of the T-DNA insertion in the fry1-3 and fry1-6 mutants alleles are indicated by triangles. The nature of the untagged fry1 alleles is indicated: Lines indicate point mutations, numbers show the position of amino acids, asterisks indicate stop codons. (B) CDS of the FRY1 locus and structure of different splice variants identified in the fry1-7 mutant. In one of the splice variants, the second intron (i2) has been conserved due to the point mutation in fry1-7. The 3rd exon is marked (e3) to clarify the interpretation of the figure. The protein length indicated for each splice variant includes the 54 amino acids of the transit peptide. Asterisks indicate stop codons. doi:10.1371/journal.pone.0016724.g002 Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect This suggested that the reduction of Pi level is probably limited. In addition, such variation should be restricted to the root stele and masked by the accumulation of vacuolar Pi in external root cell layers such as cortex and epidermis. It is therefore not surprising that such specific Pi discrepancies could not be detected by available techniques and only visualized by the use of sensitive reporter genes or by PCR techniques. We tested whether the fry1 mutation stimulates the expression of other genes related to PHT1-4. This phosphate transporter belongs to a multigenic family (the PHT1 gene family) that exhibits a tight co-regulation (in particular during Pi deficiency [12]). We found that PHT1;1 and PHT1;2 (revealed by a common pair of primers), PHT1;7 and PHT1;8 transcripts were also induced in fry1-7 (Fig. 3A, B) as compared to the wild type control. Reduced root growth can result from a defect in cell elongation and/or from a decrease in meristem activity. Measuring cortical cell length did not reveal any differences between fry1 alleles and wild type controls (Fig. 4C). However, PR cell number in the proximal meristem (PM) at 7dpg [15] was mildly reduced, although statistically significant, in the fry1-6 and fry1-7 mutants when compared to the wild type PM size (Fig. 4D). These results show that the modified PR growth observed in fry1 is due to a defect in maintenance and/or activity of the root apical meristem. Reduced root growth can result from a defect in cell elongation and/or from a decrease in meristem activity. Measuring cortical cell length did not reveal any differences between fry1 alleles and wild type controls (Fig. 4C). However, PR cell number in the proximal meristem (PM) at 7dpg [15] was mildly reduced, although statistically significant, in the fry1-6 and fry1-7 mutants when compared to the wild type PM size (Fig. 4D). These results show that the modified PR growth observed in fry1 is due to a defect in maintenance and/or activity of the root apical meristem. The fry1 mutation also reduced the LR length (Fig. 4E), the LR density (Fig. 4F) and the LR primordia number (Fig. 4G). Thus, it is likely that the altered root architecture of fry1 mutants is not only due to a delay in growth. PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect Interestingly, LR cortical cell length and PM cell number were comparable among fry1-7 and fry1-6 alleles and the corresponding wild type plants when measured at 14 dpg (data not shown), suggesting that an independent factor limits LR initiation or progression. Auxin is a good candidate for such a The fry1 mutation also reduced the LR length (Fig. 4E), the LR density (Fig. 4F) and the LR primordia number (Fig. 4G). Thus, it is likely that the altered root architecture of fry1 mutants is not only due to a delay in growth. Interestingly, LR cortical cell length and PM cell number were comparable among fry1-7 and fry1-6 alleles and the corresponding wild type plants when measured at 14 dpg (data not shown), suggesting that an independent factor limits LR initiation or progression. Auxin is a good candidate for such a PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 3 FRY1 Activity Unlinked to XRNs Quantitative real time PCR on the PHT1;7 and PHT1;8 loci in fry1-7 and Ws plantlets. (C) Quantitative real time PCR on the At1G73010 and Pho1H1 loci in fry1-7 and Ws roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Ws leaves, pale grey bars to fry1-7 leaves, dark grey bars to Ws roots and black bars to fry1-7 roots. Standard deviations are shown. Figure 3. Expression of phosphate induced genes in leaves and roots of the fry1-7 mutant. (A) Quantitative real time PCR of the PHT1;1&PHT1;2 and PHT1;4 transcripts in fry1-7 and Ws plantlets. (B) PLoS ONE \| www plosone org doi:10.1371/journal.pone.0016724.g003 doi:10.1371/journal.pone.0016724.g003 factor as the fry1 mutant has reduced auxin sensitivity at the level of LR initiation [5]. Nevertheless, this auxin response defect could not explain all fry1 root traits as the fry1 PR exhibited auxin sensitivity similar to wild type (data not shown). The 39,(29),59-bisphosphate nucleotide phosphatase activity complements the root mutant phenotype of fry1 as well as the PHT1;4:GUS induction FRY1 is a bifunctional enzyme whereas AHL (Arabidopsis HAL2-like, At5g54390) is a FRY1 paralog encoding a protein with only the 39,(29),59-bisphosphate nucleotide phosphatase activity in vitro [7]. In order to test whether the 39,(29),59-bisphosphate nucleotide phosphatase activity is sufficient to recover wild type root and PHT1;4 induction level, we used the AHL gene harboring only this activity (i.e. not the inositol polyphosphate 1-phosphatase activity). Overexpression of AHL complemented the root pheno- type of the fry1 mutant (Fig. S1D), indicating that the altered root growth of fry1 mutants is likely to be due to the lack of the FRY1 39,(29),59-bisphosphate nucleotide phosphatase activity. In the AHL overexpressor lines, wild type PHT1-4, Pho1H1 and At1g73010 phosphatase transcript levels were re-established (Fig. S3), further confirming the complementation of the fry1 phenotype by AHL activity. As expected, the overexpression of AHL was able to complement the PHT1;4:GUS induction in fry1 (data not shown). These results strongly suggest that the lack of only the 39,(29),59-bisphosphate nucleotide phosphatase activity is respon- sible for all the phenotypes analyzed in the current study. Interestingly, Kim and von Arnim [4] showed that the 35S:AHL construct complements the aerial phenotypes of fry1-6. In vivo analysis of PAP and IP3 levels in Col and fry1 mutants (Estavillo and Pogson, personal communication) confirm our conclusion that only the lack of the 39 (29),59-bisphosphate nucleotide phosphatase activity of FRY1, and the concomitant PAP accumulation, are responsible for all fry1 mutant phenotypes described here. In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems Grafting experiments reveal two modes of action for FRY1 Five weeks after grafting, we observed that wild type roots remained small like fry1 roots (Fig. 6G). Conversely, the fry1 roots grew like wild type when grafted on a wild type shoot (Fig. 6H). These grafting experiments indicate that the root growth defect of the fry1 mutant is complemented by the wild type shoot. We can hypothesize that a mobile component produced only in leaves is necessary in the root pericyle to exhibit normal root growth. When the aerial part of a graft is unable to synthesize this mobile component (fry1 scion), the roots are less sensitive to auxin and therefore initiate less LR. In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems The PHT1;4:GUS expression in the internal cell layers of fry1 roots (Fig. 1B) suggests that FRY1 may be expressed in these tissues. To verify this hypothesis, we transformed the PHT1;4:GUS/ fry1-7 mutant line with a GFP-tagged FRY1 genomic construct (pFRY1:FRY1-GFP). This construct is functional because it comple- mented the root development defects of fry1-7 (data not shown). In the mature part of the roots, the FRY1-GFP fluorescence was detected in all cell layers, except the epidermis (Fig. 5A), with strongly enhanced expression in the pericycle and stele regions of the mature part of the PR. The fusion protein was strongly expressed in the PR meristem and the root cap (Fig. 5B). It was also detected in the LR primordia (Fig. 5C), emerged LR (Fig. 5D) and LR meristems (data not shown). Therefore, the overall FRY1 expression pattern largely overlaps with the PHT1;4:GUS expression pattern observed in a fry1-7 mutant background (Fig. 1B). This suggests that the role of FRY1 on PHT1;4 expression is tissue- specific, as the induction appears limited to the regions where FRY1 shows the highest expression level in planta. Figure 3. Expression of phosphate induced genes in leaves and roots of the fry1-7 mutant. (A) Quantitative real time PCR of the PHT1;1&PHT1;2 and PHT1;4 transcripts in fry1-7 and Ws plantlets. (B) PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 4 FRY1 Activity Unlinked to XRNs PLoS ONE \| www plosone org 5 February 2011 \| Volume 6 \| February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org FRY1 Activity Unlinked to XRNs FRY1 Activity Unlinked to XRNs Figure 4. Root architecture of the fry1 mutants. (A) Primary root length at 7 days post germination (dpg). (B) Growth rate of the primary root at 8, 11 and 14 dpg on MS/10 medium. (C) Primary root cortical cell length. (D) Primary root proximal meristem (PM) cell number at 7 dpg. (E) Diagram plotting total lateral root length vs primary root length of the PHT1;4:GUS line (white squares) and PHT1;4:GUS/fry1-7 mutant (grey circles). 18 to 30 plants were measured per genotype, 10 dpg. (F) LR density of fry1 (number of LR per mm PR) at 16 dpg. (G) Number of LR primordia at early stages (I- V) and late stages (VI-VII), 7 dpg. Grafting experiments reveal two modes of action for FRY1 Conversely, the fry1 roots grew like wild type when grafted on a wild type shoot (Fig. 6H). These grafting experiments indicate that the root growth defect of the fry1 mutant is complemented by the wild type shoot. We can hypothesize that a mobile component produced only in leaves is necessary in the root pericyle to exhibit normal root growth. When the aerial part of a graft is unable to synthesize this mobile component (fry1 scion), the roots are less sensitive to auxin and therefore initiate less LR. To help in the interpretation of these contrasting results, we investigated whether grafting could also restore other known characteristics of fry1 mutants. Wilson et al. [2] have shown that fry1 mutants tolerate drought stress up to 50% longer than wild type controls. We used our different graft combinations to test The expression of FRY1 and PHT1;4 in the root stele led us to examine whether the PHT1;4:GUS induction in fry1 could be complemented by a mobile component moving from the shoot. We took advantage of the PHT1;4:GUS reporter in our fry1-7 allele to examine whether FRY1 acts in a tissue-autonomous way. Micrografting experiments were set up with in vitro plantlets (Fig. 6A), using the parental line (PHT1;4:GUS) and the mutant line (PHT1;4:GUS/fry1-7). As expected in high Pi media, we did not observe any GUS expression in roots of the control PHT1;4:GUS//PHT1;4:GUS grafts (Fig. 6B), whereas those of the control PHT1;4:GUS/fry1-7//PHT1;4:GUS/fry1-7 grafts showed strong GUS staining in the central cylinder and the pericycle (Fig. 6C). Grafting a PHT1;4:GUS scion on a PHT1;4:GUS/fry1-7 root stock (Fig. 6D) generated roots with the PHT1;4:GUS/fry1-7 GUS expression pattern, whereas grafting of a PHT1;4:GUS/fry1-7 scion on a PHT1;4:GUS root stock resulted in roots with the GUS pattern of PHT1;4:GUS plants (Fig. 6E). Therefore, a wild type FRY1 in the shoot does not complement the mutant expression pattern of PHT1;4:GUS in the fry1-7 root stock. The complementation of the fry1 root development phenotype and drought resistance by a wild type scion and the non- complementation of the PHT1;4:GUS induction by the wild type scion indicates that FRY1 regulates different aspects of plant physiology by two different mechanisms. Presumably, a mobile component produced by leaves expressing FRY1 is moving to roots and regulating root development but not PHT1 expression. Then, we tested whether a wild type shoot could complement the root growth phenotype of fry1 (Fig. 6F–I). In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems The wild type and the mutant in A, B, D and G are significantly different (P,0.01) (Student’s t-test). For A–D and F the white bars correspond to the PHT1;4:GUS parental line, the PHT1;4:GUS/fry1-7 mutant appears in pale grey, Col in dark grey and the fry1-6 mutant in black, as detailed in panel B. For all the analyses, at least three independent experiments gave similar results. Standard deviations are shown. doi:10.1371/journal.pone.0016724.g004 Grafting experiments reveal two modes of action for FRY1 whether this tolerance depends on the root system or on the shoot. Fig. 6J shows than when a wild type scion is grafted on a fry1 root it is just as tolerant to drought as when it is grafted on a wild type root (p.0.1). In contrast, wild-type root-stocks did not adversely affect the tolerance of fry1 scions compared to their endogenous roots (p.0.1). By day 12, whatever the grafting combination, most of the wild type scion plants were dead whereas the fry1 scions survided an additional 3 days on average (p.0.1). These experiments demonstrate that the root genotype does not determine the drought tolerance of the aerial part of the plant, indicating that the lack of FRY1 in the leaves is sufficient for drought tolerance. We therefore investigated whether the drought tolerance phenotype of fry1 was due to the lack of FRY1 39 (29),59- bisphosphate nucleotide phosphatase activity. The fry1-6/ 35S::AHL overexpression line displayed a wild type level of drought tolerance (Fig. 7A), indicating that the drought tolerance of fry1 is due to the lack of 39 (29),59-bisphosphate nucleotide phosphatase activity. FRY1 The expression of FRY1 and PHT1;4 in the root stele led us to examine whether the PHT1;4:GUS induction in fry1 could be complemented by a mobile component moving from the shoot. We took advantage of the PHT1;4:GUS reporter in our fry1-7 allele to examine whether FRY1 acts in a tissue-autonomous way. Micrografting experiments were set up with in vitro plantlets (Fig. 6A), using the parental line (PHT1;4:GUS) and the mutant line (PHT1;4:GUS/fry1-7). As expected in high Pi media, we did not observe any GUS expression in roots of the control PHT1;4:GUS//PHT1;4:GUS grafts (Fig. 6B), whereas those of the control PHT1;4:GUS/fry1-7//PHT1;4:GUS/fry1-7 grafts showed strong GUS staining in the central cylinder and the pericycle (Fig. 6C). Grafting a PHT1;4:GUS scion on a PHT1;4:GUS/fry1-7 root stock (Fig. 6D) generated roots with the PHT1;4:GUS/fry1-7 GUS expression pattern, whereas grafting of a PHT1;4:GUS/fry1-7 scion on a PHT1;4:GUS root stock resulted in roots with the GUS pattern of PHT1;4:GUS plants (Fig. 6E). Therefore, a wild type FRY1 in the shoot does not complement the mutant expression pattern of PHT1;4:GUS in the fry1-7 root stock. Then, we tested whether a wild type shoot could complement the root growth phenotype of fry1 (Fig. 6F–I). Five weeks after grafting, we observed that wild type roots remained small like fry1 roots (Fig. 6G). The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root It has been proposed that XRN activity is inhibited in a fry1 background [9], likely because of the accumulation of the XRNs inhibitor PAP (Estavillo and Pogson, personal communication). Accordingly, both fry1 and the xrn mutants accumulate RNA intermediates of miRNA-directed post-transcriptional regulation and share common traits [9]. To further analyze the role of XRN in the fry1 phenotype, we generated an xrn2 xrn3 xrn4 triple mutant that was fertile, unlike the sterile xrn2 xrn3 double mutant. Thus To help in the interpretation of these contrasting results, we investigated whether grafting could also restore other known characteristics of fry1 mutants. Wilson et al. [2] have shown that fry1 mutants tolerate drought stress up to 50% longer than wild type controls. We used our different graft combinations to test Figure 5. Pattern of expression of the FRY1-GFP fusion protein in roots. Roots of a fry1-7 mutant complemented with a pFRY1:FRY1:GFP construct were observed by confocal microscopy. (A) Mature root. (B) PR meristem. (C) LR primordium. (D) Emerged LR. Scale bars are 75 mm in A, B and C, and 150 mm in D. doi:10.1371/journal.pone.0016724.g005 Figure 5. Pattern of expression of the FRY1-GFP fusion protein in roots. Roots of a fry1-7 mutant complemented with a pFRY1:FRY1:GFP construct were observed by confocal microscopy. (A) Mature root. (B) PR meristem. (C) LR primordium. (D) Emerged LR. Scale bars are 75 mm in A, B and C, and 150 mm in D. doi:10.1371/journal.pone.0016724.g005 February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org 6 FRY1 Activity Unlinked to XRNs Figure 6. FRY1 in shoot complements the root growth defects of fry1 but not the expression of the PHT1;4:GUS reporter gene. (A) The grafting junction. Arrow indicates the silicon ring. (B–E) The different graft combinations (scion/root) between the PHT1;4:GUS line and the PHT1;4:GUS line are indicated. Below are the corresponding pictures of a grafted root after the overnight GUS staining. (F–I) Shoot and root phenotypes of the different graft combinations (scion/root) between the wild type and the fry1 mutant, after 4 weeks of growth in soil. Note that the root growth of fry1 is complemented by the wild type shoot (H), but wild type roots display a fry1 phenotype when grafted with a fry1 scion (G). (J) Survival rate after withholding watering of plants with different grafting combinations show that the drought tolerance phenotype of fry1 is determined by the scion genotype. The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root Data are from one representative experiment out of three. Error bars represent standard error. doi:10.1371/journal.pone.0016724.g006 Figure 6. FRY1 in shoot complements the root growth defects of fry1 but not the expression of the PHT1;4:GUS reporter gene. (A) The grafting junction. Arrow indicates the silicon ring. (B–E) The different graft combinations (scion/root) between the PHT1;4:GUS line and the PHT1;4:GUS line are indicated. Below are the corresponding pictures of a grafted root after the overnight GUS staining. (F–I) Shoot and root phenotypes of the different graft combinations (scion/root) between the wild type and the fry1 mutant, after 4 weeks of growth in soil. Note that the root growth of fry1 is complemented by the wild type shoot (H), but wild type roots display a fry1 phenotype when grafted with a fry1 scion (G). (J) Survival rate after withholding watering of plants with different grafting combinations show that the drought tolerance phenotype of fry1 is determined by the scion genotype. Data are from one representative experiment out of three. Error bars represent standard error. doi:10.1371/journal.pone.0016724.g006 shape, drought tolerance), they do not mimic the induction of the PHT1;4 locus. In addition, a qRT-PCR analysis of the xrn2 xrn3 xrn4 triple mutant confirmed that the XRN activities are not responsible for the up-regulation of PHT1 genes (Fig. S4). Indeed, the assayed mutants (xrn4-6 and the xrn2 xrn3 xrn4) showed the same level of PHT1;4, PHT1;7, Pho1H1 and AT1g73019 transcripts as the Col control. Thus, this analysis further confirmed that the xrn mutations do not mimic the induction of the PHT1;4 locus, nor the general induction of phosphate-starvation genes observed in the fry1 background. the triple xrn2 xrn3 xrn4 mutant facilitated in vitro root analysis without antibiotic selection, which has negative consequences on root development. Although the mechanism for the partial phenotypic rescue is unclear, it suggests that xrn4 mutations act to partially suppress the xrn2 xrn3 phenotypic effects. We found that the lateral root phenotype of the xrn2 xrn3 xrn4 triple mutant was similar to that of fry1 (Fig. 7B), whereas the primary root of the triple mutant was not significantly reduced compared to wild type (Fig. 7C). We also found that the xrn2 xrn3 xrn4 triple mutant tolerated a drought stress like the fry1 mutants (Fig. 7A). Altogether, these results suggest that the pleiotropic phenotype of the fry1 mutants results, at least in part, from a general perturbation in XRN activities. The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root The inability of xrn mutants to induce PHT1;4 transcription argues in favor of a model whereby FRY1 has two physiological roles for the 39,(29),59-bisphosphate nucleotide phosphatase activity (Modeled in Fig. 8). On one hand, the PAP accumulation in the mutant represses XRN activity, altering various phenotypes linked to the deregulation of the silencing machinery (root architecture, drought tolerance, leaf shape, hypocotyl sensitivity to red light, hormonal sensing and signaling). Indeed, fry1 late flowering, short petioles and hypocotyl hypersensitivity to red light phenotypes are largely mimicked by the xrn2 xrn3 double mutant [4]. The root architecture of the xrn4 single mutant has been described as being similar to that of fry1 [5]. However, only the xrn2 xrn3 xrn4 triple mutant presents fry1-like lateral root architecture defects in our conditions (Figs. 7B, C). The xrn4 mutant presents wild type LR development (Fig. S5A) and a PR PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs We investigated whether the xrn mutations could mimic the induction of PHT1;4:GUS observed in fry1. For this, we crossed the PHT1;4:GUS parental line to the different single, double and triple xrn mutant lines. We confirmed the crosses by checking that the GUS marker was active in L of the F2 when plants were grown in phosphate deficient media (Table 1). Interestingly, in plantlets grown in complete media, we never observed GUS-stained roots (Table 1) demonstrating that although the xrn mutations can mimic many of the fry1 mutant phenotypes (root architecture, leaf February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 7 FRY1 Activity Unlinked to XRNs Figure 7. The xrn2 xrn3 xrn4 triple mutant mimics fry1 drought tolerance and root architecture phenotypes. (A) Dehydration experiment on 4 week-old soil grown plants of the indicated genotypes. Two independent experiments, with 6 to 17 plants per genotype per experiment, gave the same results. (B) Root architecture phenotype of the xrn2 xrn3 xrn4 triple mutant compared to the wild type Col and the fry1-6 mutant at 11 dpg. Scale bar is 20 mm. (C) Primary root length of the same plantlets, at the same age. Note that the length of Col and xrn2 xrn3 xrn4 primary roots are not significantly different. doi:10.1371/journal.pone.0016724.g007 Figure 7. The xrn2 xrn3 xrn4 triple mutant mimics fry1 drought tolerance and root architecture phenotypes. (A) Dehydration experiment on 4 week-old soil grown plants of the indicated genotypes. Two independent experiments, with 6 to 17 plants per genotype per experiment, gave the same results. (B) Root architecture phenotype of the xrn2 xrn3 xrn4 triple mutant compared to the wild type Col and the fry1-6 mutant at 11 dpg. Scale bar is 20 mm. (C) Primary root length of the same plantlets, at the same age. Note that the length of Col and xrn2 xrn3 xrn4 primary roots are not significantly different. doi:10 1371/journal pone 0016724 g007 length intermediary between the Col and the fry1-6 PR lengths (Fig. S5B). In this mutant, the levels of the phosphate-starvation markers that appear induced in fry1 are comparable to the control levels (Fig. S4). In addition, the xrn4 single mutant is not drought tolerant (Estavillo and Pogson, personal communication). The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs Inter- estingly, the xrn2 xrn3 drought tolerance level is intermediary between the wild type and the fry1 drought tolerance levels (Estavillo and Pogson, personal communication), whereas the rosette phenotype of the double mutant is similar to that of the fry1-6 mutant [9]. Moreover, both fry1 mutants and the xrn2 xrn3 xrn4 triple mutant tolerate a drought stress that is lethal for the wild type controls (Fig. 7A), even though the rosette shape of the triple mutant is quite different from the fry1 rosette (compare the petiole length in fry1-6 and the xrn2 xrn3 xrn4 triple mutant in Fig. 7A). Thus, the drought tolerance observed in both fry1 and xrn2 xrn3 xrn4 triple mutants is not linked to a reduced leaf biomass Table 1. PHT1;4:GUS expression in different xrn backgrounds. Number of F2 plantlets stained/total nb of plantlets assayed Genetic cross on Pi depleted media on Pi complete media xrn2 X PHT1;4:GUS 16/24 0/24 xrn3 X PHT1;4:GUS 17/24 0/24 xrn4 X PHT1;4:GUS 34/45 0/204 xrn2 xrn4 X PHT1;4:GUS 39/58 0/474 xrn2 xrn3 xrn4 X PHT1;4:GUS 18/22 0/347 The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 Table 1. PHT1;4:GUS expression in different xrn backgrounds. The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org 8 FRY1 Activity Unlinked to XRNs Figure 8. Model accounting for the dual mode of action of FRY1. The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs The expression of FRY1 in the shoot is essential for root growth, drought resistance, and likely many other developmental aspects. This systemic mode of action relies on the XRN activities. By contrast, FRY1 has a local (i.e. not systemic) effect on the expression of the PHT1 genes and other phosphate starvation markers in roots; this effect depends on Pi accumulation but not on the XRN activities. doi:10.1371/journal.pone.0016724.g008 Figure 8. Model accounting for the dual mode of action of FRY1. The expression of FRY1 in the shoot is essential for root growth, drought resistance, and likely many other developmental aspects. This systemic mode of action relies on the XRN activities. By contrast, FRY1 has a local (i.e. not systemic) effect on the expression of the PHT1 genes and other phosphate starvation markers in roots; this effect depends on Pi accumulation but not on the XRN activities. doi:10.1371/journal.pone.0016724.g008 and transpiration, but rather to reduced XRN activity. All of the phenotypes linked to perturbations in XRN activities can be complemented by grafting, suggesting the presence of a systemic signal (Fig. 8, left). On the contrary, the induction of phosphate starvation markers is likely linked to a local effect of FRY1 expression (Fig 8, right). It is not complemented by a wild type scion and is not mimicked by the xrn mutations or linked to the root architecture phenotype. example, the BYPASS1 locus is required in Arabidopsis to prevent constitutive production of a root-derived graft-transmissible signal that is sufficient to inhibit leaf initiation, leaf expansion and shoot apical meristem activity [17]. We demonstrate here that FRY1 in shoots controls root development in Arabidopsis. We have identified a novel FRY1 function modulating the transcription of several Pi starvation markers in the root stele. This is the first fry1 mutant phenotype reported to be independent of XRN activities. Instead, it is likely depending on FRY1 impact on the root cytosolic Pi pool, in stele and pericycle cell layers. Interestingly, this phenotype is not complemented by a wild type scion and therefore acts locally. example, the BYPASS1 locus is required in Arabidopsis to prevent constitutive production of a root-derived graft-transmissible signal that is sufficient to inhibit leaf initiation, leaf expansion and shoot apical meristem activity [17]. We demonstrate here that FRY1 in shoots controls root development in Arabidopsis. We have identified a novel FRY1 function modulating the transcription of several Pi starvation markers in the root stele. Plant material and growth conditions Plant material and growth conditions The PHT1;4:GUS line (originally referred to as pht1;4-1 in [10]) was isolated from a T-DNA mutagenized A. thaliana ecotype Wassilewskija (Ws) seeds collection, obtained from INRA [18]. fry1-6 (SALK_020882), xrn2-1 (SALK_041148), xrn3-3 (SAIL_ 1172C07) and xrn4-6 (SALK_014209) mutants as well as the xrn2 xrn3, xrn2 xrn4 and xrn3 xrn4 double mutants have been described before [9]. Because XRN3 and XRN4 are genetically linked on chromosome 1, whereas XRN2 is on chromosome 5, the xrn2 xrn3 xrn4 triple mutant was generated by crossing xrn2 xrn4 to xrn3 xrn4 so that 1/16 of the F2 plants would be homozygous for the three mutations (xrn2 and xrn3 being genetically independent). The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs This is the first fry1 mutant phenotype reported to be independent of XRN activities. Instead, it is likely depending on FRY1 impact on the root cytosolic Pi pool, in stele and pericycle cell layers. Interestingly, this phenotype is not complemented by a wild type scion and therefore acts locally. We propose that this effect could be a result of the by-products of FRY1 activity, more specifically the result of the conversion of PAP into AMP + Pi. A reduction of FRY1 activity likely leads to a slight reduction of AMP and phosphate levels (along with an accumulation of PAP) in the tissues where FRY1 is normally very active (the root pericycle, central cylinder and meristems). The reduction of Pi availability would lead to the transcriptional induction of several phosphate starvation genes (including the PHT1;4:GUS marker) in these cell layers. This effect is not complemented by a wild type scion and is not mimicked by the xrn mutations or linked to the root architecture phenotype. February 2011 \| Volume 6 \| Issue 2 \| e16724 Mutant complementation and tissue localization of FRY1 Mutant complementation and tissue localization of FRY1 The FRY1 genomic fragment (1960 bp) and an additional 753 bp upstream region was PCR cloned by standard molecular techniques in the Ws accession. After sequencing in the pENTR/ D-TOPO (Invitrogen, Carlsbad, USA), an LR clonase (Invitrogen, Carlsbad, USA) reaction was used to clone the genomic fragment in the binary vector pGWB4 [25]. Then, the Arabidopsis fry1-7 mutant was transformed by a simplified floral dip method [26]. Similar construct were built with the FRY1 cDNA (1221 bp) with or without a C-terminal GFP fusion, under the control of the 35S promoter. Primary transformants were selected in medium containing 50 mg/L hygromycin. Their progeny was screened for root phenotype and GFP expression in standard in vitro growing conditions using a Leica SP2 AOBS inverted confocal microscope (Leica Microsystems, Germany) equipped with an Argon ion laser. Prior to confocal observation, plantlets were stained 3 min in 100 mg/mL propidium iodide (PI). Leaf shape, flowering time and GFP expression in mature plants were screened in soil-grown plants, both in short and long days conditions. To analyze mRNA splice variants, 10 mg of total RNA from roots and leaves were treated with DNase1 (Roche Diagnostics, Meylan, France) for 15 min at 37uC and were used for the reverse transcription reaction using the AMV Reverse Transcriptase (Roche Diagnostics, Meylan, France) according to the manufac- turer’s instructions. Specific primers (sequence available on request) were used to amplify FRY1 transcripts, both in the wild type and the mutant backgrounds. DNA cloning and sequencing were performed by standard procedures [31]. Mutagenesis, screening conditions and histology Approximately 3000 seeds of PHT1;4:GUS were mutagenized with a 0.3% solution of Ethyl methane sulfonate (EMS) as described [20]. Seeds were sown and the M1 plants were cultivated to obtain the M2 generation. Around 30 seeds of each M2 line (1400 lines) were sown in 6-well Petri dishes (NUNC) containing a modified Hoagland medium (1 mM MgSO4, 2 mM Ca(NO3)2, 1.7 mM KNO3, 1.6 mM Fe, 46.2 mM H3BO3, 9.1 mM MnCl2, 0.87 mM ZnSO4, 0.32 mM CuSO4, 1.03 mM Na2MoO4, 0.5 mM NH4H2PO4). After 10 days, seedlings were screened for their GUS expression as described [10]. Histological analysis were performed as described [21]. Grafting experiments y p g The mutant line was backcrossed three times to the parental line (PHT1;4:GUS) to test the linkage of the phenotype to a single Mendelian recessive mutation. For mapping purposes, a mutant plant (Ws ecotype) was crossed with a wild-type Col plant. Linkage analysis was performed with the F2 progeny of this cross as described [22]. DNA from F2 seedlings displaying the mutant phenotypes (GUS staining of a root piece from seedlings grown on complete media) was prepared as described [23]. Single Sequence Length Polymorphism markers [24] distributed on the five chromosomes and polymorphic between the Ws and Col accessions were tested on the extracted DNA. Thermal cycling consisted of an initial denaturation at 94uC for 2 minutes, followed by 38 cycles of denaturation step at 94uC for 15 seconds, annealing at the respective Tm of each oligonucleotide pair for 20 seconds, and extension at 72uC for 45 sec. At the end of the reactions, the PCR products were allowed to extend for 2 minutes at 72uC. Grafting was performed according to [30]. Parental and mutant lines were sown in vitro on a MS/10 medium. Four days after sowing they were cut at the hypocotyl level to separate the aerial and root parts. A 0.3 mm diameter silicon ring (Silastic Laboratory tubing, Dow Corning, USA) was used to maintain the aerial seedling scion and the rootstock together to allow fusion. After five days, successful grafts were transferred to fresh medium for 48 hours, followed by GUS staining for 16 h at 37uC. Alternatively, established grafts were put on soil, either on large soil-filled plates or in pots and grown in the greenhouse for 4 weeks in order to assess the root architecture and the drought tolerance of the grafts. For drought tolerance, plants were either cultured in long days (12 h light, 12 h dark), before watering was withheld, then survival of the plants was determined as described [19] or cultured in short days (8 h light, 14 h dark) using a mix of J soil and L sand and an immersion watering per day. Phenotype was assessed after 13 days without watering followed by three days were watering of the individual pots was resumed. To identify the mutant locus on chromosome 5, the Gramene Simple Sequence Repeat Identification Tool (SSRIT, http:// www.gramene.org/db/markers/ssrtool) was used to generate new markers in the area surrounding FRY1. Conclusion Long-distance signaling is used by plants to coordinate shoot and root development. Despite the importance of such coordina- tion, only a few genes have been shown to regulate root development in a systemic way. For example, in Lotus japonicus, the use of reciprocal and self-grafting studies with the hyperno- dulating mutant har1 have shown that the shoot genotype is responsible for the negative regulation of nodule development. Therefore, HAR1 in shoots mediates systemic regulation of nodulation [16]. There are also few examples of root genes regulating shoot development by long-distance signaling. For For physiological analyses and RNA extractions, seeds of Col-0, Ws, PHT1;4:GUS, and the different fry1 mutant alleles were cultivated as described before [10]. For drought tolerance tests, plants were grown in individual pots in short days for 4 weeks with standard watering conditions (once a day). Watering was stopped PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 9 FRY1 Activity Unlinked to XRNs for 13 days and the pots were then rehydrated for 3 days before the observations. Alternatively, after the onset of wilting, survival of the plants was quantified by measuring chlorophyll fluorescence as described [2,19]. software (http://rsb.info.nih.gov/ij/). To determine the speed of growth of the main root, photographs were taken at 8, 11 and 14 days post germination (dpg) from which PR length was measured. The daily growth was calculated accordingly. To measure single cell length above the differentiation zone, roots were briefly stained with ruthenium red and observed with a bright field microscope (Leica DMRXA, 20x objective). At least 30 cells for each of 12 different roots per genotype were measured using a micrometric lens. To estimate the size of the proximal meristem (PM), the number of undifferentiated cells in the cortex was measured in at least 30 roots per genotype as described [27,28]. PI-stained roots (3 min in 100 mM PI) were observed by confocal laser scanning microscopy. PI was excited at 514 nm and imaged using a custom 610–720 nm band pass emission filter. To determine the number of LR primordia at different stages of development, we used a Nomarsky optical microscope, as described [29]. All experiments were performed at least three times. Molecular and gene expression analysis For gene expression analysis, total RNA was extracted from rosettes and roots of 10 day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, grown in MS/ 10 medium as described previously [12]. cRNA was prepared using the manufacturer’s instructions (www.affymetrix.com sup- port technical manual expression_manual.affx). Labeling and hybridization on the ATH1 microarray and data analysis were performed according to [12]. Microarray data has been deposited at the EMBL database with the accession number E-MEXP-2483 (www.ebi.ac.uk/arrayexpress) and was used in the present work to identify candidate genes during the positional cloning of the mutant locus. Analysis of root architecture RTqPCR analyses were performed after reverse transcription (kit from GE Healthcare) and amplification (Applied ABI7000). Primer efficiency factors were measured for each gene and GapC Seedlings were photographed at different times after germina- tion and PR and LR length were measured with the ImageJ PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 10 FRY1 Activity Unlinked to XRNs and ROC3 were used as reference genes. Primer sequences are available upon request. Figure S3 Expression of phosphate-starvation induced genes in the 35S:AHL complemented line. (A) Quantitative real time PCR of the PHT1;4 transcripts in Col, fry1-6 and fry1-6/ 35S::AHL roots. (B) Quantitative real time PCR on the Pho1H1 transcript in Col, fry1-6 and fry1-6/35S::AHL roots. (C) Quanti- tative real time PCR on the At1g73010 phosphatase transcript in Col, fry1-6 and fry1-6/35S::AHL roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Col roots, grey bars to fry1-6 roots and black bars to fry1-6/35S::AHL roots. Standard deviations are shown. (EPS) Supporting Information Figure S1 Mutant complementation assays and leaf phenotype of PHT1;4:GUS/fry1-7 mutant. (A) Complemen- tation of the fry1-7 mutant. The progeny of a plant heterozygous for a T-DNA carrying a 35S::FRY1 cDNA construct is shown. Asterisks indicate non-complemented fry1-7 mutant plantlets that, presumably, did not inherit the transgene. (B) Picture of the rosette of the 3 week-old PHT1;4:GUS line (left) and the PHT1;4:GUS/ fry1-7 mutant (right) grown on soil under short day conditions. (C) 6 week-old PHT1;4:GUS line (left) and the PHT1;4:GUS/fry1-7 mutant (right) grown in long day conditions. (D) Complementation of the fry1-6 mutant with a 35S::AHL cDNA. The Col control (left), the fry1-6 mutant (middle) and the complemented line (fry1-6/ 35S::AHL) (right) are shown. White scale bars are 20 mm. (EPS) Figure S5 Root development and primary root length of the xrn4-6 mutant. (A) The general in vitro development of Col, xrn4-6 and fry1-6 mutants 11 dpg. Scale bars are 20 mm. (B) PR length was measured at 11 dpg. White bars correspond to Col, grey bars to fry1-6 and black bars to xrn4-6 mutant. Standard deviations are shown. (EPS) Production of Arabidopsis transformants expressing pAT1G73010::LUC A DNA fragment corresponding to 2001 bp of the promoter driving the expression of the AT1G73010 gene (ending right before the ATG) was PCR amplified and cloned into the pENTER-D-TOPO vector. The fragment was recombined into the pBGWL7 vector [32] using LR clonase. After sequencing confirmation, the vector was introduced into C58C1 Agrobacterium tumefaciens cells. Arabidopsis plants were transformed using a modified floral dip method [26], and transformed plants were selected using Basta (T1). Figure S4 Expression of phosphate-starvation induced genes in the fry1-6, xrn4 and xrn2 xrn3 xrn4 mutants. Quantitative real time PCR of the PHT1;4 transcript (A), the PHT1,7 transcript (B), the Pho1H1 transcript (C) and the At1g34010 phosphatase transcript (D) in Col, fry1-6, xrn4-6 and xrn2 xrn3 xrn4 roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Col, pale grey to fry1-6, dark grey to xrn4-6 and black bars to xrn2 xrn3 xrn4 mutant. Standard deviations are shown. Bioluminescence detection was performed on the T2 generation (8 day-old plantlets) using a UPLSAPO 4X dry objective (N.A. 0.16) or a LUCPLFLN 40X dry objective (N.A. 0.6) mounted on an Olympus LV200 Luminoview microscope coupled to an ANDOR iKon-M DU934 camera. Images were acquired with an exposure time of 2 min (4X objective) or 4 min (40X objective). Contrast and brightness of the images were adjusted in ImageJ. Bioluminescence detection was performed on the T2 generation (8 day-old plantlets) using a UPLSAPO 4X dry objective (N.A. 0.16) or a LUCPLFLN 40X dry objective (N.A. 0.6) mounted on an Olympus LV200 Luminoview microscope coupled to an ANDOR iKon-M DU934 camera. Images were acquired with an exposure time of 2 min (4X objective) or 4 min (40X objective). Contrast and brightness of the images were adjusted in ImageJ. Acknowledgments The authors are very grateful to Drs B.-H. Kim and A.G. von Arnim (University of Tennessee, USA) who provided seeds of their transgenic lines and to Dr. B. Albaud for performing array hybridizations (Affymetrix platform, Curie Institut, Paris, France). We acknowledge TAIR (http:// arabidopsis.org) as a source of data and the Arabidopsis Biological Resource Center (ABRC) for the fry1-6 mutant. We are grateful to Nathalie Pochon for technical help and to Dr. Brandon Loveall for English proofreading of the manuscript. Figure S2 The pAT1G73010::LUC construct reveals the stele specificity of gene expression in Arabidopsis roots and the phosphate starvation induction. (A) Transmitted light image and (B) bioluminescence signal of pAT1G73010::LUC plantlets grown for 4 days on P depleted medium then for 4 days on complete medium (plantlet on the left) or for 8 days on P depleted medium (plantlet on the right). Scale bar, 1 mm. (C) Close up of a mature part of a root from a plantlet grown for 8 days on P depleted medium (overlay of transmitted light and bioluminescence signal). The bioluminescence signal is only detected in the central cylinder. Scale bar, 100 mM. (EPS) Author Contributions Conceived and designed the experiments: JH GME BJP LN EM. Performed the experiments: JH JM PAC PD VB HJ SC ACM MD HV EM. Analyzed the data: JH JM PAC PD ACM AM HV MC TD MCT LN EM. Contributed reagents/materials/analysis tools: JM ACM AM EM. Wrote the paper: MC TD LN EM. 7. Gil-Mascarell R, Lopez-Coronado JM, Belles JM, Serrano R, Rodriguez PL (1999) The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase. Plant J 17: 373–383. 6. Quintero FJ, Garciadeblas B, Rodriguez-Navarro A (1996) The SAL1 gene of Arabidopsis, encoding an enzyme with 39 (29),59-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell 8: 529–537. 8. 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(2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104: 34–41. 13. Stefanovic A, Ribot C, Rouached H, Wang Y, Chong J, et al. (2007) Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways. Plant J 50: 982–994. 26. Logemann E, Birkenbihl RP, Ulker B, Somssich IE (2006) An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2: 16. 14. Thibaud MC, Arrighi JF, Bayle V, Chiarenza S, Creff A, et al. References 1. Xiong L, Lee B, Ishitani M, Lee H, Zhang C, et al. (2001) FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev 15: 1971–1984. 5. Chen H, Xiong L (2010) The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation. Plant Cell Environ. 2. Wilson PB, Estavillo GM, Field KJ, Pornsiriwong W, Carroll AJ, et al. (2009) The nucleotidase/phosphatase SAL1 is a negative regulator of drought tolerance in Arabidopsis. Plant J 58: 299–317. 6. Quintero FJ, Garciadeblas B, Rodriguez-Navarro A (1996) The SAL1 gene of Arabidopsis, encoding an enzyme with 39 (29),59-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell 8: 529–537. 3. Robles P, Fleury D, Candela H, Cnops G, Alonso-Peral MM, et al. (2010) The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis. Plant Physiol 152: 1357–1372. 4. Kim BH, von Arnim AG (2009) FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 39 (29),59-bisphosphate nucleotidase activity. Plant J 58: 208–219. 4. Kim BH, von Arnim AG (2009) FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 39 (29),59-bisphosphate nucleotidase activity. Plant J 58: 208–219. PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 11 FRY1 Activity Unlinked to XRNs (2010) Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J 64, 775–789. 27. Casamitjana-Martinez E, Hofhuis HF, Xu J, Liu CM, Heidstra R, et al. (2003) Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance. Curr Biol 13: 1435–1441. 15. Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, et al. (2008) A genetic framework for the control of cell division and differentiation in the root meristem. Science 322: 1380–1384. 28. Cazale AC, Clement M, Chiarenza S, Roncato MA, Pochon N, et al. (2009) Altered expression of cytosolic/nuclear HSC70-1 molecular chaperone affects development and abiotic stress tolerance in Arabidopsis thaliana. J Exp Bot 60: 2653–2664. 16. Nishimura R, Hayashi M, Wu GJ, Kouchi H, Imaizumi-Anraku H, et al. (2002) HAR1 mediates systemic regulation of symbiotic organ development. Nature 420: 426–429. 17. Van Norman JM, Frederick RL, Sieburth LE (2004) BYPASS1 negatively regulates a root-derived signal that controls plant architecture. Curr Biol 14: 1739–1746. 29. Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124: 33–44. 30. Turnbull CG, Booker JP, Leyser HM (2002) Micrografting 30. Turnbull CG, Booker JP, Leyser HM (2002) Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant J 32: 255–262. 18. Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobaterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Life Sciences 316: 1194–1199. long-distance signalling in Arabidopsis. Plant J 32: 255–262. 31. Marin E, Divol F, Bechtold N, Vavasseur A, Nussaume L, et al. (2006) Molecular characterization of three Arabidopsis soluble ABC proteins which expression is induced by sugars. Plant Science 171: 84–90. 19. Woo NS, Badger MR, Pogson BJ (2008) A rapid, non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence. Plant Methods 4: 27. expression is induced by sugars. Plant Science 171: 84–90. 32. Karimi M, De Meyer B, Hilson P (2005) Modular cloning in plant cells. Trends Plant Sci 10: 103–105. 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W4254023444.txt	null	en		Management, regulation and environmental impacts of nitrogen fertilization in Northwestern Europe under the Nitrates Directive; a benchmark study	null	2,012	cc-by	13,775	Biogeosciences, 9, 5143–5160, 2012 www.biogeosciences.net/9/5143/2012/ doi:10.5194/bg-9-5143-2012 © Author(s) 2012. CC Attribution 3.0 License. Biogeosciences Management, regulation and environmental impacts of nitrogen fertilization in northwestern Europe under the Nitrates Directive; a benchmark study H. J. M. van Grinsven1 , H. F. M. ten Berge2 , T. Dalgaard3 , B. Fraters4 , P. Durand5 , A. Hart6 , G. Hofman7 , B. H. Jacobsen8 , S. T. J. Lalor9 , J. P. Lesschen10 , B. Osterburg11 , K. G. Richards9 , A.-K. Techen11 , F. Vertès5 , J. Webb12 , and W. J. Willems1 1 PBL Netherlands Environmental Assessment Agency, Department: Water, Agriculture and Food, Bilthoven, The Netherlands Research International, Wageningen University and Research Centre, The Netherlands 3 Aarhus University, Department of Agroecology, Foulum, Denmark 4 National Institute for Public Health and the Environment, Bilthoven, The Netherlands 5 INRA, UMR1069, Sol Agro and Hydrosysteme, 35000 Rennes, France 6 Environmental Agency, Olton, UK 7 Ghent University, Department of Soil Management, Belgium 8 Institute of Food and Resource Economics, University of Copenhagen, Denmark 9 Teagasc, Crops Environment and Land Use Programme, Johnstown Castle, Wexford, Ireland 10 Alterra, Wageningen University and Research Centre, The Netherlands 11 Institute of Rural Studies, Johann Heinrich von Thünen-Institut (vTI), Bundesforschungsinstitut für Ländliche Räume, Wald und Fischerei, Germany 12 AEA Energy and Environment, Didcot, UK 2 Plant Correspondence to: H. J. M. van Grinsven (hans.vangrinsven@pbl.nl) Received: 21 May 2012 – Published in Biogeosciences Discuss.: 22 June 2012 Revised: 16 November 2012 – Accepted: 19 November 2012 – Published: 14 December 2012 Abstract. Implementation of the Nitrates Directive (NiD) and its environmental impacts were compared for member states in the northwest of the European Union (Ireland, United Kingdom, Denmark, the Netherlands, Belgium, Northern France and Germany). The main sources of data were national reports for the third reporting period for the NiD (2004–2007) and results of the MITERRA-EUROPE model. Implementation of the NiD in the considered member states is fairly comparable regarding restrictions for where and when to apply fertilizer and manure, but very different regarding application limits for N fertilization. Issues of concern and improvement of the implementation of the NiD are accounting for the fertilizer value of nitrogen in manure, and relating application limits for total nitrogen (N) to potential crop yield and N removal. The most significant environmental effect of the implementation of the NiD since 1995 is a major contribution to the decrease of the soil N balance (N surplus), particularly in Belgium, Denmark, Ireland, the Netherlands and the United Kingdom. This decrease is accompanied by a modest decrease of nitrate concentrations since 2000 in fresh surface waters in most countries. This decrease is less prominent for groundwater in view of delayed response of nitrate in deep aquifers. In spite of improved fertilization practices, the southeast of the Netherlands, the Flemish Region and Brittany remain to be regions of major concern in view of a combination of a high nitrogen surplus, high leaching fractions to groundwater and tenacious exceedance of the water quality standards. On average the gross N balance in 2008 for the seven member states in EUROSTAT and in national reports was about 20 kg N ha−1 yr−1 lower than by MITERRA. The major cause is higher estimates of N removal in national reports which can amount to more than 50 kg N ha−1 yr−1 . Differences between procedures in member states to assess nitrogen balances and water Published by Copernicus Publications on behalf of the European Geosciences Union. 5144 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe quality and a lack of cross-boundary policy evaluations are handicaps when benchmarking the effectiveness of the NiD. This provides a challenge for the European Commission and its member states, as the NiD remains an important piece of legislation for protecting drinking water quality in regions with many private or small public production facilities and controlling aquatic eutrophication from agricultural sources. 1 Introduction The main aim of the Nitrates Directive (1991: Directive 91/676/EEC; hereafter referred to as NiD) is to reduce water pollution caused or induced by nitrate and phosphorus from agricultural sources. The NiD is the most important piece of European (EU) regulation for reducing environmental impacts of fertilizer and manure and for increasing nitrogen use efficiency. The gross nitrogen balance, or nitrogen surplus, (Schröder et al., 2004; Vries et al., 2011) is an important indicator to evaluate the environmental impacts of the Nitrates Directive, particularly for the water compartment. This makes the NiD an important supporting instrument for other EU directives i.e. the Drinking Water Framework Directive (98/83/EC), the Water Frame Directive (2000/60/EC) and the Marine Strategy Framework Directive (2008/56/EC). The NiD legally restricts annual farm application of manure to 170 kg ha−1 of nitrogen, or in case of derogation to inputs up to 250 kg ha−1 (Oenema, 2004). The tenacious problem of regional nitrogen (and phosphorus) surpluses can be resolved by manure transport to other regions and by manure processing. In the case of the Netherlands and the Flemish region, part of the (processed) manure is exported to other countries. Agricultural practices in general, and more specifically application rates and management of chemical fertilizers and animal manures, vary greatly between and within EU member states. This makes it interesting to compare nitrogen management and regulation between countries and relate this to the observed states and trends of nitrate concentrations in groundwater and surface water. Since the introduction of the NiD in 1991, EU member states have implemented several action programs and have delivered several monitoring reports. The EU Commission obliges member states to report on the results of these action programs. It also charged synthesizing studies on these national reports but these reports are not publicly available. However, the EU Commission did publish summaries of the national data and reports in 2007 and 2011. In addition, Fraters et al. (2011) evaluated the effectiveness of environmental monitoring programs for the NiD. However, overall insight into the effectiveness of the NiD in the EU is still limited and rarely published in peerreviewed journals. Together with the submission of the next set of national monitoring reports for the NiD, this paper could increase this insight and help to improve implementation of the NiD across the EU. Biogeosciences, 9, 5143–5160, 2012 The combination of environmental directives and the Common Agricultural Policy should provide food security and a healthy natural environment in Europe while maintaining a level playing field for the agricultural entrepreneurs (De Clercq et al., 2001). This is particularly true for agriculture in northwestern EU member states as they compete to provide food to consumers in the so-called “London-Berlin-Paris triangle”. The purpose of this paper is to compare, evaluate and benchmark the implementation of the Nitrates Directive in the northwestern member states of the EU. The objective is to relate differences in implementation to differences in structure, intensity and practices of the agricultural sector and to sensitivity of soil water systems to nitrate pollution. Key issues of the NiD addressed in the benchmark are application rates of N in manure, the balance between applied N and crop requirements and water quality in relation to the ni−1 trate target of 50 mg NO− 3 L . The comparison is restricted to Denmark, Germany, the Netherlands, Belgium, the United Kingdom, Ireland and the northern part of France. Crop and fodder production potential per hectare on comparable soils in these countries are similar. Note however, that within the United Kingdom there are four separate governments and in Belgium two, which implement the Nitrates Directive in differing ways. Moreover, all these countries have regions with high livestock densities, causing feed requirements to exceed regional feed production, and manure production to exceed regional crop demands. 2 2.1 Materials and methods Data sources This analysis combines various existing studies on implementation of the Nitrates Directive (van Dijk and Berge, 2009; ten Berge and Dijk, 2009), gross nitrogen balances from Eurostat (2012), monitored nitrate concentrations in groundwater and surface water in synthesizing reports (European Commission, 2007, 2011; Fraters et al., 2011) and various national reports on implementation and evaluation of the Nitrates Directive for the last reporting period (Anonymous, 2008a, b, c, d; Desimpelaere et al., 2008; Zwart et al., 2008). A complication when comparing water quality data among EU member states (and sometimes within a single member state) to evaluate the NiD are the large differences in monitoring procedures, e.g. with regard to sampling density (Table 1), monitoring frequency and groundwater sampling depth (Fraters et al., 2011; European Commission, 2011), and data and procedures for calculation of nitrogen balances (Panten et al., 2009). In 2007 the total number of sampling sites for groundwater was 31 000 and for surface water 27 000. www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe Table 1. Density of groundwater and surface water sampling for the whole land surface in monitoring programs for the NiD (European Commission, 2011). Density of groundwater sampling stations (points/1000 km2 ) Density of surface water sampling stations (points/1000 km2 ) 99 3 34 5 1 33 13 38 1 5 3 3 13 33 Belgium Germany Denmark France Ireland Netherlands United Kingdom 2.2 Nitrogen balance In this study, calculation of the gross nitrogen balance (GNB) was based on the OECD method (OECD, 2007). In addition the soil N balance (SNB) is used which sometimes is confused with the soil surface N balance (SSNB). The GNB represents the total potential loading of nitrogen from primary agricultural production to the environment, but excluding N emissions from fossil fuel combustion for energy requirements for e.g. fertilizer manufacturing, housing, transport and soil and crop management and correcting for export and processing of manure. SNB or soil N surplus represents the total potential loading from nitrogen use on agricultural soil, while SSNB represents the total net nitrogen loading to the soil and water compartment. GNB: fertilizer + manure production + other inputs − net manure export − crop removal SNB: GNB − N-loss housing − N-loss storage SSNB: SNB − N-loss manure application Other inputs include N deposition and biological N fixation (BNF), where N deposition is the result of NH3 and NOx emissions from both agricultural and other sources, mainly transportation and energy generation. Choosing one of the balance indicators for monitoring and evaluation of NiD effects is determined mainly by data availability. Data requirements for GNB are lowest, but GNB does not correct for environmental measures reducing ammonia emission following from other EU directives like the National Emission Ceilings (NEC) directive (2001/81/EC) and the Integrated Pollution Prevention (IPPC) directive (96/61/EC). However, different calculation procedures, particularly for determining manure input and nitrogen removal by crops, and also inclusion or exclusion of N-losses during housing and storage (difference between gross and net soil balance) and of smaller input items, may need to be taken into account when comparing national or regional nitrogen balances. For this reason the use of a model for determining the nitrogen balance is an additional valuable tool to evaluate the www.biogeosciences.net/9/5143/2012/ 5145 effectiveness of the NiD. Model approaches are inherently more consistent regarding calculation schemes, but without sound ground validation, have a risk of not accounting for regional differences in response of crop removal and water quality to nitrogen fertilization. For example, in the UK a model approach is used to estimate nitrogen loading as part of the NiD assessments. Loadings are calculated using the NEAP-N model (Lord and Anthony, 2000) along with an urban estimation model (Lerner, 2000). Leip et al. (2008) coupled the economic model CAPRI and the mechanistic biochemical model DNDC for evaluation of the effects of agrienvironmental policies on the European environment, for example on groundwater pollution with nitrate. Here we use the model MITERRA-EUROPE to apply a consistent methodology to all countries. 2.3 MITERRA-EUROPE The model MITERRA-EUROPE (referred to as MITERRA hereafter) was used to quantify the nitrogen balances and nitrate leaching from agriculture on both EU-27 level, country level, and regional level. By applying a uniform calculation scheme as in MITERRA we could scrutinize results in the national reports and benchmark nitrogen surpluses and nitrate concentration at the more appropriate sub-national level. MITERRA consists of an input module with activity data and emission factors, a set of measures to mitigate ammonia and greenhouse gas emission and nitrate leaching, a calculation module, and an output module (Velthof et al., 2009; Lesschen et al., 2011). The database of MITERRA is on national and regional level (NUTS2, according Nomenclature of Territorial Units for Statistics in the EU) and includes data of N inputs, N outputs, livestock numbers, land use, crop types, soil type, and emission factors for NH3 , N2 O, and NOx , and leaching factors for NO3 . For this paper we used an updated version of MITERRA as described in Velthof et al. (2011). Crop areas were derived from EUROSTAT at NUTS2 level and crop yields from FAOSTAT at national level as the EUROSTAT data was incomplete. Grassland yields and N contents of grassland were estimated using the methodology of Velthof et al. (2009), because grassland yields are not available from statistics. The number of livestock in each year was derived from EUROSTAT. Data on annual N fertilizer consumption were collected from FAOSTAT. The N excretion of all livestock categories except dairy cows were obtained from the GAINS model (Klimont and Brink, 2004). A method was developed to estimate the N excretion from dairy cows on regional level based on milk yields, grassland yields, and N inputs (Velthof et al., 2011). The total manure N production was calculated at the NUTS2 level from the number of animals and the N excretion per animal and then corrected for gaseous N losses from buildings and storage. A method was developed to distribute the manure over crops taking account of the maximum Biogeosciences, 9, 5143–5160, 2012 5146 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe Table 2. Precipitation surplus and fraction of nitrogen surplus leaching to groundwater, the fraction leaching to surface waters and the runoff fraction of N in applied fertilizer, grazing and manure, used in the MITERRA model. Belgium-Flemish Belgium-Walloon Denmark Northern France Germany Ireland Netherlands United Kingdom Precipitation surplus mm Fraction leaching to groundwater % Fraction leaching to surface water % Fraction in surface runoff % 396 479 280 356 295 554 420 450 23 11 24 13 13 10 17 11 9 12 6 10 10 8 7 10 3 4 2 5 4 3 3 3 annual manure application of 170 kg N ha−1 or higher in case of a derogation. Nitrogen fertilizer was distributed over crops relative to their nitrogen demand, taking account of the amount of applied manure and grazing manure and their respective fertilizer equivalence (Velthof et al., 2009). Further nitrogen inputs include biological N fixation, which is estimated as a function of land use and crop type (legumes) and nitrogen deposition that is derived at NUTS2 level from EMEP (EMEP, 2010). Nitrogen leaching in MITERRA is calculated by multiplying the soil N surplus by a region specific leaching fraction, which is based on soil texture, land use, precipitation surplus, soil organic carbon content, temperature and rooting depth (Table 2). Surface runoff fractions are calculated based on slope, land use, precipitation surplus, soil texture and soil depth (Velthof et al., 2009). These parameters are derived from more detailed spatial data sources, and weighted average values for agricultural land are used at the NUTS2 level. The nitrate concentration in leaching water is calculated by dividing the amount of nitrogen leaching from agriculture by the total water flux, which is calculated as the precipitation surplus, derived from the EuroPearl model (Tiktak et al., 2006), minus surface runoff. The MITERRA model has been used in several EU studies and outcomes have been compared with other model results and national reported values. De Vries et al. (2011) compared several models, including MITERRA, on nitrogen budgets, and showed that MITERRA outcomes are in line with other model results. The distribution of calculated mean NO3 concentrations in NUTS 2 regions of EU-15 according to MITERRA agreed very well with the distribution of the means of measured NO3 concentrations in the EU-15, according to measured data from 2000–2003 (Velthof et al., 2009). Biogeosciences, 9, 5143–5160, 2012 3 3.1 Results Characteristics of agriculture and nutrient use in northwestern EU Mean annual temperatures range between 8 and 12 ◦ C, with minimum daily temperatures in January around 0 ◦ C and maximum daily temperatures around 20 ◦ C in July. Mean annual precipitation ranges from values exceeding 1000 mm per year in western coastal regions to 500 mm per year in central France, and eastern UK and Germany (Tiktak et al., 2006). The combination of favorable climatic conditions, good agricultural practices and high inputs of fertilizer and manure allow high yields of cereals, potato, sugar beet, forage grass and maize and of milk, that generally exceed average values for the EU27 (Table 3). Yield differences per hectare in northwestern EU member states are largest for milk and ruminant meat because of large differences in shares of grazing beef and dairy cattle, areas of marginal grassland, grass in arable rotations (e.g. Denmark) and grazing intensity. Ireland, the UK and France hold large areas of less productive grassland on wet, peaty or mountain soils. All countries considered are net importers of substantial amounts of fodder and feed stuff, in the range of 200–400 kg per livestock unit (LSU; reference unit for livestock species based on feed requirement) in the period between 2000 and 2007 (FAOSTAT), with the exception of France (120 kg LSU−1 ). These differences explain a minor part of differences in milk and ruminant meat yield per hectare. Mean national livestock densities in the considered member states range between 0.9 LSU per hectare in northern France, which is near to the average in the EU27, to 3.4 LSU per hectare in the Netherlands (Table 4; using LSU definition according to Eurostat). The share of dairy cows (one dairy cow represents one Livestock Unit; LSU) ranges from 10 % in Denmark to 22 % in Ireland. Regional livestock densities can be much higher, with 8.9 LSU ha−1 in the southeastern part of the Netherlands, 6.0 LSU ha−1 in Flemish Region-Belgium and 3.7 LSU ha−1 in Brittany-France, and www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe 5147 Table 3. Mean annual yields in northwestern member states of the EU for cereals, forage maize, potato and sugar beet (Sources: FAOSTAT mean crop data are for the period 2000–2007; EFMA (2008), mean data for 2006–2009), and the sum of ruminant meat +0.1 × total milk production as a proxy for ruminant productivity per hectare of permanent grassland (Sources: production from FAOSTAT, data 2008, and grassland areas from Eurostat (2011), data 2007). FAO Belgium Denmark France Germany Ireland Netherlands United Kingdom EU27 FAO FAO 2000–2007 FAO FAO 2008 EFMA EFMA 2006–2009 EFMA Wheat ton ha−1 Forage maize ton ha−1 Potato ton ha−1 Sugar beet ton ha−1 Meat + 0.1 × Milk ton ha−1 grass land All cereals ton ha−1 Potato ton ha−1 Sugar beet ton ha−1 8.2 7.1 6.9 7.3 8.9 8.2 7.7 11.1 43.4 39.5 41.4 40.9 35.2 43.5 41.6 67.9 57.3 76.5 59.1 48.6 61.6 54.7 1.09 1.67 0.50 0.85 0.36 1.85 0.25 0.43 8.8 5.9 7.2 6.5 7.0 8.2 7.1 5.0 46.0 44.7 45.7 40.1 32.8 46.3 41.6 29.0 65.0 55.7 82.5 58.0 8.6 8.8 11.2 63.2 61.7 62.1 Table 4. Main characteristics of agricultural sector in northwestern member states of the EU in 2007 (Eurostat, 2011). Belgium Denmark France North-centralb Germany Ireland Netherlands United Kingdom EU27 Agricultural area (UAA) mln ha Livestock density a LSU ha−1 Permanent Pasture % of UAA Farm size ha UAA/holding 1.4 2.7 27.5 17.8 16.9 4.1 1.9 16.1 172.5 2.8 1.7 0.8 0.9 1.1 1.4 3.4 0.9 0.8 37 8 29 21 29 76 43 62 33 29 60 53 – 46 32 26 65 13 a In the EUROSTAT definition one LSU corresponds to the feed requirement of one adult dairy cow producing 3000 kg of milk annually. b All departments above the line “Nantes-Dijon”. are always associated with the presence of a large pig and/or poultry sector. Farm sizes per holding in the northwestern member states are much higher than the EU27 average. Nitrogen from manures constitutes a substantial proportion of total nitrogen fertilization, ranging between 40 % in Germany and Northern France, to 60–65 % in Belgium, Ireland and the Netherlands. In the Netherlands and the Flemish Region the net nitrogen excretion (after subtracting ammonia emission from housing and storage) exceeds the application limit of 170 kg ha−1 set by the NiD, by 40 and 12 kg ha−11 respectively, based on MITERRA results. These two countries require a combination of derogation, on the one hand, and export and processing of manure on the other hand, to be able to comply with the NiD at a national level. The sum of nitrogen excretion plus fertilizer use per hectare of utilized 1 Unless indicated otherwise the unit kg ha−1 refers to annual fluxes. www.biogeosciences.net/9/5143/2012/ agricultural area (UAA) in the period 2005–2008 ranges between 138 kg ha−1 in France to 377 kg ha−1 in the Netherlands (Table 5) and exceeds mean values for EU12 (old member states) and EU27. 3.2 Application standards for nitrogen from manure and fertilizer The most important restriction following from the NiD is the application limit for nitrogen from animal manure. Other restrictions following from the NiD are mandatory minimum manure storage capacities, prohibition periods for nutrient application, restrictions for nutrient application near water courses, on slopes and on frozen, waterlogged or snowcovered soils (van Dijk and ten Berge, 2009; Table 6). These restrictions should facilitate the achievement of the overall objective of the NiD to establish a balance between nutrient application and crop requirements. There are large Biogeosciences, 9, 5143–5160, 2012 5148 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe Table 5. Average annual inputs, crop removal and gross balance of nitrogen in 2005–2008 in northwestern member states of the EU (Eurostat, 2012). Fertilizer Inorganic manure Gross Other inputs Removal Gross N balance 191 101 112 125 155 194 111 98 89 119 98 52 93 55 210 101 58 50 kg N ha−1 Belgium Denmark France Germany Ireland Netherlands United Kingdom EU15∗ EU27 101 75 76 103 78 140 94 67 61 168 100 62 74 117 236 87 63 54 41 24 26 42 15 28 31 26 25 ∗ EU15: member states between 1 January 1995 and 30 April 2004. Table 6. Restrictions for application of fertilizer and manure in national implementations of the Nitrates Directive (Adapted from Dijk and Berge, 2009). DK BFL FR GE1 UK NL IRL yes yes yes yes2 yes yes yes yes yes yes2 yes yes yes yes yes6 yes yes yes yes yes4 yes yes yes yes4 yes4 yes yes yes yes yes yes yes yes Farm measures Fertilizer planning Keeping records Soil analysis Fertilization Closed periods for manure/fertilizers3 Low emission application No manure application on frozen, snow covered and waterlogged land Unfertilised zones along surface water5 yes yes7 Post-harvest measures Catch crops No tillage in autumn yes4 yes yes yes yes8 Other Policy Measures Max limit for livestock yes Maximum limits on N and P use Manure Total N (manure + fertilizers) Maximum N and P surpluses Maximum soil mineral N in autumn yes yes yes yes yes yes4 yes yes9 yes yes yes yes yes yes yes yes yes1 DK = Denmark, BFL = Belgium Flemish Region, FR = France, GE = Germany, UK = United Kingdom, NL = The Netherlands, IRL = Ireland 1 Implementation varies between states (Länder) of Germany, e.g. maximum soil mineral N autumn only in Baden Wurtemberg. 2 For NL in case farm has derogation. For BFL from 2013, on fields exceeding the threshold value of maximum soil mineral N in autumn. 3 For liquid manures generally between September/October and February. 4 In some departments within the NVZ’s. E.g. catch crops in western regions (Brittany and Normandy); Anonymous (2008a). 5 With large variation in width and length of unfertilized zones. 6 Increased from 2 m to 10 m from 2012 onwards. 7 No fertilizer within 2 meters of surface water. 8 Ploughing between July and November if green cover emergence of planted crop within 6 weeks of ploughing. 9 In small highly sensitive areas (e.g. coastal areas with green tides). Biogeosciences, 9, 5143–5160, 2012 www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe 5149 Table 7. Overview of area in Nitrate Vulnerable Zones and derogations for grassland (mostly dairy) farms in 2009 (European Commission, 2011). Nitrate Vulnerable Zones area (%) Belgium Flemish Region Walloon Region Denmark France Germany Ireland Netherlands United Kingdom 68 100 422 100 45 100 100 100 39 Application limit for manure (kg N ha−1 ) Share of Agricultural land (%) Share of farms (%) 12 10 4 0 <1 8 45 1.5 3.2 0 <1 8 32 1.3 250/2001 230 170 230 250 250 250 1 Also a derogation for some arable crops. 2 Situation in 2007 (Anonymous, 2008b). discrepancies between countries regarding the way these restrictions are translated into national law and applied in practice. Large discrepancies exist for methods of estimation of N emissions by livestock (including volatilization coefficients for ammonia), definitions of periods when and areas where manure application is restricted, procedures for enforcement of regulations can be very different and hamper a strict comparison of environmental impacts of the NiD between countries. With the exception of France, all member states have negotiated with the EU Commission an extension of the application limit in the NiD of 170 kg N ha−1 for manure from ruminants (a so-called derogation; Table 7). These derogations are based on proof that this extension will not increase the −1 risk for exceeding the critical nitrate limit of 50 mg NO− 3 L in groundwater and surface water. Derogations are granted at farm level (except in the Flemish Region) and mostly apply to farms with at least 70–80 % of farm land in use for grassland (or roughage in Denmark). The Flemish Region has a derogation at field level and includes some arable crops. For grassland and forage maize followed by one cut of grass or cut rye the application limit is 250 kg N ha−1 as cattle manure or treated pig manure and 200 kg N ha−1 for beet and winter wheat followed by a catch crop (Table 7). Denmark has implemented a maximum application limit for arable land of 140 kg ha−1 of nitrogen from pig manure and on organic farms (Kronvang et al., 2008), which is beyond the requirements of the NiD. The Netherlands has the largest derogation both regarding the extension of the application limit itself, and regarding the area where this extension applies. Only the NiD action programs of the Netherlands, Denmark and the Flemish Region have introduced crop and soil type-dependent applications standards for total N inputs, from manures and mineral fertilizers (van Dijk and ten Berge, 2009). Application standards in the Netherlands and Denmark apply to fertilizer equivalent (FE) N (Table 8). In Denmark, Ireland, the Netherlands and the UK for some www.biogeosciences.net/9/5143/2012/ crops, standards are differentiated with actual yield level and target. For cereals different standards may apply to baking, malting and fodder qualities, for potato to cultivars for use as ware, french fry, starch and seed. In the Flemish Region farmers can choose between a fixed total nitrogen amount or FE N values for organic fertilizers per crop. This new system with some new limits has been introduced in 2011 (Anonymous, 2011). In Denmark, Ireland and the UK application standards also depend on the soil N status and cropping history. Differences between total FE N application standards for the Flemish Region, the Netherlands and Denmark can be quite considerable. While standards for forage maize and winter wheat on sandy soils are quite comparable, differences between standards for other crops and clay soils are higher, amounting to 110 kg N ha−1 for ware potato on clay between the Netherlands and Denmark (Table 8). As a whole, the standards are the highest in the Netherlands for most crops mentioned in Table 8. For grassland without clover, standards are highest in Denmark, however, grass with clover is predominant in Denmark, and has lower standards. Standards for winter wheat and, to a lesser extent, for forage maize in Denmark and the Flemish Region are comparable. On the other hand, the standards for potato and sugar beet are lower for Denmark compared to the Flemish Region while this is the reverse for grassland. One would expect application standards in Denmark to be lower than in the Flemish Region in view of a lower yield potential (Table 3) and taking into account that in Denmark the fertilization limits are set at 90 % of the economic optimum N-fertilization. The consequence for Denmark, the Flemish Region, and the Netherlands of having a legal system of application standards based on total FE nitrogen is the introduction of fixed statutory values for the fertilizer equivalency of manures. Also the UK and Ireland have statutory values for the FE of manure in their NiD action programs. When statutory FE values are lower than actual values they provide an incentive Biogeosciences, 9, 5143–5160, 2012 5150 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe Table 8. Nitrogen application standards (kg N ha−1 yr−1 ) for some major crops in the 4th action programs for the NiD expressed either as fertilizer equivalent N (FE) or total N. Soil Grass: graze and cut Forage maize Winter wheat Potato (ware) Sugar beet Netherlands FE FE sand clay 260 310 150 185 160 220 245 250 145 150 Denmark1,2 FE FE sand clay 3105 3305 150 155 3 150 4 180 140 140 110 120 Flemish Region FE8 FE8 total total sand clay sand clay 235 245 350 360 135 150 205 220 160 175 200 215 190 210 260 280 135 150 205 220 United Kingdom total all 330 150 220 270 120 Ireland6 total all 7 306 140 180 145 155 1 0–5 % clay, not irrigated, 2 > 15 clay, not irrigated, 3 fodder quality, 4 baking quality, 5 for grass with clover 62–227 kg N ha−1 , depending on % clover, 6 soil nitrogen index 2 for arable crops, 7 for stocking rate between 170 and 210 kg ha−1 N per year, 8 valid from 2011 and without catch crop. to farmers to increase the nitrogen efficiency of the organic manure. Low fertilizer equivalencies for manure are typically caused by gaseous losses of ammonia, N oxides and dinitrogen, leaching losses of nitrate outside the growing season and slow N release within the growing season. FE’s can be increased by using low emission manure application techniques and by improved management of manure and soil (Dalgaard et al., 2011), for example by replacing autumn application of manure by spring application. Increasing legal FE may provide a strong incentive to apply these techniques and to improve management of manure. Generally speaking, a legal system based on FE is more comparable to the system for N recommendation than a system based on total N and therefore provides the farmer more direct insight into whether he needs to improve his N management to ensure sufficient N supply to crops. The statutory FE values do not always correspond to FE used in fertilizer recommendations (ten Berge and Dijk, 2009). For slurry, statutory FEs range from about 20 % in the UK to 75 % in Denmark. The small values quoted for the UK imply that the manures are not applied using techniques to reduce ammonia emission. For solid poultry, manure FEs range from 30 % in the UK, the Flemish Region and Germany to 55–65 % in Denmark and the Netherlands (Webb et al., 2013; Table 9). In Ireland maximum FE for manure of 40 % have been reported (Hoekstra et al., 2011). In Germany there are no legal N application limits for total or FE nitrogen. Instead, there is a restriction on net N surplus at farm level in combination with statutory FE values. The farmers have the responsibility to plan fertilization in such a way that the three year average of the N surplus does not exceed 60 kg N ha−1 from 2009 onwards. This surplus conBiogeosciences, 9, 5143–5160, 2012 straint has been introduced stepwise since 2006 (Wolter et al., 2011). France does not prescribe application standards in its action program for zones vulnerable to nitrate leaching (NVZ’s). For France FE values vary with crops (spring versus winter) and application period but have no legal status (COMIFER, 2011). Total N inputs are limited only in areas where nitrate concentrations in ground or surface water are high and where that water is used for drinking water. This limit is 210 kg N ha−1 in parts of Brittany, while in some watersheds with nitrate in surface water exceeding 50 mg L−1 total N inputs are restricted to values as low as 140 kg N ha−1 (van Dijk and ten Berge, 2009). Restrictions for use of fertilizers (and other agrochemicals like pesticides) in drinking water abstraction areas are common in Europe, also before the introduction of the NiD. 3.3 Nitrogen balance Complete official reports to the EU of the effect of the national action plans for the NiD are available for the 3rd (2000–2003) and 4th (2004–2007) reporting period and summarized by the European Commission (2011). A high gross nitrogen balance (GNB) is always associated with high gross inputs of manure (Table 5). In all countries considered, the GNB decreased between 2000 and 2008 (Fig. 1). The decrease of GNB between 2000 and 2004 is larger than between 2004 and 2008. The decrease in the Netherlands was 80 kg ha−1 and largest, but the GNB in 2008 is still higher than for other countries. The relative decreases of the GNB between 2000 and 2008 in Belgium (31 %), Ireland (25 %) and the United Kingdom (23 %) are comparable to www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive 1 Figuresin northwestern Europe 5151 2 3 Table 9. Statutory nitrogen fertilizer equivalency (%) for application of most common manure types (after deduction of gaseous losses from buildings and storage; taken from Webb et al., 2013). Netherlands Flemish Region Denmark France∗ Germany United Kingdom Ireland Cattle slurry Pig slurry Layer solid manure Broiler solid manure 60 60 70 50–60 50 20/35 40 60–70 60 75 50–75 60 25/50 50 55 30 65 45–65 30 20/35 50 55 30 65 45–65 30 20/30 50 ∗ No legal status. 4 5 6 the decrease in the Netherlands (30 %). The major cause for a decrease of the GNB is the decrease of the use of chemical fertilizer. In Denmark and the Netherlands this decrease was instigated to a large extent by increased utilization of manure N (Mikkelsen et al., 2010; Dalgaard et al., 2012). Nitrogen balance calculations using MITERRA provide insight in soil inputs and outputs underlying the differences in the N balance (Table 10). MITERRA results for N removal (R 2 0.92), GNB (R 2 0.94) and even more so SNB (R 2 0.96) are significantly correlated with total N input from manure and fertilizer but results for individual countries may deviate from the average relation. This is the case for Ireland in view of dominant grazing sector. In the Netherlands and the Flemish Region the difference between total N excretion and actual manure application is larger than for other countries because of substantial net export and processing of manure from pigs and poultry, amounting to 18 kg N ha−1 and 54 kg N ha−1 in 2008, respectively. Flemish pig manure is mostly processed by waste water treatment where N is removed by denitrification. In the Netherlands the five provinces with an intensive pig and poultry sector export on average 127 kg N ha−1 to the other seven provinces and a small part (10–20 kg N ha−1 ) abroad, mainly to Germany. Comparing nitrogen surpluses at national level for the northwestern EU member states is not very informative because of large differences in agricultural structure and livestock intensity within these countries (Table 4). Therefore, nitrogen use and balance by MITERRA model at NUTS2 level were recombined to generate results for regions with similar UAA (Fig. 2). Eleven regions had an SNB exceeding 100 kg N ha−1 . In addition to the Netherlands and Belgium, Brittany in France is standing out while several regions in the UK and single regions in Germany, Ireland and France have an SNB modestly exceeding 100 kg N ha−1 . Zooming further into MITERRA results for the Netherlands and Belgium, we find greatest surpluses for 2008 in the Province of Antwerp (241 kg N ha−1 ), and the southeast of the Netherlands (mean value 191 kg N ha−1 and maximum value of 197 kg N ha−1 in the province of Noord Brabant). These regions with the www.biogeosciences.net/9/5143/2012/ Figure 1. Gross annual nitrogen balance between 2000 and 20082000 (Eurostat, Fig. 1. Gross annual nitrogen balance between and2011). 2008 (Eu- rostat, 2011). 40 1 2 3 4 5 6 Fig. Annual N balance (soilandNNsurplus) Nand inputs from Figure2. 2. Annual soil soil N balance (soil N surplus) inputs fromand manure fertilizer in manure and fertilizer by MITERRA for regions north2008 by MITERRA for regionsinin 2008 northwestern Europe of comparable UAA and in N surplus western Europe of comparable UAA andUAA N insurplus exceeding 100 kgN/ha (NUTS1 level or clusters of NUTS2; 1000 ha inexceeding between 100 kg N ha−1 (NUTS1 level or clusters of NUTS2; UAA in 1000 brackets). ha in between brackets). greatest N surplus are also most sensitive to nitrate leaching with MITERRA leaching fractions of 18 % in Brittany, 22 % in the Flemish Region (26 % in Province of Antwerp), 24 % in southeast of the Netherlands (33 % in the province of Noord Brabant). GNB by MITERRA for the seven considered countries in 2008 is on average 19 kg ha−1 higher than GNB in Eurostat and fairly well correlated (R 2 0.74). Major outliers are Belgium and Ireland with differences of 38 and 58 kg ha−1 , respectively, the possible causes41of which will be addressed in the discussion. 3.4 Water quality In view of different monitoring procedures and differences in hydrology, geology and soils in the considered member Biogeosciences, 9, 5143–5160, 2012 5152 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe Table 10. Annual N inputs, removal of soil N balance in 2008 in northwestern member states of the EU according to MITERRA ranked with SNB. UAA mln ha Netherlands Belgium Flemish R. Region Walloon R. Ireland North. France United Kingdom Denmark Germany France EU27 1 2 3 4 5 6 7 8 1.9 1.3 0.7 0.7 4.1 17.8 14.3 2.5 16.7 30.1 172.5 Total N excretion Applied manure Grazing Applied fertilizer kg N ha−1 Total N soil input N removal SNB 264 187 281 114 138 65 70 95 79 57 57 140 76 109 51 46 29 23 67 49 24 27 67 54 63 47 81 24 35 11 13 23 19 110 107 107 107 81 75 64 69 93 67 61 356 272 314 240 228 154 143 170 186 137 127 179 149 166 135 132 87 72 106 122 80 67 176 124 147 105 94 66 66 65 64 56 59 tries mainly have hydrogeochemical causes like the presence of relatively deep soils, high groundwater tables and high organic matter contents (in part as peaty soils) promoting denitrification. Some areas in the UK have deep unsaturated extents through which the travel time for nitrate may be several decades (Wang et al., 2012). Analysis of lag times required for improvements of groundwater nitrate levels in Ireland showed that the achievement of good water quality status for some water bodies may be too optimistic but improvements are predicted within subsequent 6- and 12-yr cycles (Fenton et al., 2011). Analyzing a 50 yr time series of SNB and nitrate concentration in groundwater in Fig. 3. Percentage of groundwater samples in monitoring programs Denmark, −1 Figure 3. Percentage of groundwater samples in monitoring programs for the Nitrates Hansen et al. (2011) found that nitrate concentrafor the Nitrates Directive exceeding 25 mg NO3 L for the 2nd nd rd tions have been decreasing since 1980. They found that the and 3rd reporting * For Ger-(European Commission, Directive exceedingperiod 25 mg(European NO3/l for Commission, the 2 and 3 2011). reporting period frequency of downward nitrate trends in groundwater sammany only data for the agriculture monitoring network ** For the 2011). ples clearly increased with lower recharge age, providing reporting period 2000–2003 United Kingdom reported only stations within for Germany only agriculture monitoring network England. data Forfor thethe reporting period 2000–2003 Denmark proof that younger groundwater responds fastest to decreasprovided results. ingwithin trendsEngland. of SNB. Hansen et al. (2012) further found that ni for the aggregated reporting period 2000-2003 United Kingdom reported only stations trate concentration decreased significantly more in areas with *** for the reporting period 2000-2003 Denmark provided aggregated results a high livestock density. Reported nitrate concentrations in Germany are higher than in the other northwestern EU memstates, reports to the EU Commission of nitrate concentraber states because sampling is restricted to agricultural soils tions in groundwater exceeding a policy target (in this case and focused on polluted regions. Changes in monitoring prothe nitrate limit for drinking water) do not provide direct incedures and densities do not allow solid conclusions on nisight in the effectiveness of NiD action programs or in the trate trends between the 3rd and 2nd reporting period based impact of differences of nitrogen balances. This is perhaps on the total dataset of groundwater observations. However, most strikingly illustrated in the Netherlands where mean nithe overall picture appears to be that nitrate concentrations trate concentrations in groundwater are low (Fig. 3) while did not change between 2000 and 2007. In shallow groundthe GNB is highest (Figs. 1 and 2). In part differences in water, which responds most directly to NiD action programs, the nitrate response between reporting periods and between 60 % of all samples in the EU27 were below 25 mg NO3 L−1 , countries are artifacts of different monitoring procedures and and 20 % above the NiD target of 50 mg NO3 L−1 (European data selections. For example the apparent increase of niCommission, 2011). More insight into trends may be obtrate concentrations in Denmark and the Netherlands betained by selecting data for shallow phreatic groundwater ditween 2000–2003 and 2004–2007 in the EU dataset (Eurorectly from official national NiD reports for the Netherlands pean Commission, 2011) is an artifact of inclusion of obser(Zwart et al., 2008), the Flemish Region (Desimpelaere et vations in the uppermost groundwater in the 2004–2007 EU al., 2008), Walloon region, Ireland, Germany and Denmark dataset. But differences in the nitrate response between counBiogeosciences, 9, 5143–5160, 2012 www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe 5153 1 2 3 Fig. 4. Percentage of shallow phreatic groundwater samples in monitoring programs for the Nitrates Directive for the 3rd reporting peNitrates Directive forexceeding the 3rd reporting riod (2004–2007) 25 or period 50 mg(2004-2007) NO3 L−1 . exceeding 25 or 50 mgNO3/l. Figure 4. Percentage of shallow phreatic groundwater samples in monitoring programs for the 4 1 2 (Anonymous, 2008b, c, d, e), (Fig. 4). Here differences of 3 nitrate concentration between countries appear to be more in accordance with differences of the nitrogen balance (Fig. 1). 4 In countries with a long running monitoring network 5 for nitrate in the upper, sometimes shallow, groundwater in sandy phreatic aquifers (Fig. 5) a slow to moderate decrease of nitrate concentration can be observed. The mean decrease of the nitrate concentration in the monitoring period is largest in the Netherlands (6 mg NO3 L−1 per year), followed by Denmark (2 mg NO3 L−1 per year), Germany (0.6 mg NO3 L−1 per year), Flemish Region (0.7 mg NO3 L−1 per year) and finally the Walloon region with a small increase (0.3 mg NO3 L−1 per year). These trends do not only reflect the effect of the measures from implementation of the NiD, but also on changes in agricultural practices and effects of implementation of other policies, e.g., measures for reducing ammonia emission. Trends further depend on sampling depth and travel time of infiltrating water which differ spatially within countries and between countries. Observed nitrate exceedance in the period 2004–2007 (Fig. 4) and nitrate concentrations between 2005 and 2010 (Fig. 5), both in upper levels of phreatic groundwater, agree fairly well with modeled nitrate concentrations in leaching 43 water in 2008 using MITERRA (Figs. 6 and 7). Some level of disagreement is to be expected considering that nitrate concentrations in leaching water will tend to be higher than in groundwater, and that monitoring data are not always representative for nitrate concentration in total UAA. In Germany, observed concentrations are higher than MITERRA results in view of the intended focus of the monitoring program on areas with high nitrate concentrations (Anonymous, 2008d). MITERRA results for NUTS2 regions with mean area weighted nitrate concentrations exceeding 50 mg NO3 L−1 are found only in the Netherlands, the Flemish Region, the western part of Germany and in Brittany (Fig. 7). SNB values exceeding 100 kg N ha−1 in regions in the UK and Ireland (Fig. 2) do not lead to exceedance of the nitrate target www.biogeosciences.net/9/5143/2012/ Fig. 5. Trend of nitrate concentrations in upper levels of phreatic groundwater in sandy soils, catchments or aquifers in monitoring soils, catchments or aquifers monitoring programs theFraters NitratesetDirective (Data tak programs for the NitratesinDirective (Data taken for from al., 2011). from Fraters et al. 2011). Figure 5. Trend of nitrate concentrations in upper levels of phreatic groundwater in sand of the NiD as a result of relatively low nitrate leaching fractions in these regions. However, the risk of exceedance of ecological limits for nitrate or nitrogen in surface water will be higher in regions with high SNB. The EU Water Framework Directive gives room to member states to define and differentiate national standards for good ecological status or potential. A nitrate limit concentration of 10 mg NO3 L−1 (2 mg N L−1 ) was used as a proxy for the nitrate limit in fresh waters (Cardoso et al., 2001). Surface waters with mean nitrate concentration greater than 10 mg NO3 L−1 ranged from 20 % in Ireland to 60 % in Germany (Fig. 8). Between 2000 and 2007 the percentage of surface water samples exceeding 10 mg NO3 L−1 shows a small decrease, when looking to the total population of fresh surface water samples reported to the EU Commission (Fig. 8). Differences between countries do not seem to have a clear relation with observed exceedance in groundwater. Again, in part these differences reflect different response mechanisms and response times and nitrate attenuation during transport from groundwater to surface water (Fenton et al., 2009). 44 will be less than for However, differences in response time deeper groundwater bodies. In particular, response of surface water nitrate to restrictions on how and when to apply manure and fertilizer (Table 6) should be faster, due to the shorter transport pathways compared to deeper aquifers, while full response to restrictions on application levels may take decades. Biogeosciences, 9, 5143–5160, 2012 5154 1 2 3 4 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe 1 Fig. 8. Percentage of surface water samples in monitoring programs Fig. 6. Mean nitrate concentration (UAA and precipitation surplus 2 for Figure Percentage of surface water10 samples the 8. Nitrates Directive exceeding mg NOin3 monitoring L−1 for theprograms 2nd and for the Nitrat Figure 6. Mean nitrate concentration (UAA and precipitation surplus weighted) in leaching weighted) in leaching water from agricultural soils in northwestern nd rd 3rd reporting period (European Commission, 2011). * NO 3 Directive exceeding 10 mgNO /l for the 2 and 3 reporting period (European Com 3 data for 3 water soils inmodel. northwestern EU in 2008 by MITERRA model. EU infrom 2008agricultural by MITERRA 2000–2003 were not available. 4 2011). 5 *NO3 data for 2000-2003 were not available 6 1 2 3 4 view of the similar yield potentials. However, it is difficult to compare fertilizer recommendations as different countries apply different systems (ten Berge and van Dijk, 2009). The Flemish Region, Denmark and the Netherlands use systems based on dose–effect trials, while Germany and France use a balance approach. All countries use calculation schemes to correct N recommendations for yield level and N deliveries from soil, and cropping history and manure application. These schemes are not standard, and may depend on the local advisors, which leads to significant variability in the recommendations. In general nitrogen application standards in NiD action programs for Denmark and for fodder maize on dry sandy soils in the Netherlands tend to be lower than the Nfertilizer recommendation. In the Danish case the legal application standards are now 10 % under the economic optimum for all crops. With the recently introduced standards, this is partly also the case for the Flemish Region. The overall effects of these differences on the N balance and on water quality are difficult to judge, as standards are implemented at farm level and crops are cultivated in rotations. Denmark has far less permanent grassland than the Netherlands and grassland contains more clover while temFigure 7. Mean nitrate concentration in leaching water from the root-zone in 2008 atporary NUTS2grassland is part of the crop rotation. Such differences level7. byMean the MITERRA model. 45 water from the rootFig. nitrate concentration in leaching in rotations to some extent may level out environmental efzone in 2008 at NUTS2 level by the MITERRA model. fects of differences between standards for individual crops. A more elaborate analysis is needed to assess whether differ47 ences in recommendations between countries are justified in 4 Discussion economic terms, and whether differences in application standards are justified from the environmental viewpoint. This is 4.1 Application standards beyond the scope of our contribution. The theoretical or empirical basis of differences between nitrogen application standards in national regulations for NiD implementation in northwest European countries is not always clear (Table 8). Differences between standards to a large extent derive from differences in fertilizer recommendation in the northwestern members states (Table 11). One may expect more comparable fertilizer recommendations in 4.2 Nitrogen balance There are considerable differences between estimates of GNB in EUROSTAT, by MITERRA and in national reports (Table 12). Precise comparison of results for GNB was difficult because results were not always available for the same years and because data underlying GNB for a specific 46 Biogeosciences, 9, 5143–5160, 2012 www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe 5155 Table 11. Ranges of N recommendations in different regions for sandy to loamy soils with no effect of previous crop and a medium level of soil nitrogen supply (SNS). Relatively high N-recommendations are found in The Netherlands and Denmark, relatively low values in France and the UK (sources: Dijk and Berge, 2009; for FL Bodemkundige Dienst van België, 2012; for UK DEFRA, 2010 for FR COMIFER, 2011; for IRL Coulter and Lalor, 2008). NL DK FL GE FR UK IRL1 50–2502 70–160 10–210 100–160 100–140 180–340 50 70–120 60–160 80 40–3062 110–180 120–2103 120–170 120–195 kg N ha−1 Grass Fodder maize Winter wheat Potato – ware Sugar beet 285–385 150–175 190–230 245–250 150 365–405 160–190 180–210 155–180 125–150 250–300 150–175 150–190 200–225 130–160 200–300 150–160 130–220 70–140 90–150 1 Rates shown for non-grassland correspond to a soil N Index range of 1 to 3. 2 Rates of N application on grassland vary depending on stocking rate and usage for grazing and/or cutting. 3 Assuming 9 t ha−1 yield of winter wheat (additional N is recommended for higher yields). Table 12. Annual N removal, and gross N balance (GNB) by MITERRA in 2008, compared to values in Eurostat and national reports in the period 2004–2009. MITERRA 2008 UAA mln ha removal GNB EUROSTAT 2005–2008 National 2004–2009 removal removal kg N ha−1 EU27 Belgium Flemish R 172.5 1.4 0.7 67 149 166 70 156 200 Walloon R Denmark France North. France Brittany Germany Ireland Netherlands United Kingdom 0.7 2.5 30.1 17.8 1.6 16.7 4.1 1.9 14.3 135 106 80 87 89 122 132 179 72 122 82 67 79 215 81 108 213 84 GNB kg N ha−1 kg N ha−1 93 49 1911 213–2232 2201 1631 1153 1204 1171 572 631 571 793 504 92 50 188 93 138 1316 155 2097 1378 795 916 53 1787 918 191 118 101 112 125 155 194 111 GNB 1 Gybels et al., 2009, for period 2004–2006. 2 Lenders et al., 2012, for period 2007–2009. 3 Grant et al., 2010, period 2006–2008. 4 Anonymous 2008a, period 2004–2006; GNB inferred from SNB using gaseous N loss by MITERRA. 5 Agreste, 2012; mean for 2006, 2008 and 2012. SNB value converted to GNB using gaseous N loss by MITERRA (48 kg N ha−1 ). 6 Anonymous, 2008c, period 2004–2006. 7 CBS statline, http://statline.cbs.nl, downloaded January 2012. 8 Fernal and Murray, 2009, period 2005–2007. year are regularly modified. GNB for 2008 calculated by MITERRA is on average 19 kg N ha−1 higher than reported to the EU Commission (EUROSTAT) and to a lesser extent than reported by the OECD (Velthof et al., 2009). Differences are most marked for Belgium and Ireland. N removal and, to a lesser extent, N excretion (not shown) are major sources of difference between GNB estimates. National use of chemical fertilizer in general is fairly accurate, but values for specific years in national reports, e.g. Belgium, show quite some variation, and in part reflect the absence of reliable registration www.biogeosciences.net/9/5143/2012/ systems for fertilizer purchase. Different estimates of UAA play a minor role. On average, estimates of N removal in MITERRA (2008) for the seven member states are 22 kg N ha−1 lower than estimates for EUROSTAT (2005–2008) and could fully account for the mean difference of GNB (Table 12). Estimates in national reports for some countries tend to be somewhat higher than values reported to EUROSTAT, but this in part may be due to comparing different periods. The uncertainty of N removal in crops is further illustrated by results from Leip et Biogeosciences, 9, 5143–5160, 2012 5156 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe al. (2008), that were on average nearly 28 kg N ha−1 higher than in EUROSTAT, using a more deterministic European model approach. N removal from grassland for fodder likely is the major source of difference in estimates of total N removal (Velthof et al., 2009). MITERRA excretion (2008) on average is 7 kg N ha−1 higher than in EUROSTAT (2005– 2008). For the Flemish Region Lenders et al. (2012) estimate N removal at about 320 kg N ha−1 based on grassland yields of 10.5 ton ha−1 for permanent grassland and 11.5 ton ha−1 for temporary grassland, and an N content of 3 %. MITERRA estimates N removal from permanent grassland at about 220 kg N ha−1 . Differences are caused by lower estimates of effective dry matter yield for mixed system of grazing and cutting, and of lower N contents. Estimates of mean N removal from grassland in the Netherlands, with practices and N intensity comparable to that in the Flemish Region, are around 260 kg N ha−1 . So overestimation of N removal from grassland (36 % of UAA) could explain a major part of the difference between GNB estimates by MITERRA and national reports. GNB in 2008 by MITERRA for Brittany in France is more than twice the regional estimate for 2006–2010 (Agreste, 2012). Again this can be largely (> 50 %) explained by a much higher regional estimate of N removal, and to lesser extent by lower estimates of manure input (about 20 %) and chemical fertilizer (about 10 %). Regional data would suggest an overall nitrogen use efficiency (N removal over total N input from fertilizer and manures) of 80 %, which does not seem realistic. Nitrogen use efficiency in Brittany by MITERRA is about 40 %, as compared to 60 % for EU27. For Ireland, total N removal in MITERRA in 2008 is 23 kg N ha−1 lower than the average N removal between 2005 and 2008 in EUROSTAT and national reports. In Ireland 3.9 mln ha of UAA (95 %) is grassland. Mean N removal on grassland is estimated for EUROSTAT at 155 kg N ha−1 , while MITERRA calculates about 130 kg N ha−1 . Part of this difference may be due to different assumptions on reduction of yields and N removal for grazing as compared to cutting, and to different assumptions on shares of intensively and extensively managed grassland. Differences in N removal per hectare between intensive and extensive grassland can amount to a factor of two (Velthof et al., 2009). Another major source of discrepancy for Ireland between MITERRA results and national reporting is a higher gross input of N in manure. In Ireland almost 90 % of N production in manure is from cattle. Irish national reports use an N excretion value of 85 kg N per dairy cow (Anonymous, 2010), while MITERRA uses a value of 105 kg N per dairy cow (Velthof et al., 2011; Annex 1). The high value is based on a more dynamic approach accounting for regional differences in milk yields, grassland yields, and N inputs, while the low value is mainly a function of milk yield. Estimates of N removal for fodder and N excretion are related, as fodder is the major N input and manure N is the major output. For Ireland, N removal in Biogeosciences, 9, 5143–5160, 2012 EUROSTAT (and national reports) is more than 30 % higher than N excretion. Even when taking into account N removal in milk and meat and N imports of feed concentrates, the large difference between N removal and N excretion may be an indication that either N removal is overestimated or N excretion is underestimated. On the other hand, excretion estimates by MITERRA do not seem to match with a relatively modest average milk yield in Ireland around 5000 kg per cow per year. Germany is the only country that has established targets for the surplus of N (90 kg ha−1 for 2006–2008) and phosphate (20 kg ha−1 in a six-year average); and managed to achieve these targets in 2008. The stricter targets of 60 kg N ha−1 as a three-year average from 2009–2011 onwards may also be achieved, but some intensive livestock farms and other farms with higher N surplus still have to increase their N efficiency. Infringements of these restrictions are not directly subject to fines, but will lead to administrative procedures with increasing obligations for farmers to adapt to the maximum surplus levels. Recent national census data indicate that since 2008 the use of chemical fertilizer in Denmark, Germany and the Netherlands is still decreasing, and along with that, probably also the soil surplus of nitrogen. The decrease of the purchase of chemical N fertilizer coincides with the increase in fertilizer prices since 2008 (Fig. 9). This price increase is not compensated by an increase of prices of agricultural commodities. Between 1990 and 2011 the price of nitrogen fertilizer in Europe has increased twice as fast as the price of wheat, but since 2007 both prices have become very volatile. In view of the high fertilizer prices farmers may tend to reduce or postpone fertilizer purchases. The latter hypothesis is supported by a decrease of purchase of chemical N fertilizer in Germany in 2009 and 2010. In Denmark and the Netherlands the purchase of N fertilizer was hardly affected, which can be explained by the presence of legal N application standards that are below the economic optimum. So changes of nitrogen use and surpluses since 2008 in part can be price effects which interfere with effects of the NiD. This price effect is more apparent for the use of inorganic phosphate fertilizer which increased since 2009 in all three countries. 4.3 Implications for the NiD Monitoring and evaluation of the implementation and effects of NiD is crucial for its success. At a national level it is a requirement to maintain support from farmers and their local advisors, as the main actors involved, and for national governments to optimize policies. The main activities for monitoring and evaluation are registrations of farm resources and activities (fertilizer, livestock, UAA), monitoring of water quality and using calculation procedures and models to assess environmental loads and relate this to farm measures and water quality. These evaluation activities take place at the national level, with varying levels of detail and sophistication, www.biogeosciences.net/9/5143/2012/ H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe and in a more harmonized and generalized manner at the European level. For the latter, the European Commission uses institutes like the European Environment Agency (EEA) and the Joint Research Centers (JRC) and has initiated various service contracts, to improve datasets of agricultural activities, and develop and apply models to relate activities to N emissions and water quality (RAINS, GAINS, CAPRI, MITERRA). In spite of recent progress it is difficult to judge to what extent national implementation and evaluation of the NiD benefits from joint activities and what are major caveats in data and knowledge about the effects and effectiveness of the NiD. A typical conclusion from national evaluations is that the1 NiD has made a major contribution to reduction of the N sur-2 plus. Evaluation of the Danish Aquatic Plan II concluded that3 between 1998 and 2004 the reduction of N application stan-4 dards contributed 13 mln kg (32 %) to the total reduction of5 the soil N surplus (SSNB) of 80 mln kg, while increasing le-6 gal FE for N in manure contributed 10 mln kg (26 %) and re-7 duced N in feeding 4 mln kg (10 %) (Mikkelsen et al., 2010).8 Evaluation of the Dutch second action program concluded that between 1998 and 2004 the Mineral Accounting System (MINAS) led to an overall reduction of the net SSNB by 78 mln kg N (van Grinsven et al., 2005). Here the combination of reducing N-loss standards, and more efficient N management by better insight from keeping mineral accounts at farm level, contributed about 100 mln kg (67 %), while reduced N in feeding contributed 14 mln kg (19 %) and reducing livestock and increasing manure export 11 mln kg (14 %). In the Netherlands the dairy sector contributed most to reduction of the use of chemical fertilizer, and this reduction was both a learning effect of applying mineral accountancy at farm level and of enforcement of N-loss standards. In spite of various efforts at the European level to streamline procedures for monitoring and evaluation of the NiD, implementation and insight into the effectiveness still vary considerably. A first logical step is to further harmonize procedures for monitoring water quality and for assessing the nitrogen balance, while recognizing country specific monitoring needs to, for example, show the effectiveness of specific measures in an Action Program (Fraters et al., 2011). Another major source of difference among member states is how manure N is taken into account in recommendations as well as in the regulation of allowable N input. Nitrogen emissions from agricultural sources, particularly manures, are a major source of environmental pollution and welfare loss (Sutton et al., 2011). A logical next step for improving harmonization and effectiveness of the NiD is to demand stricter accounting of nitrogen in manures, e.g. by imposing a compulsory time path for increasing nitrogen fertilizer equivalencies for different types of manures in application limits (Csathó and Radimszky, 2009). However, such steps require knowledge sharing, e.g. in defining codes of Good Agricultural Practice and adopting techniques to improve nitrogen efficiency in manures. Without that, a too fast and too strict regulation of www.biogeosciences.net/9/5143/2012/ 5157 Fig.9.9.Trends Trends 1990 nitrogen and Figure sincesince 1990 of pricesofof prices nitrogenof fertilizer and offertilizer wheat in the EU,ofand trends wheat in the EU, and trends of total use of inorganic nitrogen fertilizer in agriculture in Germany (http://www.bmelv-statistik.de; N statistik.de; N fertilizer use in 1990 was 130 kgN/ha), Denmark (http://www.statbank.dk/; N fertilizer use in 1990 was 130 kg N ha−1 ), Denmark (http://www. fertilizer use in 1990 was 150 kgN/ha) and The Netherlands (http://statline.cbs.nl/StatWeb/; statbank.dk/; N fertilizer use in 1990 was 150 kg N ha−1 ) and the N Netherlands fertilizer use in (http://statline.cbs.nl/StatWeb/; 1990 was 220 kgN/ha) (downloaded October 31, 2012). N fertilizer use in 1990 −1 Note: MacSharry in 1992 and later reduced theNote: price support for cereals wasThe 220 kg N ha reform ) (downloaded 31 reforms October 2012). the MacSharry reform 1992 and later reforms reduced the price support and therefore also theinprice of wheat. for cereals and therefore also the price of wheat. of total use of inorganic nitrogen fertilizer in agriculture in Germany (http://www.bmelv- nitrogen in manures may decrease the willingness of arable farmers to accept manure from livestock farmers, because of fear of insufficient N supply. In the future, increasing prices of nitrogen fertilizer may provide an additional economic incentive to reduce the use of chemical fertilizer and to increase the efficiency of manures. The NiD and the national implementation of restrictions on where, when and how much nitrogen in fertilizer and manure can be applied to agricultural land, will remain a major instrument to reduce nitrogen pollution in waters. However, we should also recognize that agricultural sources of nitrate are only part of the nitrogen burden. In 2005, diffuse agricultural sources in the EU on average contributed 55 % to the N load to surface waters,48the remainder coming from communal, industrial and natural sources. The agricultural shares for northwest European countries tend to be higher, ranging from 50 to 60 % in the UK, Germany, France and Belgium to 70–85 % in the Netherlands, Denmark and Ireland (inferred from Bouraoui et al., 2011). So even when all the measures under NiD have taken hold, it is unlikely that nitrate concentrations in surface water, and to a lesser extent in groundwater, will return to pre-industrial levels (Howden et al., 2011). For the immediate future the importance of the NiD for protecting drinking water may be best seen in those areas with private or small public drinking water facilities, using groundwater from shallow aquifers, as is the case in Denmark (van Grinsven et al., 2010). In order to protect their coastal waters, member states in deltas or estuaries of large cross boundary rivers, like the Netherlands and Romania, depend on the NiD, particularly when national implementation of the Water Framework Directive is limited to reducing nonagricultural sources of N. A problem when implementing the Biogeosciences, 9, 5143–5160, 2012 5158 H. J. M. van Grinsven et al.: Benchmarking the Nitrates Directive in northwestern Europe NiD for this purpose is that the limit value of 50 mg L−1 does not apply to fresh waters and coastal waters (Nimmo Smith et al., 2007). Nonetheless, the NiD requires member states to protect such bodies at risk of eutrophication. The lack of a single standard along with the range of influences that bear on eutrophication can cause some confusion. For control of coastal eutrophication, e.g. in Brittany, a limit value around 5–10 mg NO3 L−1 would be more appropriate. 5 Conclusions The most significant effect of the implementation of the NiD since 1995 in the northwest of the EU is a major contribution to the decrease of the nitrogen soil N balance and by that of the gross N load to the aquatic environment. This effect of the NiD has not yet manifested in a convincing decrease of nitrate concentrations in EU monitoring in groundwater and fresh surface waters since 2000. However, before 2000, introduction of Good Agricultural Practices for fertilization has decreased median and extreme nitrate concentration in many surface water systems in e.g. the Netherlands, Denmark and the Flemish Region. Only countries that operate long running monitoring programs in shallow groundwater in agricultural areas, viz. Denmark and the Netherlands, can detect a convincing decrease of nitrate concentrations. Without good opportunities to evaluate the effectiveness of NiD, it is difficult for the EU community to improve the NiD and implementation in member states may lose momentum. This benchmark study indicates that differences in calculation and data procedures between member states in northwestern EU for determining the nitrogen balances are such that comparison of effects of NiD on the N balance between countries is not yet possible. In particular the calculations methods for N excretion and N removal vary considerably among countries. Regarding compliance with application limit for N in manure also the definition of farm area differs between countries ranging from total farm area to the area where manure actually is applied. Harmonization of the rationale of national fertilizer recommendation systems is important for deriving N application standards that can lead to balanced fertilization, as required by the NiD, and eventually to create a transparent policy debate about balancing economic and environmental goals across the EU. Improved guidelines and procedures for monitoring water quality and registration of fertilizer use also would improve the evaluability of the NiD. Better selections of, and access to the collective monitoring results in EU synthesis reports and data facilities can help to improve the efficiency of our monitoring effort to evaluate the NiD. Implementation of the NiD in member states in the northwest of the EU is fairly comparable regarding restrictions for application of fertilizer and manure, but can be quite different regarding application standards for total N fertilization. Nitrogen application standards in national implementations Biogeosciences, 9, 5143–5160, 2012 of the NiD are closely linked to national nitrogen fertilizer recommendations. However, differences in national systems for nitrogen recommendations are substantial and resulting recommendations for specific combination of crops and soils and do not bear a clear relationship with differences in yield per hectare. At some point in the future, when the first and relatively easy environmental improvements by the present implementations of NiD are achieved, the NiD may need adjustment to become more effective, notably through more specific regulation of nitrogen in manure and through differentiation of targets with respect to water quality. This will also help to achieve the targets set in the Water Frame Work Directive. However, there is an immediate need to improve our data procedures to allow evaluation and benchmarking of adequacy and effectiveness of NiD implementation. Edited by: S. 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https://openalex.org/W3033448535	https://europepmc.org/articles/pmc7283349?pdf=render	English	null	Combining Physiological and Neuroimaging Measures to Predict Affect Processing Induced by Affectively Valent Image Stimuli	Scientific reports	2,020	cc-by	9,698	www.nature.com/scientificreports www.nature.com/scientificreports OPEN Kayla A. Wilson, G. Andrew James, Clint D. Kilts & Keith A. Bush ✉ The importance of affect processing to human behavior has long driven researchers to pursue its measurement. In this study, we compared the relative fidelity of measurements of neural activation and physiology (i.e., heart rate change) in detecting affective valence induction across a broad continuum of conveyed affective valence. We combined intra-subject neural activation based multivariate predictions of affective valence with measures of heart rate (HR) deceleration to predict predefined normative affect rating scores for stimuli drawn from the International Affective Picture System (IAPS) in a population (n = 50) of healthy adults. In sum, we found that patterns of neural activation and HR deceleration significantly, and uniquely, explain the variance in normative valent scores associated with IAPS stimuli; however, we also found that patterns of neural activation explain a significantly greater proportion of that variance. These traits persisted across a range of stimulus sets, differing by the polar-extremity of their positively and negatively valent subsets, which represent the positively and negatively valent polar-extremity of stimulus sets reported in the literature. Overall, these findings support the acquisition of heart rate deceleration concurrently with fMRI to provide convergent validation of induced affect processing in the dimension of affective valence. The importance of affect processing to human behavior has long driven researchers to pursue its measurement. Affect processing impacts all facets of an individual’s life, from their emotional responses to their perceptual processing1 and affect processing responses have been measured using a variety of modalities, from analyses of self-report to neuroimaging. However, in the past researchers have been forced to choose a single modality, thus constraining the interpretation of affective responses2. With more recent focus on validity and generalizability, researchers have begun measuring affect processing according to multiple, independent modalities3. With hopes to add to this literature, this study examines the simultaneous measurement of perceived affective valence by both neuroimaging and physiology. Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 Materials and Methods Study overview. We analyzed data acquired from two studies that share a common image set and order- ing of tasks: the Intrinsic Neuromodulation of Core Affect (INCA) study and the Cognitive Control Theoretic Mechanisms of Real-time fMRI-Guided Neuromodulation (CTM) study. Both of these studies are functional neuroimaging explorations of affect perception, unguided affect regulation, and real-time fMRI-guided volitional affect regulation. Details of the relevant tasks have been previously reported in Bush et al.30. To aid study rigor and reproducibility, we have elaborated below the experimental details for the combined studies related to their task design, image selection algorithm, and data processing methodologies. All study procedures were conducted in the Brain Imaging Research Center (BIRC) at the University of Arkansas for Medical Sciences (UAMS). All participants provided written informed consent. All study proce- dures were conducted with approval and oversight by the UAMS Institutional Review Board in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations. Participation demands were nearly identical across both studies, occurring in two sessions on separate days, save the following differences. CTM included the Perceived Invalidation of Emotion Scale (PIES) assessment as well as facial EMG measurement concurrent with fMRI. INCA required a negative urine screen prior to day two activities while CTM required a negative urine screen prior to day one and day two activities. During Session 1, participants provided written informed consent, were screened for clinically relevant exclusionary criteria via structured clinical interview (SCID-I/NP), and completed behavioral surveys and questionnaires. Neuroimaging was conducted during Session 2 and lasted approximately 1 hour. This analysis includes only data acquired during the first two functional image acquisitions (i.e., scans) of the session, comprising the System Identification Task (detailed below).h The authors have made the source code used to conduct this analysis publicly available at: https://github.com/ kabush/HR. Participants. The participant sample (n = 50) used for this analysis completed the System Identification Task within either the INCA (n = 19) or CTM (n = 31) studies. The sample had the following demographic character- istics: age [mean(s.d.)]: 33.7(13.6), range 18‒63; sex: 29 (58%) female; race/ethnicity: 43 (86%) self-reporting as White or Caucasian, 6 (12%) as Black or African-American, 1 (2%) as Hispanic or Latino; education [mean(s.d.)]: 16.5(2.8) years, range 12‒23; WAIS-IV IQ [mean(s.d.)]: 106.6(13.8), range 74‒137. Aims of Present Studyh y The aim of this study is to extend the comparison between physiological (i.e., heart rate change) and neural acti- vation measurements of affective valence induction. Specifically, we perform this comparison on a stimulus set representing a broad continuum of conveyed affective valence, i.e., including a range of neutral and weakly valent stimuli in addition to the polar-extremes of positive and negative valent stimuli as have been commonly reported in the literature. Heart Rate Change as a Measure of Affective Valence gf Heart rate deceleration (also termed HR change and abbreviated throughout this work as ΔHR) is calculated as the difference in heart rate from a baseline value, which can vary by experiment. Recent literature has shown a correlation between HR change, measured relative to the time of affective stimuli, and the valence ratings of those stimuli. Specifically, negatively valent stimuli have been associated with immediate, dramatic deceleration in heart rate7,13–18. In contrast, positively valent stimuli have been associated with a triphasic trajectory of HR change, con- sisting of an immediate deceleration followed by a slight acceleration before decelerating again14,15,17,38. Psychophysiological and behavioral measures of affect processing y p y gf p g Multiple measures exist that detect weak, but reliable, physiological and behavioral responses to affectively coded stimuli. For example, the galvanic skin conductance response (SCR) has been widely employed as a measure of autonomic arousal responses to affectively laden stimuli4–6. Meanwhile, activity in the zygomaticus (smile expression) and corrugator (frown expression) muscles, detected via facial electromyography, has been linked, respectively, with observations of positively and negatively valent stimuli7–12. Similarly, physiological responses to positively and negatively valent stimuli have been characterized by variations in heart rate deceleration following stimulus onset7,13–18. Due to the variation in the dimensions of affect that these psychophysiological measures detect, the use of multiple modalities would contribute to a broader understanding of affective processing. Brain Imaging Research Center, University of Arkansas for Medical Sciences, Little Rock, USA. ✉e-mail: kabush@ uams.edu Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Linking Neural Processing Correlates of Affect Processing with Psychophysiological Measuresh The advent of functional magnetic resonance imaging (fMRI) of blood oxygen-level dependent (BOLD) sig- nal acquisitions enabled the inference of functional neuroanatomical circuit responses to affective stimuli19–25. However, a critical methodological step in these experiments, often omitted, is the verification of affect induction through independent measurement. To this end, patterns of neural activation have been compared to both car- diac responses to categorical emotional faces26 and skin conductance responses during reappraisal of negative affectively valent stimuli27. Heller et al.3 demonstrated that concurrent recording of facial EMG could objectively measure the affective valence induced by image stimuli when reporting functional neuroanatomical activations.f f y g p g More recently, multivoxel pattern analysis (MVPA) has been proposed as a means of characterizing affect induction. MVPA of BOLD signal28 was shown to accurately predict the affective content of previously unseen stimuli, based solely on the temporally succinct neural activation patterns that represent specific cognitive pro- cesses within the brain29. Indeed, growing empirical evidence describes reproducible30, distributed30–33, and predictive29,30,33–35 neural activation patterns that are central to affect processing31–33. Indeed, the power of neu- ral activation patterns to predict affect processing in human subjects, particularly in un-tasked experimental domains lacking ground truth36,37, calls for rigorous validation of MVPA as a measure of affect induction against long-established measurement standards rooted in physiology and behavior. To address this need, a recent study by the authors35 validated affective arousal prediction efficacy of MVPA models versus the galvanic skin conduct- ance response (SCR). Ideally, validation of MVPA predictions would extend to the orthogonal affective dimension of valence. With valence shown to be well-characterized by heart rate deceleration7,13–18, and, as heart rate may be recorded via photoplethysmogram (PPG), an optical instrument that minimally interacts with the MRI envi- ronment, PPG offers a reliable, widely-used, measure of heart rate that may be acquired concurrently with MRI. Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 Materials and Methods (D,E) PPG processing: PPG signals were processed using Kubios 3.1.0 to detect beat times. (F) Beats were assigned to pre- and post-stimulus intervals, then inter-beat intervals (IBIs) were computed between successive beats and assigned to the time point of the second beat. (G,H) Pre- and post-stimulus IBIs were combined to construct a heart rate change time course with respect to the mean pre-stimulus heart rate. (J) Group-mean HR change time courses were computed for each stimulus and the overall post-stimulus peak deceleration, tpsmax, is determined. HR change at tpsmax of each time course was then combined with machine learning predictions as fixed effects of a general linear mixed-effects model (GLMM) where random slope and intercept effects were modeled subject-wise. interview (APA, 1994)), no current reported usage of psychotropic medication, and produced a negative urine screen for drugs of abuse (cocaine, amphetamines, methamphetamines, marijuana, opiates, and benzodiazepines) immediately prior to the MRI scan. Additionally, all participants’ vision was corrected to 20/20 during the MRI scan using a lens system and color-blindness was exclusionary. interview (APA, 1994)), no current reported usage of psychotropic medication, and produced a negative urine screen for drugs of abuse (cocaine, amphetamines, methamphetamines, marijuana, opiates, and benzodiazepines) immediately prior to the MRI scan. Additionally, all participants’ vision was corrected to 20/20 during the MRI scan using a lens system and color-blindness was exclusionary. System identification task. The purpose of this task was to induce affect processing concurrently with fMRI and psychophysiological signal acquisition for subsequent construction, independent validation, and comparison of predictive measures of affect processing. Specific details of this task were previously reported in Bush et al.30 with the following summary of the task design provided for experimental context. Ninety image stimuli were selected from the International Affective Picture Set (IAPS) to maximally span the normative affect representa- tion subspace. Image stimuli were presented for 2 s (stimulation) followed by a visual fixation cross presented for a random inter-trial interval (ITI) sampled uniformly from the range of 2–6 s. IAPS image presentations were balanced across two 9.25 min scan runs according to the images’ normative valence and arousal scores in order to minimize the correlation of general linear model regressors that would be used to identify their functional neural activations by post-hoc analysis. The image order was then fixed for all participants. A graphical representation of the image stimuli and their presentation scheme is depicted in Fig. Materials and Methods All participants were right-handed, native-born United States citizens (a requisite condition for applying IAPS normative scores), med- ically healthy, without current Axis I psychopathology including mood disorders (assessed via SCID-IV clinical Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Figure 1. Methodological Overview. Experiment Design: (A) Ninety images drawn from the International Affective Picture System (IAPS) were presented for 2 s each interleaved with random inter-trial intervals uniformly randomly sampled on the range of 2–6 s during concurrent recording of both fMRI BOLD and photoplethysmogram (PPG) signals. fMRI processing: (B) fMRI BOLD signal was preprocessed to remove noise and motion artifacts, transformed into whole-brain neural activation patterns via General Linear Modeling according to the beta-series method43, and then applied to predict normative valence scores accordin to intra-subject leave-one-out cross validated linear support vector machine learning as reported previously by Bush et al.35. (C) Raw PPG signal was captured at 2000 Hz from the left index finger. (D,E) PPG processing: PPG signals were processed using Kubios 3.1.0 to detect beat times. (F) Beats were assigned to pre- and post-stimulu intervals, then inter-beat intervals (IBIs) were computed between successive beats and assigned to the time point of the second beat. (G,H) Pre- and post-stimulus IBIs were combined to construct a heart rate change time course with respect to the mean pre-stimulus heart rate. (J) Group-mean HR change time courses were computed for each stimulus and the overall post-stimulus peak deceleration, tpsmax, is determined. HR change at tpsmax of each time course was then combined with machine learning predictions as fixed effects of a general linear mixed-effects model (GLMM) where random slope and intercept effects were modeled subject-wise. Figure 1. Methodological Overview. Experiment Design: (A) Ninety images drawn from the International Affective Picture System (IAPS) were presented for 2 s each interleaved with random inter-trial intervals uniformly randomly sampled on the range of 2–6 s during concurrent recording of both fMRI BOLD and photoplethysmogram (PPG) signals. fMRI processing: (B) fMRI BOLD signal was preprocessed to remove noise and motion artifacts, transformed into whole-brain neural activation patterns via General Linear Modeling according to the beta-series method43, and then applied to predict normative valence scores according to intra-subject leave-one-out cross validated linear support vector machine learning as reported previously by Bush et al.35. (C) Raw PPG signal was captured at 2000 Hz from the left index finger. Materials and Methods These neural activations served as features on which to conduct MVPA as depicted in Fig. 1, panel B. Heart rate acquisition and preprocessing. We recorded psychophysiological measures of heart rate using a BIOPAC MP150 Data Acquisition System (BIOPAC Systems Inc., Goleta, CA) combined with the AcqKnowledge software and TSD200-MRI pulse photoplethysmogram (PPG), which has been shown to detect inter-beat intervals with accuracy comparable to electrocardiogram44. The PPG was placed on the left index fin- ger. Respiration and skin conductance response were also recorded for all subjects. For those subjects that were part of the CTM study (n = 31), facial electromyography was concurrently recorded. This work explores the PPG data only.t y Using Kubios heart rate variability analysis software (version 3.1.0), we processed all of the raw PPG signals using the automatic correction parameters for artifact removal and exported the processed data as a Matlab structure (Fig. 1, Panel D). We then extracted the heart beat times detected by Kubios from this structure for the remaining analysis (Fig. 1, panel E). Heart rate change analysis. As there is not a consensus methodology by which to analyze heart rate change induced by affective image stimuli, we extracted from the literature a set of thematic methodological elements that mapped onto our experiment design, allowing us to first reproduce and then extend prior work. Prior research has often focused on the comparison between distributions of HR changes induced by subsets of IAPS images representing the polar-extremes of positive and negative valence. Prior methods of analysis have also centered on the time course of HR change post-stimulus onset. HR change, typically, is measured in units of deceleration from rest where the resting heart rate is estimated over a time period occurring immediately prior to stimulus onset or from a separate resting state portion of the experiment7,17,18. HR change is also often reported in units of average beats per unit time (e.g., beats per minute, bpm), which is the inverse of the inter-beat interval (IBI), the time elapsing between successive heart beats. Our methodology has attempted to honor all of these methodological conventions while remaining grounded within the principles of data-driven prediction-based analysis. HR change time course construction. We computed time courses of HR changes with respect to the System Identification Task’s inter-trial intervals (brief periods of rest uniformly randomly sampled from the range 2–6 s). Materials and Methods 1, panel A. MR image acquisition and preprocessing. We acquired all imaging data using a Philips 3 T Achieva X-series MRI scanner (Philips Healthcare, Eindhoven, The Netherlands) with a 32-channel head coil. MR acqui- sition parameters for both anatomic and functional images used for both INCA and CTM studies have been reported previously in Bush et al.30. Standard image correction and preprocessing pipelines, previously detailed in Bush et al.30, were applied to both anatomic and functional images. In brief, we acquired anatomic images with a MPRAGE sequence (matrix = 256 × 256, 220 sagittal slices, TR/TE/FA = 8.0844/3.7010/8°, final resolu- tion = 0.94 × 0.94 × 1 mm3). We acquired functional images with EPI sequence parameters: TR/TE/FA = 2000 ms/30 ms/90°, FOV = 240 × 240 mm, matrix = 80 × 80, 37 oblique slices, ascending sequential slice acquisition, slice thickness = 2.5 mm with 0.5 mm gap, final resolution 3.0 × 3.0 × 3.0 mm3. We processed all MRI data Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ using AFNI (Version AFNI_18.2.06)39 unless otherwise noted. Anatomical data were processed via skull strip- ping, spatial normalization to the icbm452 brain atlas, and segmentation via FSL40 into white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF). Functional images underwent the following processing sequence: despiking; slice-time correction; deobliquing; motion correction; transformation to the spatially normalized ana- tomic image; regression of the mean timecourse and temporal derivative of the WM and CSF masks as well as a 24-parameter motion model41,42; spatial smoothing (6-mm FWHM Gaussian kernel); and scaling to percent sig- nal change. Gray matter (GM) masks for each subject were created using anatomical segmentation. A group-level GM mask was constructed incorporating only GM voxels present in ≥50% of the individual subject GM masks. The group-level GM mask was used to report group-level neural activation localizations related to the fit hyper- planes (see 2.12 Construction of Neuroanatomical Encoding Parameters). BOLD beta-series construction. As reported in Bush et al.30 we exploited the beta-series method43 to construct whole-brain, task-related neural activation maps corresponding to each individual IAPS image. To summarize, we constructed a general linear model (GLM) that includes hemodynamic regression functions for each individual experimental stimulus as well as additional functions related to motion and drift artifacts. We then solved this GLM, and the resulting parameter set associated with each stimulus regressor constitutes the neural activations induced by that stimulus. Materials and Methods We chose the SVM architecture for this analysis based on its established performance in domains having small sample sizes47, fast analytical solution, and previ- ously demonstrated ability to predict normative valence scores from high-dimensional GM neural activations30. The architecture was trained according to intra-subject (i.e. within subject) leave-one-out-cross-validation (LOOCV). That is, for a set of subjects, N, and feature-label pairs, M, the jth prediction (j ∈ M) for the ith subject (i ∈ N) was made using an SVM regression model trained on the M-1 disjoint feature-label pairs (k ∈ M-1; k ≠ j). All beta-series features were GM masked to the individual subject, i. Mixed-effects modeling. We estimated prediction effect sizes of our affective measures via general linear mixed effect model (GLMM). The measure of interest was set to be the normative valence scores of the experi- mental stimuli. Affective measures (i.e., HR deceleration and SVM-predicted valence) were incorporated as fixed effects. Random slope and intercept effects were modeled subject-wise. GLMMs were solved using Matlab’s fitlme function. Effect-sizes (R2) were calculated from the resultant model residuals. The significance of the random effects was tested by constructing two models for each test (including and excluding the random effects) and comparing the fit of the model variants using a likelihood ratio test (implemented as Matlab’s compare function). Only significant random effects were incorporated into estimates of effect size. Controlling for stimulus set affective valence via neutral stimuli thresholding. One challenge for the analytical methodology employed in this work, which arises from our IAPS image sampling process (see Section 2.3 System Identification Task as well as Fig. 1, panel A), was that our selected image set exhibited nor- mative affect rating scores that maximally (and continuously) spanned the arousal-valence plane. Therefore, our stimulus set provided us with no clear demarcation between polar-extreme positively and negatively valent cate- gories by which to group the concurrent HR changes. Further, a review of the literature found no accepted meth- odology on which to base the determination of positive and negative valence. Indeed, prior studies used clusters of polar-extreme positively or negatively valent stimuli rather than a natural demarcation, such as the middle Likert score of the IAPS image set (i.e., valence = 5). A summary of the valence and arousal scores exhibited by image sets employed in prior HR change analyses are provided in Supplemental Table S1. Materials and Methods Pre-stimulus beats were identified within a one second interval pre-stimulus onset9,10. Post-stimulus beats were identified within a four second interval post-stimulus onset (depicted in Fig. 1 panel F). Pre- and post-stimulus beats were converted to IBIs by calculating the difference between the times of successive beats, using, if neces- sary, beats falling outside the strict time intervals to form a beat pair. We assigned the IBI value to the time of the second beat of the pair. We then computed the average over the pre-stimulus IBIs to use as a baseline for deter- mining HR deceleration post-stimulus onset. g p We formed time courses of HR change by calculating, at each half-second post-stimulus, the current heart rate relative to the pre-stimulus average heart rate9. To construct these half-second bpm estimates, we identified the time-fractionally-weighted average of each IBI that fell within the half-second interval of interest, converted this average from IBI (s) to bpm and then subtracted the mean pre-stimulus average heart rate (converted to bpm). By performing this computation for half-second intervals spanning 0–4 s post-stimulus onset, we formed smooth time courses of HR change for each IAPS stimulus for each subject (depicted graphically in Fig. 1, panel H). Identifying the time of measurement for HR deceleration. One challenge for our methodology was determining a point at which to measure deceleration. The literature provides a wide range of parameters. We settled on the following methodology (driven by our data). First, we compute the group mean time-course for each of the 90 IAPS stimuli. We then divide these data into two sets, positive and negative stimuli (using the normative valence scores of the stimuli as the label and partitioning based on the middle Likert score of 5). Based on widely reported patterns of deceleration of negative stimuli7,13,16–18 we used the time-point of the minimum of the mean trajectory over the negative stimuli as the point at which to measure stimuli (i.e., maximum deceleration, denoted tpsmax; see Supplemental Fig. S1). Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Intra-subject multivariate regression training and cross-validation. We conducted multivariate pattern analysis (MVPA) using a linear support vector machine (SVM) regression architecture45, fitrsvm, param- eterized by the default settings of the Matlab Statistics Toolbox46. Materials and Methods g p y p g y p pp To address this confound in the literature, we performed our primary analysis across a range of stimulus sets in which we incrementally remove stimuli from a region of neutrality (symmetrically about the middle Likert score) in order to control the polar-extremity of the positively and negatively valent stimuli in the modeled stimulus set. Starting with the baseline condition (all stimuli), we classified the image stimuli according to their pre-defined normative valence scores (calculated as mean Likert scores from a 9 point scale) relative to the middle Likert score (5). We then conducted GLMM modeling of SVM and ΔHR fixed effects for the stimulus set as well as secondary calculations of the mean and standard deviation of the normative scores of the positively and negatively valent stimuli as well as the fraction of stimuli kept (relative to the baseline condition). We then repeated this modeling process at small increments of the threshold used to classify positively and negatively valent stimuli as follows. For each threshold value (on the range, 0–3, at increments of 0.2) we stratified the stimuli to exclude those falling within a normative valence score range of 5+/−threshold. We then labeled those stimuli falling outside the threshold as positively or negatively valent. Results R d Reproducing canonical HR change in response to affectively valent stimuli. We qualitatively reproduced the structure of valence-specific HR deceleration in response to IAPS stimuli within the fMRI envi- ronment. Consistent with prior studies13–18,38, we report a relatively large post-stimulus HR deceleration for neg- atively valent stimuli, depicted graphically in Supplemental Fig. S1. However, in contrast with earlier work7,15 we report neither deceleration nor a triphasic response to positively valent stimuli. Reproducing canonical functional neuroanatomical encoding of affective valence. We found significant inter-analysis consistency between the affective valence encodings detected in this work and the encodings detected in earlier work that used a substantially smaller (n = 19) set of subjects30. Encoding parame- ters derived from the intra-subject MVPA models fit in this experiment explain 85% of the variance of those prior encoding parameters and no encoding parameter (out of 2524 voxels that survive joint-analysis permutation testing) changed sign, depicted graphically in Supplemental Fig. S2. Neural activation patterns are stronger measures of affective valence induction than heart rate change. Both HR change (p = 0.036; F-test; null: β = 0) and SVM-predicted valence (p < 0.001; F-test; null: β = 0) significantly contribute fixed effects to the GLMM prediction of normative valence scores of the stimuli, as depicted in Supplemental Fig. S3. Random effects were not significant. Moreover, SVM-predicted valence contributed an order of magnitude more explanation of variance (R2 = 0.049) in comparison to the contribution of HR change (R2 = 0.001). Neural Activation patterns measure valence induction across a broad range of stimulus sets. A critical limitation in relating these combined prediction findings to the broader literature linking HR change to affective valence is the selection of stimuli, which varies widely in the literature. To control for this potential confound, we repeated the GLMM modeling reported in Section 3.3 along a continuously varying set of stimuli (see Materials and Methods Section 2.12) in which the most neutrally valent stimuli (according to IAPS normative valence ratings) are excluded from analysis, thus generating stimulus sets of incrementally greater Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Figure 2. Summary of the effects of polar-extremity in the normative valence scores of the stimulus on modeling performance. (A) Estimation of the contribution of fixed effects, separately for heart rate deceleration and SVM predictions as a function of the width of the threshold used to exclude “neutral” stimuli. Results R d This threshold is reported in units of the 9-point Likert scale within which the IAPS image set was originally acquired. Denotes a non-significant fixed effect at that level of thresholding. (B) Mean normative valence scores of the positively and negatively valent stimulus sets created by excluding neutrally valent images as a function of the exclusion threshold. Note, the black dots depict the mean normative valence scores of the positively and negatively valent image sets analyzed in Bradley et al.7. The neutral valence threshold representative of this stimulus set in our dataset was transferred to panels A and C, respectively, for interpretation of these findings. Also note, horizontal gray lines denote mean valence scores for positively and negatively valence stimulus sets reported for other prior work reporting heart rate change discrimination of valenced image stimuli (see Supplemental Table 1 for reference). (C) Fraction of full stimulus set included as a function of scaling. Figure 2. Summary of the effects of polar-extremity in the normative valence scores of the stimulus on modeling performance. (A) Estimation of the contribution of fixed effects, separately for heart rate deceleration and SVM predictions as a function of the width of the threshold used to exclude “neutral” stimuli. This threshold is reported in units of the 9-point Likert scale within which the IAPS image set was originally acquired. Denotes a non-significant fixed effect at that level of thresholding. (B) Mean normative valence scores of the positively and negatively valent stimulus sets created by excluding neutrally valent images as a function of the exclusion threshold. Note, the black dots depict the mean normative valence scores of the positively and negatively valent image sets analyzed in Bradley et al.7. The neutral valence threshold representative of this stimulus set in our dataset was transferred to panels A and C, respectively, for interpretation of these findings. Also note, horizontal gray lines denote mean valence scores for positively and negatively valence stimulus sets reported for other prior work reporting heart rate change discrimination of valenced image stimuli (see Supplemental Table 1 for reference). (C) Fraction of full stimulus set included as a function of scaling. polar-extremity. The resultant prediction effect sizes and related analyses are depicted in Fig. 2. In support of the findings in Section 3.3, SVM-predicted valence contributes more to the explanation of variance than does heart rate change at every level of thresholding (Fig. 2, panel A). Discussionh The primary aim of this study was to extend the comparison of the relative validity of physiological and neural measurements to the domain of affective valence induction. A key contribution of this study is that the compari- son was performed on a stimulus set representing a broad continuum of conveyed affective valence, i.e., including a range of neutral and weakly valent stimuli in addition to the polar-extremes of positively and negatively valent stimuli as have been commonly reported in the heart rate change literature. Another contribution of this work is that we demonstrated a significant empirical relationship between the neural activations derived in this study with an earlier effect-size comparison between physiological and neural measurements of affective arousal induc- tion. We believe this type of analysis and reporting will contribute to better generalization of our findings to mul- tiple, independent dimensions of affect processing. Finally, to our knowledge, this is the first study to explore the combined predictive effect of physiological and neural measurements and the first to clearly rank their individual contributions in predicting normative valence scores of IAPS stimuli. Limitations. The combined predictions of HR change and neural activations explained less than 6% of total variance of the normative valence scores of our stimulus set (up to approximately 17% of total variance for a highly thresholded, and therefore, valently polar-extreme stimulus set). We attribute part of the remaining unex- plained variance to the demographics of our sample. Previous research showed that IAPS normative scores may differ depending upon a participant’s age48–50 as well as their sex51–53. As the age and sex of our sample differs from the sample from which the IAPS normative valence scores were initially derived54, we anticipate that individ- ual variation in affect processing strategies and their neural activations (with respect to the IAPS image stimuli included in this study) contributes to unexplained variance, a study limitation that could have been addressed by collecting each subject’s self-reported affect stimulus rating scores. g j pf g We also suspect that our experiment design, which was not optimized for post-hoc HR change analysis, may also contribute to unexplained variance. Our inter-trial intervals of 2–6 s were not sufficient for all subjects to fully re-acquire basal HR and neural activations. Discussionh Therefore, when measuring HR deceleration relative to a baseline HR estimated from a short pre-stimulus interval, the baseline HR may be influenced by residual responses related to the affective response to the previous stimuli. Our use of beta-series to construct SVM predictions of valence, however, attenuates the contribution of prior stimuli to stimulus-bound neural activations, likely contributing to some of the differences in prediction effect-size between the measures by introducing a bias towards the neural activation measure. Within this same line of reasoning, it is possible that our stimulus presentation time was not sufficient to optimize HR changes. Prior work has shown that the length of stimulus presentation (on the range of 0.03 to 12 s) alters HR deceleration17. This could potentially weaken the predictive effect-size of HR changes with respect to neural activation; however, we point out that the beta-series method captures the affective content of the stimuli, making it suitable for these relatively fast presentation formats. Further, temporally structured artifacts in the BOLD signal, notably the effect of respiration not specifically attributable to motion55, may also contribute to prediction effect size differences in our measurement modalities and were not specifically modeled in our MRI processing pipeline. in our MRI processing pipeline. Finally, we acknowledge that the fit MVPA models may be exploiting aspects of the neural activations induced by the image stimuli that are incidentally correlated with the valence property of affect based on biases in the IAPS dataset (e.g., colors and shapes typical of infant photos being overly represented within images labeled as inducing positive affect). We previously explored this possibility in Bush et al.30, and found no direct evidence for these correlations. Rather, Bush et al.30 showed that SVM model encodings of affective properties were highly reproducible across affect induction experiments which differed by both image stimuli and subjects. However, the definitive test of MPVA-based prediction of affect processing prediction remains to be reported and represents an ongoing goal for this research team. Future directions. Prior work30 presented evidence that neural activations for affectively polar-extreme stimuli exhibit greater inter-subject consistency than less polar-extreme stimuli. Following this line of evidence, we formed a theoretical model describing the relationship between the prediction effect-size of HR change and individual differences in affect processing, depicted graphically in Fig. 3. Results R d With a complex feature space such as the brain, the stronger SVM-prediction compared to heart rate change is not surprising. Moreover, this difference between SVM prediction is robust to methodological differences of stimulus selection that may exist between this work and related reports in the literature. p As would be expected, based on how the base stimulus set maximally spans the arousal-valence plane of normative affect scores (see Fig. 1, panel A), mean valence of the resulting sets of positively and negatively valent stimuli scales linearly with thresholding (see Fig. 2, panel B); moreover, the scaling rate is dictated by sign (pos- itive stimulus sets become more positively valent with thresholding and vice versa). In combination with the increases in prediction efficacy shown in Fig. 2 (panel A), this scaling suggests that neural activation patterns also become more consistently predictive at polar-extremes of affective valence, which confirms (in a regression modeling paradigm under highly controlled thresholding) an earlier report of this phenomenon in a classification paradigm30. It is also useful to note that in Fig. 2 (panel C), the threshold that achieves comparable polar-extremes of valence to the experiment reported in Bradley et al.7 requires removal of approximately 33% of our study’s stim- uli, supporting the conceptualization of the middle third of the IAPS image set as neutrally valent, which com- ports with an intuitive, but unreported, tertiary categorization of IAPS stimuli in the affective valence dimension. Heart rate change and neural activations uniquely predict affective valence. A critical inference to be drawn from Fig. 2 (panel A) is that HR change remains a weak, but reliable predictor of affective valence across a broad range of stimulus sets. To understand the unique contribution of HR change we attempted to pre- dict, via GLMM, SVM-based predictions using ΔHR-based prediction (see Supplemental Fig. S4). This test did not yield a significant model (p = 0.07; F-test; null: β = 0). Further, the normative valence score of the stimulus Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ is highly predictive of both the SVM-based prediction error (p < 0.001; F-test; null: β = 0) and ΔHR-based prediction error (p < 0.001; F-test; null: β = 0); moreover, ΔHR-based prediction error is a strong predictor of SVM-based prediction error (p < 0.001; F-test; null: β = 0), depicted in Supplemental Fig. S5. Results R d Combined, these findings suggest that HR change, with respect to MVPA, captures unique information concerning the induction of affective valence in our experiment. Discussionh Single subject perceived valence scores of the stimuli are depicted as gray numbers above and to the right of the circles. (A) Theoretical Case 1: a subject’s perceived valence of the stimuli exactly agrees with the normative valence scores. The resulting direction of prediction for fixed effects of heart rate deceleration (blue arrow) and predictions of a fit support vector machine (red arrow) mutually agree. (B) Theoretical Case 2: a subject’s perceived valence of the stimuli (as indicated by shifts of neural activations and associated perceived valence scores) disagrees with the normative valence scores, thereby increasing the relative predictive effect of heart rate deceleration. When image stimuli are polar-extreme, perceived affect better aligns with the stimulus set’s normative affect scores (panel A); therefore, measures of both neural activation (which is fit to, and, therefore, predicts normative affect scores) and HR change (which, in theory, measures perceived affective valence) predict along the axes of normative valence scores (the measure of interest in this experiment). However, when image stimuli are neutral, disagreements between the subjects’ perceived affect and the normative valence scores emerge. Measures of neural activation generalize to these individual differences during the learning process whereas HR change continues to weakly, but reliably, predict perceived affect, yielding relatively lower predictive effect size on the measure of interest. Conclusions Th d h This study characterized, for the first time, the combined effects conveyed by both patterns of neural activa- tion and heart rate deceleration in predicting normative valence scores of affectively valent image stimuli. Both measures were shown to exhibit significant and unique predictive effects; however, patterns of neural activa- tion were shown to explain a significantly greater proportion of variance than heart rate deceleration. These observed traits (significance and uniqueness as well as relative performance) persisted as the positive and nega- tive polar-extremity of the stimuli’s valence were incrementally increased to represent the diversity of stimulus sets reported in the heart rate deceleration literature. In sum, these findings support the acquisition of heart rate deceleration concurrently with fMRI to provide convergent validity of induced affect processing in the dimension of affective valence. Received: 26 November 2019; Accepted: 6 May 2020; Published: xx xx xxxx Received: 26 November 2019; Accepted: 6 May 2020; Published: xx xx xxxx References References 1. Gross, J. J. & Barrett, L. F. The emerging field of affective science. Emotion 13, 997–998 (2013). 1. Gross, J. J. & Barrett, L. F. The emerging field of affective science. Emotion 13, 997–998 (2013). h g gif 2. Bradley, M. & Lang, P. J. Measuring Emotion: Behavior. Feeling, and Physiology. in Cognitive Neuroscience of Emotion 25, 4 (2000). 3. Heller, A. S., Greischar, L. L., Honor, A., Anderle, M. J. & Davidson, R. J. Simultaneous acquisition of corrugator electromyography and functional magnetic resonance imaging: A new method for objectively measuring affect and neural activity concurrently NeuroImage 58, 930–934 (2011). 3. Heller, A. S., Greischar, L. L., Honor, A., Anderle, M. J. & Davidson, R. J. Simultaneous acquisition of corrugator electromyography and functional magnetic resonance imaging: A new method for objectively measuring affect and neural activity concurrently. NeuroImage 58, 930–934 (2011). 3. Heller, A. S., Greischar, L. L., Honor, A., Anderle, M. J. & Davidson, R. J. Simultaneous acquisition of corrugator electromyography and functional magnetic resonance imaging: A new method for objectively measuring affect and neural activity concurrently. NeuroImage 58, 930–934 (2011). g 4. Boucsein, W. Electrodermal Activity. (Springer US, 2012). 5. Braithwaite, J. J., Watson, D. G., Jones, R. & Rowe, M. A guide for analysing electrodermal activity (EDA) & skin conduc responses (SCRs) for psychological experiments. Psychophysiology 49, 1017–1034 (2013). p p y g p y p y gy 6. Figner, B. & Murphy, R. O. Using skin conductance in judgement and decision making research. In A handbook of process tracing methods for decision research 1–34 (Psychology Press, 2011). 6. Figner, B. & Murphy, R. O. Using skin conductance in judgem methods for decision research 1–34 (Psychology Press, 2011). 7. Bradley, M. M., Codispoti, M., Cuthbert, B. N. & Lang, P. J. Emotion and motivation I: Defensive and appetitive reactions in picture processing. Emotion 1, 276–298 (2001).f p g 8. Cacioppo, J. T., Petty, R. E., Losch, M. E. & Kim, H. S. Electromyographic activity over facial muscle regions can differentiat valence and intensity of affective reactions. J. Pers. Soc. Psychol. 50, 260–268 (1986).f yf y ( ) 9. Greenwald, M. K., Cook, E. W. & Lang, P. J. Affective judgment and psychophysiological response: Dimensional covariation in the evaluation of pictorial stimuli. J. Psychophysiol. 51–64 (1989).f f evaluation of pictorial stimuli. J. Psychophysiol. 51–64 (1989) p y p y 0. Lang, P. J., Greenwald, M. Discussionh Measures of neural activation generalize to these individual differences during the learning process whereas HR change continues to weakly, but reliably, predict perceived affect, yielding relatively lower predictive effect size on the measure of interest. l d l f l b f d h l l f ff Fi 3 C t l d l f l ti t ib ti f i i d h i l i l f ff t Figure 3. Conceptual model of relative contributions of neuroimaging and physiological measures of affect induction based on a theory of individual differences. Patterns of neural activation are represented as gray circles. Normative valence scores of the stimuli associated with these neural activations are presented as labels within the circles’ boundaries. Single subject perceived valence scores of the stimuli are depicted as gray numbers above and to the right of the circles. (A) Theoretical Case 1: a subject’s perceived valence of the stimuli exactly agrees with the normative valence scores. The resulting direction of prediction for fixed effects of heart rate deceleration (blue arrow) and predictions of a fit support vector machine (red arrow) mutually agree. (B) Theoretical Case 2: a subject’s perceived valence of the stimuli (as indicated by shifts of neural activations and associated perceived valence scores) disagrees with the normative valence scores, thereby increasing the relative predictive effect of heart rate deceleration. When image stimuli are polar-extreme, perceived affect better aligns with the stimulus set’s normative affect scores (panel A); therefore, measures of both neural activation (which is fit to, and, therefore, predicts normative affect scores) and HR change (which, in theory, measures perceived affective valence) predict along the axes of normative valence scores (the measure of interest in this experiment). However, when image stimuli are neutral, disagreements between the subjects’ perceived affect and the normative valence scores emerge. Measures of neural activation generalize to these individual differences during the learning process whereas HR change continues to weakly, but reliably, predict perceived affect, yielding relatively lower predictive effect size on the measure of interest. Figure 3. Conceptual model of relative contributions of neuroimaging and physiological measures of affect induction based on a theory of individual differences. Patterns of neural activation are represented as gray circles. Normative valence scores of the stimuli associated with these neural activations are presented as labels within the circles’ boundaries. Discussionh This model potentially explains how measurements of HR change and neural activations uniquely and significantly inform the combined prediction of the stimuli’s normative valence scores. In sum, measurement modalities differ in their detection of disagree- ments between a subject’s perceived affect and the normative valence scores, which are most likely to emerge among neutrally valent stimuli30. Measures of neural activation generalize to these individual differences during the learning process whereas HR change continues to weakly, but reliably, predict perceived affect, yielding rela- tively lower predictive effect size on the measure of interest. An experiment design which captures an additional measure of affective induction (e.g., self-reported scores of each stimulus or an additional physiological measure of valence, such as facial EMG) could test this theoretical model. Data collection is ongoing to power such an experiment. Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Figure 3. Conceptual model of relative contributions of neuroimaging and physiological measures of affect induction based on a theory of individual differences. Patterns of neural activation are represented as gray circles. Normative valence scores of the stimuli associated with these neural activations are presented as labels within the circles’ boundaries. Single subject perceived valence scores of the stimuli are depicted as gray numbers above and to the right of the circles. (A) Theoretical Case 1: a subject’s perceived valence of the stimuli exactly agrees with the normative valence scores. The resulting direction of prediction for fixed effects of heart rate deceleration (blue arrow) and predictions of a fit support vector machine (red arrow) mutually agree. (B) Theoretical Case 2: a subject’s perceived valence of the stimuli (as indicated by shifts of neural activations and associated perceived valence scores) disagrees with the normative valence scores, thereby increasing the relative predictive effect of heart rate deceleration. When image stimuli are polar-extreme, perceived affect better aligns with the stimulus set’s normative affect scores (panel A); therefore, measures of both neural activation (which is fit to, and, therefore, predicts normative affect scores) and HR change (which, in theory, measures perceived affective valence) predict along the axes of normative valence scores (the measure of interest in this experiment). 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International affective picture system (IAPS): Affective ratings of pictures and instru manual. (2008). 55. Byrge, L. & Kennedy, D. P. Identifying and characterizing systematic temporally-lagged BOLD artifacts. NeuroImage 171, 376–392 (2018). Scientific Reports \| (2020) 10:9298 \| https://doi.org/10.1038/s41598-020-66109-3 www.nature.com/scientificreports/ Acknowledgementsh g This work was funded by the Department of Psychiatry of the University of Arkansas for Medical Sciences, the National Science Foundation (BCS-1735820), and the National Institute on Drug Abuse (1T32DA022981). The authors would like to thank Bradford S Martins, Anthony Privratsky, Jennifer Payne, Emily Hahn, Natalie Morris, and Nathan Jones for their assistance in recruiting and assessing research subjects and acquiring subject data. Subject recruitment for the project was also supported by the UAMS Translational Research Institute (TRI) through the National Center for Advancing Translational Sciences (1U54TR001629-01A1). Finally, the authors would like to thank Sonet Smitherman and Favrin Smith for their efforts in gaining IRB protocol approval and maintaining human subject research compliance during the course of the study. Competing interestsh Competing interestsh p g The authors declare no competing interests. Author contributions K.W. designed, implemented, and validated the heart rate change processing pipeline. K.B. designed, implemented, and validated the MVPA analysis. K.W. and K.B. wrote the main manuscript text. K.W. and K.B. prepared figure 1 and K.B. prepared figures 2–4. K.W., K.B., C.K. and G.A.J. reviewed the manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66109-3. Correspondence and requests for materials should be addressed to K.A.B. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 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. 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https://openalex.org/W4311970588	https://aben.springeropen.com/counter/pdf/10.1186/s43251-022-00075-w	English	null	Progressive collapse analysis of prestressed concrete girder bridges using improved applied element method	Advances in bridge engineering	2,022	cc-by	6,487	© 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 mate‑ rial. 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/. Progressive collapse analysis of prestressed concrete girder bridges using improved applied element method Mohamed Magdi Abdelaziz* , Mohamed Sayed Gomaa and Hany Ahmed El‑Ghazaly *Correspondence: mma22@fayoum.edu.eg Faculty of Engineering, Fayoum University, Faiyum, Egypt Abstract In this paper, the Improved Applied Element Method (IAEM), which was originally developed as an effective analysis technique for large-scale bonded and unbonded prestressed structures, is utilized to carry out failure modeling of prestressed bridges under different hazard loads. A typical prestressed concrete girder bridge is analyzed under two hazardous loading scenarios. The first one is applying a detonation charge located in the middle of the central span, while the second scenario is a sudden failure of a column representing a truck or a vessel colliding with the bridge pier. For both scenarios, the collapse analyses of the bridge structure after damage are explored. In addition, the mechanism and severity of damage in the bridge pier and deck are investigated. Both material and geometric nonlinearities are considered in the analy‑ sis. Moreover, it considers contact-impact, re-contact, and inertia effects, and hence, it can track the collapse stages of the structure as well as debris movement until the complete collapse of the structure. The results show a strong capability for simulating the total performance of the bridges from early the stages of loading until the total col‑ lapse, with a clear graphic representation of the collapse phenomena. Faculty of Engineering, Fayoum University, Faiyum, Egypt Keywords: Improved applied element method (IAEM), Prestressed bridges, Progressive collapse, Failure analysis, Blast loading, Pier collapse Advances in Bridge Engineering Advances in Bridge Engineering Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 https://doi.org/10.1186/s43251-022-00075-w Open Access Open Access 1 Introduction Progressive collapse has become a popular research topic due to its history of causing catastrophic damage to structures and people. The terminology of “progressive col- lapse” is defined as the spread of an initial local failure from element to element, eventu- ally resulting in the collapse of an entire structure or a disproportionally large part of it. Many numerical investigations were conducted in an attempt to completely under- stand this phenomena and improve structural resistance to progressive collapse. The effect of blast loads on critical bridge components and bridge global response, as well as proposed protection strategies for mitigating blast hazards, were studied by (Yahia, 2009). Various blast scenarios were applied to a typical prestressed concrete girder, either above or below the bridge deck. A fine mesh has been used to simulate the variable cross sections of the bridge girder. The computer simulation results were Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 2 of 15 utilized to identify the susceptible bridge components during a blast danger, as well as to estimate the magnitudes and locations of maximum shear forces and bending moments. Moreover, progressive collapse analysis for post-tensioned box girder bridges was conducted under blast loads by (Ibarhim et al., 2012). The mish was chosen to be fine enough to simulate box section accurately by using 31,600 elements and 2,860,388 springs. A large vehicle bomb (LVB) placed beneath the bridge was studied in two differ- ent locations. The severity and mechanisms of damage in the bridge pier and deck were investigated. utilized to identify the susceptible bridge components during a blast danger, as well as to estimate the magnitudes and locations of maximum shear forces and bending moments. Moreover, progressive collapse analysis for post-tensioned box girder bridges was conducted under blast loads by (Ibarhim et al., 2012). The mish was chosen to be fine enough to simulate box section accurately by using 31,600 elements and 2,860,388 springs. A large vehicle bomb (LVB) placed beneath the bridge was studied in two differ- ent locations. The severity and mechanisms of damage in the bridge pier and deck were investigated. In addition, the progressive collapse of a multi-span prestressed continuous bridge due to vessel collision was studied by (Zhao et al., 2014; Jiang et al., 2017). Finer meshes were utilized to adequately capture the internal contact between various components, local deformation or buckling, and material failure as best as possible. 1 Introduction The results dem- onstrated that the bridge pier directly impacted by a vessel was collapsed in the lateral direction of the bridge span, whereas non-impacted piers were collapsed in the longitu- dinal direction of the bridge span. The progressive collapse of Hongqi Bridge, a multi-span simply-supported bridge in Zhuzhou city, was studied by (Bi et al., 2015; Seyed Khoei et al., 2020). The results indi- cated that the multi-span simply-supported bridge with wall-type piers might be vulner- able to domino-type progressive collapse due to the low shear strength of the supporting piers. Furthermore, because insufficient seating length in the abutment was identified as a key effective factor in the onset of collapse, the use of restrainers as a deck-to-abut- ment connection may reduce the likelihood of progressive collapse. The procedure of progressive collapse of prestressed voided slab bridges under earth- quakes, as well as the effects of other parameters on the propagation of collapse of regu- lar, semi-regular, and irregular bridges, were investigated by (Seyedkhoei et al., 2019). The findings revealed that domino-type progressive collapse occurred in bridges with voided slabs after the first failure of the deck at the bridge abutment seating. In addition, it was found that, the deck type, piers’ height, and ground slope had a significant impact on the progressive collapse procedure on both regular and irregular bridges with voided slab decks. The behavior of bridge decks under blast loads was studied using the finite element method by (Hassan et al., 2021). Several explosive charges were deployed at various sites to ascertain the impact of explosion magnitude and placement on the bridge. The results showed that for minor charges, the bridge was almost completely unaffected, however for big charges, the deck completely failed, causing the bridge to fall. The collapse of bridges during extreme loading conditions has prompted several con- cerns about their ability to withstand partial or whole collapses. It was reported that the progressive collapse could be initiated due to two broad categories, natural factors such as earthquakes, tsunamis, floods, mudslides, and hurricanes; and human factors such as design error and construction method, vehicle overloading or collision, fire, and ter- rorist attack (Deng et al., 2016; Zhang et al., 2022). Since the collapse process is strongly dependent on the entire structural system, non-linear numerical simulation with a rea- sonable solution time and good accuracy becomes a targeted method of investigation. 1 Introduction Recent studies have extended the Improved Applied Element Method (IAEM) to facilitate modeling prestressed concrete structures with bonded and unbonded tendons Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 3 of 15 (Abdelaziz et al., 2021a; Abdelaziz et al., 2021b). The models were validated under dif- ferent types of loadings, highlighting the reliability of the proposed model. However, the capability of the models to perform failure analysis was not examined. Therefore, the current work focuses on carrying out failure modeling of prestressed bridges under extreme loading circumstances using the IAEM to show the precision and validity of the numerical method and to demonstrate its capacity and efficiency in modelling the fail- ure of the prestressed bridges. 2 Improved applied element method The IAEM has recently been developed as an efficient analysis technique for modeling large scale framed buildings with homogeneous and non-homogeneous cross sec- tions, such as steel sections, reinforced concrete (RC) elements, retrofitting sections, and unbonded and bonded prestressed structures, up to complete failure under vari- ous hazard loads (Elkholy & Meguro, 2004; El-Kholy et al., 2012; Abdelaziz et al., 2020; Abdelaziz et al., 2021a; Abdelaziz et al., 2021b). In this technique, each structural mem- ber is divided into an appropriate number of multi-layered rigid elements composed of several layers representing the unconfined and confined concrete, the bonded non-pre- stressing reinforcing steel, the bonded prestressing tendon, and the retrofitting material, if any; as illustrated in Fig. 1 (Abdelaziz et al., 2021a). Different rectangular or non-rec- tangular sections can be simulated using the advantage of the multi-layered element type without any complications. All identical layers in the neighboring elements are con- nected together by shear and normal springs representing the material characteristics of each layer, as shown in Fig. 2 (El-Kholy et al., 2012). The main procedure of this approach is illustrated in the flowchart shown in Fig. 3. The structural equilibrium equation can be solved using both incremental methods, load control and displacement control. In addition, this approach takes into consideration P-∆ effects and large displacement during the structural analysis. The main distinction between the IAEM and the conventional Applied Element Method (AEM) is that the IAEM employs only one element to model the nonrectangular cross sections with high efficiency and reliable accuracy, resulting in a significant reduction in the solving time. fii The Okamura and Maekawa compression model (Okamoto & Maekawa, 1991), shown in Fig. 4a, is employed for both confined and unconfined concrete. The tangent modu- lus of each concrete spring is computed at each load step, whether the concrete spring is in the loading or unloading stage. In tension, the stress-strain curve is assumed to be linear up to the tensile strength. After this point, the stiffness of springs under tension is assumed to be zero. Fig. 1 The multi-layered element type (Abdelaziz et al., 2021a) 2021a) Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 4 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Fig. 2 Modeling RC framed structures with IAEM (Abdelaziz et al., 2021b) Fig. 3 Flow chart of the IAEM (El-Kholy et al., 2012) Fig. 3 Simulation of the bridge To understand the global behavior of bridges subjected to extreme loading conditions, a typical prestressed concrete girder superstructure bridge has been analyzed using the IAEM method. In this example, the bridge design, geometry, materials, dimensions, and details are in accordance with the Egyptian Code of Practice for Design and Con- struction of Concrete Structures and the Egyptian Loading Code (ECP-203, 2018; ECP- 201, 2012). The bridge consisted of three simple spans; each of 30 m long. Each span is supported by prestressed girders 2685 mm apart and a 200 mm concrete deck. Three prestressing tendons are used for each girder with a nominal diameter of 15.24 mm, a yield strength of 1674 MPa, and an ultimate strength of 1860 MPa; conforming to the British Standard BS-5896 (BS-5896 TBS, 2012). Each tendon has a total prestress- ing force of 2930 kN. The girders are supported on 200 mm elastomeric bearing pads. The supporting structure consists of a moment-resisting frame with two columns of 1400 × 1400 mm2 and a RC girder of 1500 × 2000 mm2. The utilized concrete has a com- pressive strength of 55 MPa, whereas reinforcing bars have yield and ultimate strengths of 400 MPa and 600 MPa, respectively. The bridge geometry, dimensions, and reinforce- ment details are illustrated in Figs. 5 and 6. 2 Improved applied element method 2 Modeling RC framed structures with IAEM (Abdelaziz et al., 2021b) Fig. 2 Modeling RC framed structures with IAEM (Abdelaziz et al., 2021b) Fig. 3 Flow chart of the IAEM (El-Kholy et al., 2012) Fig. 3 Flow chart of the IAEM (El-Kholy et al., 2012) Fig. 3 Flow chart of the IAEM (El-Kholy et al., 2012) (2022) 3:24 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 5 of 15 Fig. 4 Material model for (a) concrete, (b) non-prestressing reinforcement, and (c) prestressing reinforcement (Abdelaziz et al., 2021a). Fig. 4 Material model for (a) concrete, (b) non-prestressing reinforcement, and (c) prestressing reinforcement (Abdelaziz et al., 2021a). A classical elastic plastic material with strain hardening using a bilinear stress–strain relationship in both tension and compression loading conditions, shown in Fig. 4b, is employed for non-prestressing reinforcement. On the other hand, the Menegotto and Pinto stress-strain relationship (Menegotto & Pinto, 1973), shown in Fig. 4c, is employed to represent the prestressing tendons. 3.1 Structural modeling A typical girder has been selected to perform the analysis. Using the improved approach, the studied bridge has been modelled taking into account the constituent material prop- erties. With the multi-layered element feature, each bridge girder has been modeled using 25 elements, with a total number of 256 elements. The girders’ dimensions and the tendons’ varying eccentricities along the girders’ length have been considered in the model. The model and the elements’ configurations are shown in Fig. 7. The character- istic parameters for each material are listed in Table 1. The bridge model has been ana- lyzed under two extreme loading scenarios. The first one is applying a detonation charge Page 6 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Fig. 5 The geometric configuration of the typical prestressed girder Fig. 5 The geometric configuration of the typical prestressed girder The geometric configuration of the typical prestressed girder Fig. 6 Cross section of bridge Fig. 6 Cross section of bridge Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 7 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 located in the middle of the central span. The second scenario is a sudden failure of a column representing a truck or a vessel colliding with the bridge pier. At first, the design load of the structure is statically applied as an initial load in 50 load increments, while the blast loads or the sudden failure of the column are applied as loading in the time domain. The time step for blast load should be different from the time step for sudden column removal because the blast load reaches its highest value and decays to atmos- pheric pressure in milliseconds (Amr Ramadan Ibrahim, 2018). The chosen time steps are 0.0001 sec. and 0.01 sec. For the blast load condition and the column removal condi- tion, respectively. Fig. 7 IAEM model of the analyzed bridge Table 1 Material properties of the concrete, the reinforcing steel, and prestressing tendon Concrete properties Non-prestressed steel properties Prestressing Tendon properties Ec (GPa) fcu (MPa) Es (GPa) fy (MPa) fu (MPa) Ep (GPa) fpy (MPa) fpu (MPa) εi 32.8 55 200 400 600 195 1674 1860 0.0060775 Fig. 3.1 Structural modeling 7 IAEM model of the analyzed bridge Table 1 Material properties of the concrete, the reinforcing steel, and prestressing tendon Concrete properties Non-prestressed steel properties Prestressing Tendon properties Ec (GPa) fcu (MPa) Es (GPa) fy (MPa) fu (MPa) Ep (GPa) fpy (MPa) fpu (MPa) εi 32.8 55 200 400 600 195 1674 1860 0.0060775 Table 1 Material properties of the concrete, the reinforcing steel, and prestressing tendon located in the middle of the central span. The second scenario is a sudden failure of a column representing a truck or a vessel colliding with the bridge pier. At first, the design load of the structure is statically applied as an initial load in 50 load increments, while the blast loads or the sudden failure of the column are applied as loading in the time domain. The time step for blast load should be different from the time step for sudden column removal because the blast load reaches its highest value and decays to atmos- pheric pressure in milliseconds (Amr Ramadan Ibrahim, 2018). The chosen time steps are 0.0001 sec. and 0.01 sec. For the blast load condition and the column removal condi- tion, respectively. 3.2 Blast load In general, an explosion is a phenomena caused by the quick and unexpected release of a massive quantity of energy (Beshara, 1991). When an explosion occurs, the hot gases released by the explosion source forcefully push the atmosphere surrounding the explo- sion and generate a blast wave. This wave exerts significant pressure on exposed sur- faces and penetrates the structure through apertures. In a fraction of a second after the explosion, the blast wave spreads outward from the explosion site. As the shock prop- agates outward, the peak of the overpressure caused by this hemispherical explosion decays rapidly as a function of distance from the source, as shown in Fig. 8. The reflected wave’s pressure amplitude is at least twice that of the original shock wave and is propor- tional to the initial shock’s strength, which is proportional to the charge weight. When a blast wave reflects, the pressure amplitude can be substantially larger than the pressure caused by the original shock alone (Beshara, 1991). Figure 9 shows how the blast pres- sure decays exponentially and finally becomes negative. i Unlike static and seismic loading, blast loading is a high-magnitude pressure that affects just a small portion of the building. The fundamental distinction between blast loading and other forms of loading is the quick load rate, which can excite higher struc- tural modes that are typically ignored for other types of hazards, such as earthquakes. The factors influencing a structure’s behavior during explosion scenarios are classi- fied as internal and external factors. Internal factors are those that are dependent on the Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 8 of 15 Abdelaziz et al. Advances in Bridge Engineering f Fig. 8 Variation of pressure with distance (UFC-3-340-02, 2008) Fig. 9 Blast wave pressure- time history (UFC-3-340-02, 2008) Fig. 8 Variation of pressure with distance (UFC-3-340-02, 2008) properties of the structural element subjected to the blast load, such as stiffness, mass, ductility, redundancy, and overall structure continuity, while external factors are those that are affected by the explosion situation. They include the explosive material, stand- off distance, charge weight, and angle of incidence. In explosion situations, the standoff distance is the most important element. Simply extending the standoff distance by a few meters reduces the pressure significantly. Therefore, a scaled distance is used to weight Fig. 8 Variation of pressure with distance (UFC-3-340-02, 2008) Fig. 3.2 Blast load 9 Blast wave pressure- time history (UFC-3-340-02, 2008) Fig. 8 Variation of pressure with distance (UFC-3-340-02, 2008) Fig. 8 Variation of pressure with distance (UFC-3-340-02, 2008) Fig. 9 Blast wave pressure- time history (UFC-3-340-02, 2008) properties of the structural element subjected to the blast load, such as stiffness, mass, ductility, redundancy, and overall structure continuity, while external factors are those that are affected by the explosion situation. They include the explosive material, stand- off distance, charge weight, and angle of incidence. In explosion situations, the standoff distance is the most important element. Simply extending the standoff distance by a few meters reduces the pressure significantly. Therefore, a scaled distance is used to weight the energy of the blast load as follows: properties of the structural element subjected to the blast load, such as stiffness, mass, ductility, redundancy, and overall structure continuity, while external factors are those that are affected by the explosion situation. They include the explosive material, stand- off distance, charge weight, and angle of incidence. In explosion situations, the standoff distance is the most important element. Simply extending the standoff distance by a few meters reduces the pressure significantly. Therefore, a scaled distance is used to weight the energy of the blast load as follows: (1) Z = R 3√ W Z = R 3√ W (1) where Z is the scaled distance, R is the stand-off distance, and W is the equivalent TNT charge weight. This relationship illustrates that the energy delivered to different targets at the same scale distance is similar. For example, the energy given to a target by 8 ton TNT at a stand-off distance of 10 m from a specified point, is the same as the energy delivered by 64 kg of TNT at a stand-off distance of 2 m from the point (both having the same scaled distance of 0.50 m/kg1/3) (Amr Ramadan Ibrahim, 2018). Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 9 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 The blast loads acting on the bridge have been determined using a developed soft- ware, named VecTor-Blast (Miller, 2004) which calculates the pressure-time history at specified points on a three-dimensional cuboid structure for known values of charge weight and standoff distance. 3.2 Blast load This numerical tool was validated using experimental data, which demonstrated that VecTor-Blast is capable of reliably estimating pressure time histories on the structure’s front and back sides. In order to model a vehicle explosion over the bridge in the current study, several charge weights have been explored at a height of 1.5 m above the middle bridge’s mid- span until the girder collapsed. The pressure-time histories for each element in the present model have been calculated based on their stand-off distance. In addition, the pressures have been converted into nodal forces by multiplying the pressures by the tributary areas of the elements. The generated load-time histories have been used on the bridge model with a time step of 0.0001 sec. It should be noted that the negative phase’s effect has ignored during the analysis. The procedure for applying the time history of the blast load is illustrated in Fig. 10. The general differential equation of motion governing the response of the bridge is described as (Tagel-Din & Meguro, 2000): (2) [M] .. U + [C] . U + [K]{U} = f (t) + {Rm} + {RG} (2) where: [M] is the mass matrix; [C] is the damping matrix; [K] is the nonlinear stiffness matrix; {Δf(t)} is the incremental applied load vector; .. U , . U , and {ΔU} are the incremental acceleration, velocity, and acceleration vectors, Rm is the residual force vec- tor due to cracking and incompatibility between strain and stress of each spring; and RG is the residual force vector due to geometrical changes in the structure during loading. 3.3 Collapse mechanism due to blast loads When an explosion occurs over the bridge’s mid-span, the hemispherical blast waves flow towards the bridge structure. The structural elements affected by these blast waves are the girders. The failure history of the bridge due to the blast load is illus- trated in Fig. 11. Collapse of a bridge due to blast in reality is shown Fig. 12. The his- tory of failure can be summarized as follows: 1- The middle span has experienced severe vertical displacement, resulting in additional straining actions on the girder. 2- Tensile cracks have occurred at the mid-span followed by yielding of the top and bot- tom reinforcement and the tendon, as well as, crushing of the top fiber of the con- crete. 3- With the increase of the additional loads caused by the propagation of the blast wave, the bridge girder has been exposed to more severe damage until the mid-span sec- tion failed resulting in downward movement of the two failed parts of the girder behaving as two cantilevers connected to the elastomeric bearing. 4- The subsequent mode of failure depends on the type of bearing between the girder and the supporting column. Separation between the girder and the column has hap- pened when the girders are simply resting on the elastomeric bearing (free bearing), Page 10 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Fig. 10 Procedure of applying the blast load time history Fig. 10 Procedure of applying the blast load time history Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 11 of 15 Abdelaziz et al. Advances in Bridge Engineering Fig. 11 Collapse mechanism of the prestressed girder under blast loading Fig. 11 Collapse mechanism of the prestressed girder under blast loading Fig. 11 Collapse mechanism of the prestressed girder under blast loading Fig. 11 Collapse mechanism of the prestressed girder under blast loading as in the current model. On the other hand, for a typical fixed bearing connection, another mode of failure might occur depending on the tensile strength of the anchor bolts which connect the sole plate to the pier. 5- The girder has fallen down and collided with the ground, while the other side has col- lided with the adjacent column, resulting in a shear load acting on the column. 3.3 Collapse mechanism due to blast loads If this shear load is more than the shear capacity of the column, it will collapse, resulting in the collapse of the adjacent span, and this procedure will continue progressively until the complete collapse of the bridge takes place. However, in the current model, the shear capacity of the column was sufficient to sustain the shear force resulting from the girder impact on the column. 5- The girder has fallen down and collided with the ground, while the other side has col- lided with the adjacent column, resulting in a shear load acting on the column. If this shear load is more than the shear capacity of the column, it will collapse, resulting in the collapse of the adjacent span, and this procedure will continue progressively until the complete collapse of the bridge takes place. However, in the current model, the shear capacity of the column was sufficient to sustain the shear force resulting from the girder impact on the column. Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 12 of 15 Abdelaziz et al. Advances in Bridge Engineering z et al. Advances in Bridge Engineering (2022) 3:24 (2022) 3:24 Fig. 12 Collapse of a prestressed bridge due to blast scenario Fig. 12 Collapse of a prestressed bridge due to blast scenario 3.4 Sudden failure of a column The entire bridge’s integrity is vital to prevent the horizontal spread of damage to nearby areas. Progressive collapse happens when a local failure of a key structural element causes a chain reaction of structural failures, resulting in the collapse of the entire struc- ture or a disproportionately significant part of it. Proper design and details can greatly reduce the probability of such a collapse. This can be accomplished by providing struc- tural elements with enough continuity, redundancy, and energy-dissipating ability to shift loads from the locally injured region to nearby regions capable of supporting these extra loads without collapsing. In different cases, the main supporting members in the bridges are exposed to differ- ent hazardous loading conditions (e.g., bomb, earthquake, or vehicle impact). Interme- diate piers in multiple span bridges should be designed to resist the loss of one or more of their supporting columns. In addition, the cap beam should be designed for different scenarios, including column removal. To obtain full knowledge of the total response of the bridge due to the sudden failure of one supporting member, the analyzed bridge has been exposed to an internal pier removal to represent truck or vessel impacts to the bridge pier. Initially, the structure’s design load is applied as an initial load in 50 load increments followed by the unexpected failure of one supporting structural column at 0.2 sec. With a time step of 0.01 sec. 3.5 Collapse mechanism due to column removal In this case of failure, an interior column is assumed to fail suddenly to represent differ- ent cases of pier collapse, such as vehicle or vessel impact. The failure of the column is modeled by removing the springs connecting the column elements during a period of 0.20 second. The dynamic nonlinear collapse analysis using IAEM has been performed. Based on the simulation results, the mechanism of failure due to the column collapse is shown in Fig. 13. Moreover, the collapse of a bridge due to column removal in reality is shown in Fig. 14. The history of failure can be summarized as follows: Page 13 of 15 Advances in Bridge Engineering (2022) 3:24 1- Once the interior column has collapsed, the two adjacent girders have fallen down i il d h l i b i I ddi i il Fig. 13 Collapse mechanism of the prestressed girder due to column removal Fig. 14 Collapse of bridge due to column removal in practice (2022) 3:24 Page 13 of 15 Abdelaziz et al. Advances in Bridge Engineering (20 Fig. 13 Collapse mechanism of the prestressed girder due to column removal Fig. 13 Collapse mechanism of the prestressed girder due to column removal Fig. 13 Collapse mechanism of the prestressed girder due to column removal Fig. 14 Collapse of bridge due to column removal in practice Fig. 14 Collapse of bridge due to column removal in practice Fig. 14 Collapse of bridge due to column removal in practice Fig. 14 1- Once the interior column has collapsed, the two adjacent girders have fallen down acting as two cantilevers connected to the elastomeric bearing. In addition, tensile cracks, as well as, yielding of the top reinforcement, bottom reinforcement, and ten- don have occurred. 1- Once the interior column has collapsed, the two adjacent girders have fallen down acting as two cantilevers connected to the elastomeric bearing. In addition, tensile cracks, as well as, yielding of the top reinforcement, bottom reinforcement, and ten- don have occurred. 2- Due to the lack of continuity between the adjacent spans, no catenary action has been developed in the superstructure of the bridge. The two spans have continued falling dawn until one of their sides collided with the ground. 3- The type of the bearing between the girder and the supporting column affects the post-behavior of the girders. 3.5 Collapse mechanism due to column removal In the current bridge, the connection between their other sides and the elastomeric supports has failed. As a result, these sides have col- lided with the adjacent column, resulting in a shear load acting on the column. 4- As previously stated, if the shear capacity of the column is less than the action shear force, it will collapse, and the collapse of the adjacent span will take place, resulting in progres- sively collapse of the bridge. However, the shear capacity of the column in the current model is sufficient to resist the shear force caused by the girder impact on the column. Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Page 14 of 15 5- The final mode of in the current model is that one of the girder sides has rested on the ground while the other side has rested on the column. However, there is another possibility: the entire length of the bridge might fall down to the ground after the fail- ure of the connection between the girder and the elastomeric bearing. This depends on the span of the bridge, the height of the piers, and the column’s mode of failure. 5- The final mode of in the current model is that one of the girder sides has rested on the ground while the other side has rested on the column. However, there is another possibility: the entire length of the bridge might fall down to the ground after the fail- ure of the connection between the girder and the elastomeric bearing. This depends on the span of the bridge, the height of the piers, and the column’s mode of failure. Funding Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Availability of data and materials Availability of data and materials Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. 4 Conclusions In this study, the IAEM has been utilized to perform collapse analysis of prestressed bridges under various hazard loads. A typical prestressed bridge has been analyzed under two severe loading conditions; blast load and sudden failure of column. According to the presented results, progressive collapse phenomena can occur due to the lack of redundancy. Alternative load paths should be provided to avoid such catastrophic col- lapses and minimize the induced damage during blast hazards. Blast resistant bridge design should be developed and implemented by the national building codes to pro- tect bridges against terrorist events. The numerical approach, IAEM, presented in this research, demonstrates a strong capability for studying the overall behavior of pre- stressed bridges under high loading circumstances, from the early stages of loading to final collapse. The investigation has confirmed the IAEM’s dependability in simulating prestressed bridges using a minimum number of elements and, therefore, a reasonable CPU solving time. Competing interests Th th d l p g The authors declare no conflict of interest, financial or otherwise. Received: 23 October 2022 Accepted: 11 November 2022 Authors’ contributions bd l l Authors contributions M. Abdelaziz mainly contributed to the research and numerical studies. M. Gomaa and H. El-Ghazaly mainly contributed as technical leading and support. All authors have read and approved the manuscript. References US Army Corps Ofengineers Yahia MT (2009) Response of bridge structures subjected to blast loads and protection techniques to mitigat of blast hazards on bridges PHD thesis New Brunswick UFC-3-340-02 (2008) Structures to resist the effects of accidental explosions. US Army Corps Ofengineers Yahia MT (2009) Response of bridge structures subjected to blast loads and protection techniques to mitigate the effect of blast hazards on bridges. PHD thesis,, New Brunswick Yahia MT (2009) Response of bridge structures subjected to blast loads and protection techniques to mitigate the effect of blast hazards on bridges. PHD thesis,, New Brunswick g Zhang G, Liu Y, Liu J, Lan S, Yang J (2022) Causes and statistical characteristics of bridge failures: A review. 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In: Proc IABSE Symp Resist i Okamoto H, Maekawa K (1991) Nonlinear analysis and constitutive models of reinforced concrete. Gihodo Shuppan Company Seyed Khoei A, Akbari R, Maalek S, Gharighoran A (2020) Assessment of design and retrofitting solutions on the progres‑ sive collapse of Hongqi bridge. Shock Vib 2020 Seyedkhoei A, Akbari R, Maalek S (2019) Earthquake-induced domino-type progressive collapse in regular, semiregular, and irregular bridges. Shock Vib 2019 g g Tagel-Din H, Meguro K (2000) Applied element method for dynamic large deformation analysis of structures. Doboku Gakkai Ronbunshu 2000:1–10 UFC-3-340-02 (2008) Structures to resist the effects of accidental explosions. US Army Corps Ofengineers Yahia MT (2009) Response of bridge structures subjected to blast loads and protection techniques to mitigat UFC-3-340-02 (2008) Structures to resist the effects of accidental explosions. References e e e ces Abdelaziz MM, El-Ghazaly H, Gomaa MS (2020) Modelling of Prestressed concrete girders using improved applied ele‑ ment method. In: 7th Int. Conf Integrity-Reliability-Failure, pp 505–506 Abdelaziz MM, El-Ghazaly H, Gomaa MS (2020) Modelling of Prestressed concrete girders using improved applied ele‑ ment method. In: 7th Int. Conf Integrity-Reliability-Failure, pp 505–506 Abdelaziz MM, El-Ghazaly HA, Gomaa MS (2021a) Improved applied element model for bonded Prestressed concrete structures. J Struct Eng 147:4020298 Abdelaziz MM, El-Ghazaly HA, Gomaa MS (2021b) Numerical simulation of unbonded prestressed concrete beams using improved applied element method. Eng Struct 245:112962 Amr Ramadan Ibrahim (2018) Reducing progressive collapse in reinforced concrete building structures. PhD Thesis. Benha Faculty of Engineering, Benha University Beshara FBA (1991) Nonlinear finite element analysis of reinforced concrete structures subjected to blast loading. PhD Thesis -. City University London Page 15 of 15 Abdelaziz et al. Advances in Bridge Engineering (2022) 3:24 Bi K, Ren W-X, Cheng P-F, Hao H (2015) Domino-type progressive collapse analysis of a multi-span simply-supported bridge: a case study. Eng Struct 90:172–182 Bi K, Ren W-X, Cheng P-F, Hao H (2015) Domino-type progressive collapse analysis of a multi-span simply-supported bridge: a case study. Eng Struct 90:172–182 g y g BS-5896 TBS (2012) High tensile steel wire and strand for the prestressing of concrete. Specification. g y g BS-5896 TBS (2012) High tensile steel wire and strand for the prestressing of concrete. Specification. Deng L, Wang W, Yu Y (2016) State-of-the-art review on the causes and mechanisms of bridge collapse. J Perform Constr Facil 30:4015005 Deng L, Wang W, Yu Y (2016) State-of-the-art review on the causes and mechanisms of bridge collapse. J Perform Const Facil 30:4015005 ECP-201 (2012) Egyptian code for calculating loads and forces in structural work and masonry. Housing and Building ( ) gyp g y g National Research Center. Ministry of Housing, Utilities and Urban Planning, Cairo y g g ECP-203 (2018) Egyptian code for design and construction of reinforced concrete structures. Housing and Building gy g National Research Center. Ministry of Housing, Utilities and Urban Planning, Cairo gy g National Research Center. Ministry of Housing, Utilities and Urban Planning, Cairo Elkholy S, Meguro K (2004) Numerical simulation of high-rise steel buildings using improved applied element method. In: 13th World Conf. Earthq. Eng. Vancouver, BC, Canada Elkholy S, Meguro K (2004) Numerical simulation of high-rise steel buildings using improved applied element method. Publisher’s Note S i N i Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2120410300	https://www.scielo.br/j/po/a/bLBHrTZjkDWpG7NH9sn5cdx/?lang=pt&format=pdf	Portuguese	null	Mensagem do Presidente da ABPol	Polímeros	2,003	cc-by	403	M E N S A G E M D O P R E S I D E N T E D A A B P O L “Os primeiros 6 meses de 2003 já foram praticamente cumpridos e as realizações no período confirmam as previsões iniciais de um ano desa- fiador para a nossa Associação. A marcante participação na Brasilplast, em março (com o apoio do INP), o sucesso das 70ª. e 71ª reuniões das Comis- sões Técnicas, em fevereiro e abril, e o êxito do curso “Polímeros para Em- balagens”, realizado em maio, representam apenas uma amostra do que nos espera nos próximos semestre... A ABPol, além de co-patrocinar o Seminário sobre Borrachas Termoplásticas organizado pela ABTB - Associação Brasileira de Tecnologia das Borrachas, em junho, realizará no mesmo mês o 7º Seminário das Comissões Técnicas e o segundo curso “in company” do ano, em Joinville. A partir de julho, iniciam-se os preparativos para a realização de 2 seminários técnico-mercadológicos, em Manaus e Salvador, que deverão ser os embriões das futuras Regionais Nordeste e Norte da ABPol, conforme definido no planeja- mento estratégico para os biênios 2002/2003 e 2004/2005. Não bastassem essas iniciativas, atin- girão seu pico ao longo dos próximos quatro meses as atividades e esforços da Comissão Organizadora, da Comissão Científica e da Secretaria da ABPol, para a organização, estruturação e divulgação do 7º CBPol, cujo sucesso e importância estão já avalizados pelos mais de 500 trabalhos submetidos para apresentação no Congresso. Estou certo de que 2003 não será apenas desafiador, mas um ano muito gratificante para a ABPol. Domingos Jafelice E E E Polímeros: Ciência e Tecnologia, vol 13, nº 2, 2003 E D I T O R I A L Polímeros: Ciência e Tecnologia já está consolidada como principal meio de divulgação de trabalhos técnico-científicos em nosso país, em língua portuguesa. Os próximos passos estão sendo direcionados para implementar a indexação da Revista em um maior número de instituições especializadas, principalmente do exterior. O sucesso desta iniciativa está diretamente vinculado à resposta de nossa comunidade em manter nossa Revista como canal importante de divulgação de seus trabalhos e fomentar o uso de citações de artigos publicados. A colaboração e apoio dos pesquisadores levará Polímeros: Ciência e Tecnologia a conse- guir mais esta importante conquista. Comitê Editorial Comitê Editorial E1 Polímeros: Ciência e Tecnologia, vol 13, nº 2, 2003 E1 Polímeros: Ciência e Tecnologia, vol 13, nº 2, 2003
https://openalex.org/W2758496413	https://repository.ubn.ru.nl//bitstream/handle/2066/177124/177124.pdf	English	null	Development and validation of a prediction model for adenoma detection during screening and surveillance colonoscopy with comparison to actual adenoma detection rates	PloS one	2,017	public-domain	9,573	Development and validation of a prediction model for adenoma detection during screening and surveillance colonoscopy with comparison to actual adenoma detection rates Brand, E.C.; Crook, J.E.; Thomas, C.S.; Siersema, P.D.; Rex, D.K.; Wallace, M.B. 2017, Article / Letter to editor (PLoS One, 12, 9, (2017), article e0185560) Doi link to publisher: https://doi.org/10.1371/journal.pone.0185560 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/177124 Download date: 2024-10-24 Note: To cite this publication please use the final published version (if applicable). RESEARCH ARTICLE Editor: John Green, University Hospital Llandough, UNITED KINGDOM Editor: John Green, University Hospital Llandough, UNITED KINGDOM Editor: John Green, University Hospital Llandough, UNITED KINGDOM Received: June 21, 2017 Accepted: September 14, 2017 Published: September 28, 2017 OPEN ACCESS Citation: Brand EC, Crook JE, Thomas CS, Siersema PD, Rex DK, Wallace MB (2017) Development and validation of a prediction model for adenoma detection during screening and surveillance colonoscopy with comparison to actual adenoma detection rates. PLoS ONE 12(9): e0185560. https://doi.org/10.1371/journal. pone.0185560 Materials and methods Screening and surveillance colonoscopy data from the cross-sectional multicenter cluster- randomized Endoscopic Quality Improvement Program-3 (EQUIP-3) study (NCT02325635) was used. The dataset was split into two cohorts based on center. A prediction model for detection of 1 adenoma was developed using multivariable logistic regression and subse- quently internally (bootstrap resampling) and geographically validated. We compared pre- dicted to observed ADRs. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Development and validation of a prediction model for adenoma detection during screening and surveillance colonoscopy with comparison to actual adenoma detection rates Eelco C. Brand1,2, Julia E. Crook3, Colleen S. Thomas3, Peter D. Siersema4, Douglas K. Rex5, Michael B. Wallace1* Eelco C. Brand1,2, Julia E. Crook3, Colleen S. Thomas3, Peter D. Siersema4, Douglas K. Rex5, Michael B. Wallace1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, Florida, United States of America, 2 Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, the Netherlands, 3 Division of Biomedical Statistics and Informatics, Mayo Clinic, Jacksonville, Florida, United States of America, 4 Department of Gastroenterology and Hepatology, Radboud University Medical Center, Nijmegen, The Netherlands, 5 Department of Gastroenterology and Hepatology, Indiana University Medical Center, Indianapolis, Indiana, United States of America * Wallace.Michael@mayo.edu Objective The adenoma detection rate (ADR) varies widely between physicians, possibly due to patient population differences, hampering direct ADR comparison. We developed and vali- dated a prediction model for adenoma detection in an effort to determine if physicians’ ADRs should be adjusted for patient-related factors. Conclusion The substantial variation in ADRs could only partially be explained by patient-related factors. These data suggest that ADR variation could likely also be due to other factors, e.g. physi- cian or technical issues. Abbreviations: ADR, adenoma detection rate; ASA, American Society of Anesthesiology physical status class; BMI, body mass index; CI, confidence interval; C-statistic, concordance statistic; CRC, colorectal cancer; EQUIP, Endoscopic Quality Improvement Program; GIQuIC, Gastro-Intestinal Quality Improvement Consortium; NA, not applicable; NCT, clinical trial registration number of clinicaltrials.gov; OR, odds ratio; SD, standard deviation; TRIPOD, Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis. Prediction of adenoma detection rates during screening and surveillance colonoscopy Competing interests: Peter D. Siersema is a consultant for EndoAid and EndoChoice and received grant support from Pentax and Boston Scientific. Douglas K. Rex is a consultant for Olympus, received research support from EndoChoice, Medivators, EndoAid and Boston Scientific, and received honoraria from Boston Scientific. Michael B. Wallace received grant support from Boston Scientific, Olympus, Medtronic and Ninepoint, and is in possession of stocks of Cosmo pharmaceuticals within a blind trust. None of the mentioned funders had any role in the conception, planning, design, and execution of the study, neither did they have a role in the interpretation of the results, drafting of the manuscript, and decision to publish. This does not alter our adherence to PLOS ONE policies on sharing data and materials. 1.29; 95%-CI: 1.17–1.43, OR class III vs. I: 1.57; 95%-CI: 1.32–1.86), surveillance versus screening (OR: 1.39; 95%-CI: 1.27–1.53), and Hispanic or Latino ethnicity (OR: 1.13; 95%- CI: 1.00–1.27). The model’s discriminative ability was modest (C-statistic in the derivation: 0.63 and validation cohort: 0.60). The observed ADR was considerably lower than predicted for 12/66 (18.2%) physicians and 2/9 (22.2%) centers, and considerably higher than pre- dicted for 18/66 (27.3%) physicians and 4/9 (44.4%) centers. Results The derivation (5 centers, 35 physicians, overall-ADR: 36%) and validation (4 centers, 31 physicians, overall-ADR: 40%) cohort included respectively 9934 and 10034 patients (both cohorts: 48% male, median age 60 years). Independent predictors for detection of 1 ade- noma were: age (optimism-corrected odds ratio (OR): 1.02; 95%-confidence interval (CI): 1.02–1.03), male sex (OR: 1.73; 95%-CI: 1.60–1.88), body mass index (OR: 1.02; 95%-CI: 1.01–1.03), American Society of Anesthesiology physical status class (OR class II vs. I: Funding: Eelco C. Brand received an unrestricted scientific internship (16-03S) abroad grant from The Dutch Digestive Foundation (Maag Lever Darm Stichting). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 1 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Data source We used the data from the cluster-randomized cross-sectional Endoscopic Quality Improve- ment Program-3 (EQUIP-3) study (Clinical Trials Registration: NCT02325635) that ran from September 2013 until January 2015.[9] The EQUIP-3 study was approved by the Mayo Clinic Institutional Review Board and was considered minimal risk and exempt from patient-level consent.[9] In the EQUIP-3 study centers were randomized, after a lead-in phase, to receive a quality improvement program aimed at increasing the ADR or no intervention. During the lead-in phase data on all colonoscopies performed in all participating centers was collected enabling analyses of the colonoscopy quality metrics without the influence of the quality improvement program. The centers randomized to receive the quality improvement program, received the first EQUIP training as described in the EQUIP-1 study,[27] consisting of baseline measurement of ADR, followed by an in-person 1-hour powerpoint based training emphasiz- ing on improvement of adenoma detection and flat lesion recognition. Furthermore in these centers posters about EQUIP were placed in each endoscopy room, and the ADR of all endos- copists was one-on-one discussed, typically with low performers. Each center and individual then received regular follow-up ADR reports, approximately monthly during the post-inter- vention phase.[9] Data was collected through the GIQuIC-form (S1 File)[12] by the physician or nurse at the end of the procedure. Pathology results were entered subsequently when avail- able. Predictor assessment was thus blinded for the outcome. Introduction Colonoscopy combined with polypectomy, when necessary, has been shown to decrease colo- rectal cancer (CRC) incidence[1] and CRC-related mortality.[1,2] However, the protective benefit is reduced by the occurrence of interval-CRC, i.e. CRC occurring within the colonos- copy surveillance interval. Three main reasons for the occurrence of interval-CRC have been suggested in literature, namely: 1) missed lesions during colonoscopy (accounting for approxi- mately 50–60% of the cases), 2) incomplete resection, and 3) newly developed cancers.[3] The proportion of patients undergoing colonoscopy in which at least one adenoma is detected, the adenoma detection rate (ADR), has been shown to be inversely associated with the develop- ment of interval-CRC.[4,5] Quality improvement in colonoscopy therefore aims, among other things, at increasing and thereby achieving sufficient physicians’ ADRs. American and Euro- pean guidelines recommend thus ADRs to be 25%,[6,7] i.e. 30% in male and 20% in female patients.[7] The influence of several modifiable factors, such as procedural and technological factors, on ADR have been studied.[8] Moreover, training programs to improve the ADR have been developed and evaluated.[9,10] Nevertheless, ADRs vary widely between physicians,[11] possi- bly caused by patient population differences. ADR comparison between centers and physicians is therefore challenging and even more complicated by the strict domain in which the ADR should be calculated, i.e. for patients at average risk of adenoma detection in a screening popu- lation. Average risk individuals are those without additional risk factors for adenoma detec- tion, e.g. a family history of CRC, a personal history of CRC or colorectal adenomas, and are not preselected for colonoscopy through, for example, stool tests, e.g. fecal immunochemical tests, or other diagnostic tests. Several initiatives aim to provide physicians with feedback on their ADR through online databases, such as the Gastro-Intestinal Quality Improvement Con- sortium (GIQuIC).[12] The feedback could be improved if next to these unadjusted ADRs the expected ADR for a physician’s patient population could be predicted. Sample split The original dataset was split into two cohorts based on the performing center. This enabled validation of the model in geographically different centers, which is a second-best after fully external validation. In short, in the first cohort, i.e. the derivation cohort, the model will be developed and internally validated. Subsequently, the fitted model will be geographically vali- dated in the second cohort, i.e. the validation cohort. The number of patients, centers, physi- cians, and randomization to the intervention were balanced between the derivation and validation cohort. Introduction Several prediction models based on patient risk factors have been developed for the detec- tion of advanced adenomas[13–23] and a few for any adenoma.[18,20,23–25] Most of the models for any adenoma detection were developed in Asian populations,[18,20,23,24] with a generally lower adenoma prevalence than Western populations, had a moderate discriminative ability, and only included screening colonoscopies.[18,20,23–25]Application of these models is thus hampered in a Western setting and in day-to-day practice where screening and PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 2 / 18 Prediction of adenoma detection rates during screening and surveillance colonoscopy surveillance colonoscopies might both be used for ADR calculations as they are performed side-to-side. Therefore, the aim of the present study was to develop and validate a prediction model for colorectal adenoma detection based on patient risk factors in a screening and surveillance pop- ulation. The secondary aim was to compare the observed individual physicians’ and centers’ ADRs to the predicted proportion of patients with 1 adenoma based on the developed model. Materials and methods The present study has been performed and reported according to the TRIPOD statement for the reporting of multivariable prediction models (S2 File).[26] Predictors for adenoma detection Pre-colonoscopic possible predictors for adenoma detection were selected based on previously published prediction models for the detection of (advanced) adenomas.[13–25] Age and body mass index (BMI) in kg/m2 were analyzed as continuous variables. We cate- gorized race as: “African-American”, “Asian”, “other” (due to the small numbers of patients per subgroup including white, native American, Alaska native, native Hawaiian, native Pacific and patients categorized as other), and “unknown or patient declined to provide”. Ethnicity was categorized as: “Hispanic or Latino”, “not Hispanic or Latino”, and “unknown or patient declined to provide”. As a proxy for clinical condition and co-morbidities the American Society of Anesthesiol- ogy physical status (ASA) was used[28]. Because no ASA V patients and only a small number of ASA IV patients were included, we categorized this variable as: “ASA I”, “ASA II” and “ASA III or IV”. The indication of colonoscopy was surveillance, i.e. a personal history of colorectal adeno- mas or surveillance marked as indication on the GIQuIC form, or colorectal cancer screening. Family history, i.e. 1 first-degree relative <60 years diagnosed with the condition, of colorec- tal adenomas and family history of CRC were analyzed as dichotomous variables. The pre- colonoscopic risk on adenoma detection, i.e. high or low, was not included as a possible pre- dictor due to possible multicollinearity with indication, family history of CRC and family his- tory of colorectal adenomas. Outcome The outcome of the prediction model was the detection of 1 histologically confirmed colo- rectal adenoma per patient. The histological assessment was performed in daily practice, and thus not completely blinded for patient factors, e.g. sex and age of the patient. However, the pathologist did not have access to the GIQuIC form and was consequently blinded to factors such as BMI, ASA class, and race and ethnicity, and it is therefore unlikely that these factors could have influenced the pathologists’ judgment. Prediction of adenoma detection rates during screening and surveillance colonoscopy in the GIQuIC database; and 3) bowel preparation was adequate, i.e. sufficient to accurately detect polyps 6 mm. In the present study we excluded patients: 1) <50 years, because screening is recommended 50 years; 2) with a known increased risk of colorectal neoplasia who are normally discarded in ADR calculations, i.e. colonoscopy for a high-risk genetic CRC syndrome or surveillance colonoscopy because of inflammatory bowel disease; and 3) a personal history of CRC, because these patients will probably have undergone colorectal surgery. Screening and surveillance colonoscopies were both included, because these are performed side-to-side in daily practice and might both be used in ADR calculations. Furthermore, a prediction model for adenoma detection in both a screening and surveillance population potentially facilitates ADR compari- sons across physicians and centers in both screening and surveillance settings. A minuscule proportion (N = 53, 0.2%) of colonoscopies were probably performed in a patient already included in the study, we therefore excluded these second colonoscopies from the database. Participants Sixty-six physicians from nine centers in the United States, i.e. within California, Illinois, Indi- ana, New Mexico, New York, Ohio, Tennessee, and Virginia, participated. Patients undergoing outpatient colonoscopy were included in the EQUIP-3 study if 1) they did not have a history of colorectal surgery; 2) indication for colonoscopy was screening or surveillance, as indicated 3 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Statistical analysis All statistical analyses were performed with R language environment for statistical computing version 3.1.3.[29] 4 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Prediction of adenoma detection rates during screening and surveillance colonoscopy Sample size. We refer to the original paper for sample size calculations.[9] The number of patients with 1 adenoma detected exceeded ten per possible predictor considered, and should therefore be sufficient for the analysis. Sample size. We refer to the original paper for sample size calculations.[9] The number of patients with 1 adenoma detected exceeded ten per possible predictor considered, and should therefore be sufficient for the analysis. Missing data. To correct for possible errors with data-entry on the GIQuIC-form we recoded the following implausible values as missing: BMI <15 or >55kg/m2, height <140 or >220cm, weight <40 or >250kg. We assumed missing data of these variables to be missing at random, in other words the fact that the data is missing is not related to the value that is miss- ing.[30] Multiple imputation based on iterative (10 iterations) chained equations with predic- tive mean matching was performed, creating 20 multiple imputed datasets, using the MICE- package for R.[31] The multiple imputation procedure was performed based on center, physi- cian, all possible predictors and detection of: 1 adenoma, 3 adenomas, any polyp, advanced adenoma(s), adenocarcinoma(s), and serrated lesion(s).[32] The imputed values for BMI were calculated from the imputed height and weight. We assumed patient’s race and ethnicity being categorized as “unknown or patient declined to provide” to be missing not at random, i.e. we expect that there is a reason why these values are not filled out in the GIQuIC database, and we therefore retained this category in the modeling process. Descriptive statistics. The number of patients per center and per physician is presented as medians and ranges. Continuous baseline characteristics are presented as mean ± standard deviation, and categorical data as frequencies with proportions. The proportion of patients with 1 adenoma detected, i.e. the ADR, is reported per subgroup. Univariable odds ratios (ORs) including 95%-confidence intervals (CIs) for the detection of 1 adenoma were estimated for all possible predictors based on logistic regression modeling. An odds ratio was regarded statistically significant if the 95%-CI did not include ‘one’. Model development and internal validation within the derivation cohort. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Statistical analysis A multivari- able logistic regression model with detection of 1 adenoma as outcome was fitted with the following possible predictors: age, sex, BMI, race, ethnicity, ASA class, indication, family his- tory of CRC, family history of colorectal adenomas, interaction between race and sex, and interaction between ethnicity and sex. The model estimates were adjusted for randomization to a quality improvement intervention by adding the dichotomous variable “colonoscopy per- formed after intervention received versus no intervention received” to the model. The final model was selected using stepwise backwards selection based on Akaike’s information criterion. The model’s discriminative ability was assessed with the apparent C-statistic, which is equivalent to the area under the receiver operating characteristic curve. Subsequently internal validation was performed using 1000 bootstrap resamples per imputed dataset to calculate the shrinkage factor and optimism-corrected C-statistic. Regular bootstrap resampling without taking clustering into account was performed.[33] The optimism-corrected model coefficients and intercept were calculated based on the shrinkage factor. The model calibration was visually assessed with a calibration plot. Geographical validation and model-update within the validation cohort. Because we expected differences in the baseline adenoma prevalence and overall effect of predictors due to geographic variation we performed logistic re-calibration by fitting a logistic regression model with the original model’s linear predictor as the independent variable and detection of 1 adenoma as the dependent variable. The intercept and coefficient of this new logistic model are the re-calibrated intercept and slope-correction, respectively.[34] The discrimina- tive ability (C-statistic) and calibration (with a calibration plot) of the updated model were assessed. 5 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Prediction of adenoma detection rates during screening and surveillance colonoscopy Missing values No outcome data were missing. A substantial proportion (28.2–35.1%) of patients had missing values for height, weight or BMI (Fig 1). Baseline characteristics and ADR per subgroup of patients The provider and patient characteristics, and ADR per variable are summarized in Table 1. The reasons for being at high risk of adenoma detection are summarized in S1 Table. Within the derivation cohort the mean age was 60.2 years, 47.6% were male, and the mean BMI was 28.3 kg/m2. The overall ADR was 35.9%, with ADRs for female (29.6%) and male (42.9%) patients above the recommended thresholds[7] of 20% and 30% respectively. Within the validation cohort the patients were slightly older (mean age 61.0 years), almost the same pro- portion was male (47.8%), and patients had a higher mean BMI (29.2 kg/m2). The overall ADR (40.0%) was higher, and again the ADR for female (34.9%) and male (45.6%) patients was above the recommended thresholds[7]. Regarding the baseline characteristics the derivation and validation cohort differed the most with respect to the included proportion of African-American and Hispanic or Latino patients, and colonoscopies with screening as indication (Table 1). Study population The 22316 patients were divided between the derivation and validation cohort. In the deriva- tion cohort 9934 patients (1223 patients were excluded) were examined by 35 physicians in five centers (Fig 1). In the validation cohort 10034 patients (1125 patients were excluded) were examined by 31 physicians in four centers. Predicted proportion of patient with 1 adenoma detected compared to actual ADRs In both cohorts the probability for the detection of 1 adenoma per patient was predicted with the final model. The sum of the predicted probabilities of adenoma detection per patient was calculated resulting in the predicted proportion of patients with 1 adenoma per physi- cian and center. These predicted proportions were graphically compared to the physicans’ and centers’ observed ADR, i.e. the proportion of patients with 1 adenoma detected, including 95% Wilson CIs. The observed ADR was considered considerably lower than predicted if the upper bound of the 95% CI was lower than the predicted ADR, and considerably higher than predicted if the lower bound of the 95% CI was higher than the predicted ADR. Univariable association between predictors and detection of 1 adenoma In the derivation cohort statistically significant positive predictors for detection of 1 ade- noma were age (per year increase), male sex, BMI (per kg/m2 increase), ASA class II compared to class I, ASA class III or IV compared to I, Hispanic or Latino ethnicity and surveillance compared to screening as indication (Table 2). Some univariable associations within the validation cohort clearly differed from those observed in the derivation cohort. The OR of ASA class III or IV compared to I was lower and statistically non-significant. The OR of family history of CRC was above 1 in the validation cohort, but was still non-significant. The OR for African-American, Asian, and Hispanic or 6 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Prediction of adenoma detection rates during screening and surveillance colonoscopy Fig 1. Flowchart of patients in the derivation and validation cohort with an overview of the reasons for and number of excluded patients and number of implausible and missing values. BMI, body mass index; CRC, colorectal cancer; EQUIP-3, Endoscopic Quality Improvement Program-3 study; IBD, inflammatory bowel disease; N, number of patients. Fig 1. Flowchart of patients in the derivation and validation cohort with an overview of the reasons for and number of excluded patients and number of implausible and missing values. BMI, body mass index; CRC, colorectal cancer; EQUIP-3, Endoscopic Quality Improvement Program-3 study; IBD, inflammatory bowel disease; N, number of patients. https://doi.org/10.1371/journal.pone.0185560.g001 https://doi.org/10.1371/journal.pone.0185560.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 7 / 18 Prediction of adenoma detection rates during screening and surveillance colonoscopy Table 1. Provider and patient characteristics for the derivation and validation cohort and the adenoma detection rate per patient subgroup. Derivation cohort Validation cohort All patients [N = 9934] N (%)a Patients with 1 adenoma [N = 3568] N (ADR)a All patients [N = 10034] N (%)a Patients with 1 adenoma [N = 4013] N (ADR)a Provider characteristics Centerb, median N (range) 1544 (1144–3336) 537 (431–1232) 2492.5 (1228–3821) 735 (561–1982) Physicianc, median N (range) 289 (55–725) 93 (11–306) 246 (57–812) 96 (12–330) Patient characteristics Age in years, mean ± SD 60.2 ± 7.7 61.3 ± 7.8 61.0 ± 8.2 61.8 ± 8.3 Female 5209 (52.4) 1542 (29.6) 5237 (52.2) 1826 (34.9) Male 4725 (47.6) 2026 (42.9) 4797 (47.8) 2187 (45.6) BMI in kg/m2, mean ± SD 28.3 ± 5.6 [N = 6851]d 28.8 ± 5.5 [N = 2538]d 29.2 ± 5.8 [N = 6511]d 29.7 ± 5.8 [N = 3120]d ASA I 2689 (27.1) 793 (29.5) 1920 (19.1) 668 (34.8) ASA II 6455 (65.0) 2420 (37.5) 7532 (75.1) 3119 (41.4) ASA III or IVe 790 (8.0) 355 (44.9) 582 (5.8) 226 (38.8) Race Otherf 6368 (64.1) 2250 (35.3) 8306 (82.8) 3463 (41.7) African-American or black 1320 (13.3) 477 (36.1) 701 (7.0) 263 (37.5) Asian 134 (1.3) 49 (36.6) 149 (1.5) 51 (34.2) Unknown or patient declined to provide 2112 (21.3) 792 (37.5) 878 (8.8) 236 (26.9) Ethnicity Not Hispanic or Latino 5933 (59.7) 2067 (34.8) 8451 (84.2) 3517 (41.6) Hispanic or Latino 1415 (14.2) 533 (37.7) 87 (0.9) 31 (35.6) Unknown or patient declined to provide 2586 (26.0) 968 (37.4) 1496 (14.9) 465 (31.1) Indication for colonoscopyg Screening 7353 (74.0) 2409 (32.8) 6518 (65.0) 2361 (36.2) Surveillance 2581 (26.0) 1159 (44.9) 3516 (35.0) 1652(47.0) Risk assessmenth Average risk 6558 (66.0) 2168 (33.1) 6589 (65.7) 2452 (37.2) High risk 3376 (34.0) 1400 (41.5) 3445 (34.3) 1561 (45.3) ADR d d t ti t i ti f ti t ith 1 d d t t d b ASA A i S i t f A th i l h i l ADR, adenoma detection rate, i.e. proportion of patients with 1 adenoma detected per subgroup; ASA, American Society of Anesthesiology physical status class; BMI, body mass index; N, number of patients; SD, standard deviation. ADR, adenoma detection rate, i.e. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Latino patients was <1 within the validation cohort, while these variables had an OR >1 in the derivation cohort. Latino patients was <1 within the validation cohort, while these variables had an OR >1 in the derivation cohort. Latino patients was <1 within the validation cohort, while these variables had an OR >1 in the derivation cohort. proportion of patients with 1 adenoma detected per subgroup; ASA, American Society of Anesthesiology physical status class; BMI, body mass index; N, number of patients; SD, standard deviation. aUnless, stated otherwise in the beginning of the row. bFive centers were included in the derivation cohort, and four centers in the validation cohort. cThirty-five physicians were included in the derivation cohort, and thirty-one physicians were included in the validation cohort. dNumber of patients without missing values. eOnly three patients in the development cohort and no patients in the validation cohort were in ASA category IV and therefore ASA III and IV were combined. fIncluding white, native American, Alaska native, native Hawaiian, native Pacific patients and patient’s race categorized as other. gThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form. hThe number of patients categorized per reason for high risk of adenoma detection are displayed in supporting information Table 1 (S1 Table). https://doi.org/10.1371/journal.pone.0185560.t001 y p aUnless, stated otherwise in the beginning of the row. bFive centers were included in the derivation cohort, and four centers in the validation cohort. cThirty-five physicians were included in the derivation cohort and thirty-one physicians were included in the validation cohort eOnly three patients in the development cohort and no patients in the validation cohort were in ASA category IV and therefore ASA III and IV were combined. fIncluding white, native American, Alaska native, native Hawaiian, native Pacific patients and patient’s race categorized as other. gTh i di ti i id d ill f ti t ith l hi t f l t l d ill k d i di ti th fIncluding white, native American, Alaska native, native Hawaiian, native Pacific patients and patient’s race categorized as other. gThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the Including white, native American, Alaska native, native Hawaiian, native Pacific patients and patient s race categorized as other. gThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form. https://doi.org/10.1371/journal.pone.0185560.t001 8 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Prediction of adenoma detection rates during screening and surveillance colonoscopy Table 2. Univariable odds ratios of possible risk factors for the detection of 1 adenoma within the derivation and validation cohort. Possible predictors Derivation cohort Validation cohort Univariable odds ratioa [95%-CI] Univariable odds ratioa [95%-CI] Age (per year increase) 1.03 [1.02–1.04] 1.02 [1.02–1.03] Female 1.00 (ref) 1.00 (ref) Male 1.79 [1.64–1.94] 1.57 [1.44–1.70] BMI (per kg/m2 increase)b 1.02 [1.01–1.03] 1.03 [1.02–1.04] ASA I 1.00 (ref) 1.00 (ref) ASA II 1.43 [1.30–1.58] 1.32 [1.19–1.47] ASA III or IVc 1.95 [1.66–2.30] 1.19 [0.98–1.44] Race Otherd 1.00 (ref) 1.00 (ref) African-American or black 1.04 [0.91–1.17] 0.84 [0.72–0.98] Asian 1.06 [0.73–1.50] 0.73 [0.51–1.02] Unknown or patient declined to provide 1.10 [0.99–1.22] 0.51 [0.44–0.60] Ethnicity Not Hispanic or Latino 1.00 (ref) 1.00 (ref) Hispanic or Latino 1.13 [1.00–1.27] 0.78 [0.49–1.20] Unknown or patient declined to provide 1.12 [1.02–1.23] 0.63 [0.56–0.71] Indication for colonoscopye Screening 1.00 (ref) 1.00 (ref) Surveillance 1.67 [1.53–1.83] 1.56 [1.44–1.70] History of colorectal cancer No 1.00 (ref) 1.00 (ref) Familyf 0.88 [0.75–1.02] 1.13 [0.97–1.31] History of colorectal adenomas No 1.00 (ref) 1.00 (ref) Familyf 1.12 [0.93–1.35] 1.17 [0.88–1.55] ASA, American Society of Anesthesiology physical status class; BMI, body mass index; CI, confidence interval; ref, reference category. aOdds ratios based on a univariable logistic regression model with detection of 1 adenoma as outcome. 95% confidence intervals are profiled confidence intervals. , y gy p y ; , y ; , ; , g y aOdds ratios based on a univariable logistic regression model with detection of 1 adenoma as outcome. 95% confidence intervals are profiled confidence intervals. bThis association is calculated after multiple imputation. bThis association is calculated after multiple imputation. cO S S cOnly three patients in the development cohort and no patients in the validation cohort were in ASA category IV and combined. dIncluding white, native American, Alaska native, native Hawaiian and native Pacific patients and patient’s race categorized as other. eThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form. f y fFamily history is defined as a first degree relative diagnosed with the condition at an age <60 years. https://doi.org/10.1371/journal.pone.0185560.t002 Geographical validation within the validation cohort The re-calibrated model with an intercept of -2.406 and overall slope correction of 0.757 (final model estimates not shown) had a modest discriminative ability (C-statistic 0.603). The model tended to overestimate the adenoma detection among patients with a high observed adenoma detection (Fig 2B). Prediction of adenoma detection rates during screening and surveillance colonoscopy Table 3. Prediction modela for the detection of 1 adenoma per patient based on multivariable logistic regression within the derivation cohort. Factors Uncorrected multivariable OR [95%-CI] Correctedb β coefficients Correctedb multivariable OR [95%-CI] Intercept - -3.134 - Age (per year increase) 1.02 [1.02–1.03] 0.023 1.02 [1.02–1.03] Female 1.00 (ref) 0 (ref) 1.00 (ref) Male 1.76 [1.62–1.92] 0.549 1.73 [1.60–1.88] BMI (per kg/m2 increase) 1.02 [1.01–1.03] 0.017 1.02 [1.01–1.03] ASA I 1.00 (ref) 0 (ref) 1.00 (ref) ASA II 1.30 [1.18–1.44] 0.256 1.29 [1.17–1.43] ASA III or IV 1.59 [1.34–1.90] 0.451 1.57 [1.32–1.86] Ethnicity Not Hispanic or Latino 1.00 (ref) 0 (ref) 1.00 (ref) Hispanic or Latino 1.13 [1.00–1.28] 0.122 1.13 [1.00–1.27] Unknown or patient declined to provide 1.16 [1.05–1.29] 0.148 1.16 [1.05–1.28] Indication for colonoscopyc Screening 1.00 (ref) 0 (ref) 1.00 (ref) Surveillance 1.41 [1.28–1.55] 0.332 1.39 [1.27–1.53] ASA, American Society of Anesthesiology physical status class; BMI, body mass index; CI, confidence interval; OR, odds ratio; ref, reference category. aThe presented odds ratios are adjusted for EQUIP intervention (colonoscopy performed after versus no intervention received) which had an uncorrected OR of 1.24 [95%-CI: 1.13–1.36] in the final model. bCorrected after internal validation using bootstrap resampling with a shrinkage factor of 0.969. The intercept is additionally corrected by subtraction of the intercept correction of -0.017. cThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form ction of 1 adenoma per patient based on multivariable logistic regression within the derivation cohort. cThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form. https://doi.org/10.1371/journal.pone.0185560.t003 accurate, however, the model overestimated the probability of the detection of 1 adenoma among patients with a low observed and especially high observed adenoma detection (Fig 2A). Model development and validation within the derivation cohort After stepwise backwards selection the following patient-related predictors were selected in the multivariable model (Table 3): age, sex, BMI, ASA class, indication (surveillance versus screen- ing), and Hispanic or Latino ethnicity. The discriminative ability was modest (apparent C-sta- tistic: 0.630 and optimism-adjusted C-statistic: 0.626). Calibration of the model was visually PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 9 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 ASA, American Society of Anesthesiology physical status class; BMI, body mass index; CI, confidence interval; OR, odds ratio; ref, reference category. aThe presented odds ratios are adjusted for EQUIP intervention (colonoscopy performed after versus no intervention received) which had an uncorrected OR of 1.24 [95%-CI: 1.13–1.36] in the final model. bCorrected after internal validation using bootstrap resampling with a shrinkage factor of 0.969. The intercept is additionally corrected by subtraction of the intercept correction of -0.017. cThe indication is considered surveillance for patients with a personal history of colorectal adenomas or surveillance marked as indication on the GastroIntestinal Quality Improvement Consortium (GIQuIC) form. Prediction of adenoma detection rates during screening and surveillance colonoscopy Fig 2. Calibration plot of the model performance A) within the derivation cohort after internal validation (figure at the left), and B) within the validation cohort after logistic re-calibration (figure at the right). Grouped observations are grouped per decile of patients. The observed proportion of patients with 1 adenoma detected is displayed on the y-axis, and the predicted proportion of patients with 1 adenoma on the x-axis. C-statistic, concordance statistic. Fig 2. Calibration plot of the model performance A) within the derivation cohort after internal validation (figure at the left), and B) within the validation cohort after logistic re-calibration (figure at the right). Grouped observations are grouped per decile of patients. The observed proportion of patients with 1 adenoma detected is displayed on the y-axis, and the predicted proportion of patients with 1 adenoma on the x-axis. C-statistic, concordance statistic. https://doi.org/10.1371/journal.pone.0185560.g002 Predicted proportion of patients with 1 adenoma compared to observed ADR Within the derivation cohort the median observed ADR was 35.2% (range: 20.0–52.5%) per physician and 36.9% (range: 27.9–39.5%) per center. Six physicians and no center had an ADR below the recommended threshold[6,7] of 25%. The median predicted proportion of patients with 1 adenoma was 36.7% (range: 31.0–40.2%) per physician and 36.5% (range: 35.0%-38.0%) per center. The upper bound of the 95%-CI of the observed ADR was lower than the predicted proportion of patients with 1 adenoma for five physicians and one center. The lower bound of the 95%-CI of the observed ADR was higher than the predicted propor- tion of patients with 1 adenoma for five physicians and one center (Figs 3 and 4A). Within the validation cohort the median observed ADR was 41.9% (range: 4.2–65.0%) per physician and 43.4% (range: 23.5–51.9%) per center. Five physicians and one center had an PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 10 / 18 Fig 3. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the derivation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. https://doi org/10 1371/journal pone 0185560 g003 Fig 3. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the derivation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. Fig 3. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the derivation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. https://doi.org/10.1371/journal.pone.0185560.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 11 / 18 Prediction of adenoma detection rates during screening and surveillance colonoscopy Fig 4. A. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per center within A) the derivation cohort (figure at the left) and B) the validation cohort (figure at the right). The centers are ranked in ascending order of adenoma detection rate. The numbers on the x-axis denote the number of patients per center. https://doi.org/10.1371/journal.pone.0185560.g004 Fig 4. A. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per center within A) the derivation cohort (figure at the left) and B) the validation cohort (figure at the right). The centers are ranked in ascending order of adenoma detection rate. The numbers on the x-axis denote the number of patients per center. https://doi.org/10.1371/journal.pone.0185560.g004 https://doi.org/10.1371/journal.pone.0185560.g004 observed ADR <25%. The median predicted proportion of patients with 1 adenoma was 39.1% (range: 31.9–44.2%) per physician and 39.1% (range: 36.4–41.7%) per center. The upper bound of the 95%-CI of the observed ADR was lower than the predicted proportion of patients with 1 adenoma for seven physicians and one center. The lower bound of the 95%-CI of the observed ADR was higher than the predicted proportion of patients with 1 adenoma for 13 physicians and three centers (Figs 5 and 4B). Discussion This study shows that age, sex, BMI, ASA class, surveillance vs. screening, and a Hispanic or Latino ethnicity are independent patient-related predictors of colorectal adenoma detection in a screening and surveillance population. These patient risk factors only modestly account for the variation in individual physicians’ and centers’ ADR. The final multivariable model had a moderate discriminative ability within the derivation and validation cohort, which slightly increased after the addition of the performing physician as predictor. The lowest observed ADRs were lower than predicted, while the highest observed ADRs were higher than pre- dicted. Approximately one in six physicians performed colonoscopy with an ADR below that predicted by patient risk factors. Our finding that increasing age is an independent predictor of adenoma detection is in line with the recommendation to start screening colonoscopy from 50 years onwards, since ade- noma detection increases with age.[35] The increased risk (OR: 1.73, 95%-CI: 1.60–1.88) for adenoma detection in male versus female patients is consistent with the current recommended difference in adenoma detection rate for male patients (30%) and female patients (20%) that equals an OR of 1.71.[7] Increasing BMI was included as a predictor in almost every previ- ous prediction model for adenoma detection[18,20,24,25] as is the case in our prediction model. A recent Asian study found a self-reported family history of colorectal adenomas to be associated with an increased risk of adenoma detection.[36] Moreover, a family history of advanced adenomas has been shown to increase the risk of advanced adenoma detection.[37] However in the present study a family history of adenomas was not independently predictive for adenoma detection. Interestingly a family history of CRC was not found to be an indepen- dent predictor either, while a family history of CRC has been previously included in prediction models for both adenoma[18,23–25] and advanced adenoma detection.[16–19,22,23] Some possible predictors for adenoma detection that have been associated with (advanced) adenoma detection or CRC development are not collected within the GIQuIC database. Smok- ing was included in prediction models for adenoma detection.[18,23–25] Furthermore alcohol consumption has been associated with the development of (advanced) adenomas,[38] and has been included as independent predictor in two prediction models for advanced adenomas. Fig 5. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the validation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. Fig 5. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the validation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. Fig 5. Observed adenoma detection rates (including 95% Wilson confidence intervals) versus predicted proportions of patients with 1 adenoma per physician within the validation cohort. The physicians are ranked in ascending order of adenoma detection rate. The numbers on the x- axis denote the number of patients per physician. https://doi.org/10.1371/journal.pone.0185560.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 12 / 18 Prediction of adenoma detection rates during screening and surveillance colonoscopy Overall, there was no obvious trend of smaller sample sizes per physician or center at the extremes of observed ADRs. The predicted proportion of patients with 1 adenoma varied less than the observed ADR. Model performance including physician as predictive factor Given the moderate discriminative ability of the prediction model, a sensitivity analysis was performed in which the performing physician was added as a variable during model develop- ment within the derivation cohort. After stepwise backwards selection performing physician was selected in the model and ethnicity was left out which led to a modest increase in the apparent C-statistic from 0.630 to 0.647. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Discussion [21,22] Certain medication, such as aspirin has been associated with a decreased risk of CRC development[39] and could therefore have possibly added to the discrimination between patients with and without adenomas.[25] In our model ASA class, which is routinely assessed to make sedation decisions, might function as a proxy for medical condition in general. Never- theless, assignment of ASA-class might heavily depend on the assessor, and certain medical conditions, if predictive, would be less prone to observer judgment and might be more specific. Lastly, dietary components, e.g. dietary fibers have been associated with a decreased risk of PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 13 / 18 Prediction of adenoma detection rates during screening and surveillance colonoscopy colorectal adenoma development,[40] and fried food, picked food, white meat or green vegeta- ble consumption were predictive of advanced adenoma detection in a Chinese population.[15] However, dietary factor assessment in clinical practice might suffer from recall bias. These fac- tors should be considered to be included as variables within ADR monitoring databases such as GIQuIC, if addition of any of these predictors would improve prediction of adenoma detection. The moderate discriminative ability of our patient-factor based prediction model for ade- noma detection is in line with a recent study in which adjustment of the ADR for age, sex, race/ethnicity, and family history of CRC did reduce the variability in ADRs but had only a small effect on the differences of ADRs between physicians.[41] In another study excluding different patient subgroups from ADR calculations changed ADRs substantially, but had only a small effect on the ADR ranking among physicians[42] suggesting that factors, such as physi- cian-related, procedural or technological factors, are likely to influence the ADR. A physician- effect might also be part of the explanation of the ADR increase during the first ten years of the German CRC screening program, because physicians who started to perform screening colo- noscopy detected more adenomas compared to physicians who stopped performing colonos- copies.[43] Unfortunately, no physician-factors were known in our study, therefore their influence on the variation in ADR could not be assessed. Future identification of modifiable physician-factors next to for example withdrawal time could create opportunities for further quality improvement in colonoscopy. Discussion In this study, to the best of our knowledge, we describe the first prediction model for ade- noma detection in a screening and surveillance population and its application to compare the predicted proportion of patients with 1 adenoma to the individual physician’s and center’s observed ADRs. The study is strengthened by the prospective data collection. The large sample size decreased the risk of model overfitting, which was further decreased by the internal and geographical validation, however, a full external validation is still preferable before the model would be widely applied. Our study also has some limitations. Firstly, although handled in the most appropriate way with multiple imputations, the substantial proportion of missing data on BMI might have influenced our results. Moreover, data on race and ethnicity was possibly missing not at ran- dom and could therefore not be handled with multiple imputation. Data on smoking status was unknown for all patients. Secondly, the race and ethnicity sub classification was based on the possibilities within the GIQuIC form; therefore we could for example not specify whether Asian patients originally came from Asian countries with a low or high colorectal adenoma prevalence. Thirdly, due to the design of data collection the pathologists could not be blinded for all predictors during histology assessment. Fourthly, the number of patients who under- went surveillance colonoscopy earlier or later than recommended is unknown. This could have influenced the ADR in the surveillance group, however compared to screening colonos- copy more patients with 1 adenoma were detected during surveillance colonoscopy in the present study. Lastly, since adenoma detection is not considered to be perfect with reported per adenoma miss rates up to 20% of all adenomas,[44] the reference standard for detection, i.e. colonoscopy, is not perfect. If the performing physician or procedural factors are truly influencing the adenoma detection per patient, the outcome assessment might have been biased. Unfortunately, it is not possible to determine to what extent this has influenced the per- formance of our model and the selection of predictors. Conclusions We developed and validated a prediction model for colorectal adenoma detection in a screen- ing and surveillance population based on patient-related predictors, i.e. age, sex, BMI, ASA 14 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185560 September 28, 2017 Prediction of adenoma detection rates during screening and surveillance colonoscopy class, surveillance as indication, and Hispanic or Latino ethnicity, with a modest discriminative ability. Additional patient-related predictors that were not included in the database, e.g. smok- ing, alcohol use, medication and medical history, could possibly have added to the model per- formance. However, these data suggest that variation in ADR between physicians could likely be due to a combination of patient-related and other factors, e.g. physician, procedural or tech- nical issues. These data also suggest that a low individual physician’s ADR, can only partially be explained by patient mix, and efforts to improve ADR to meet current guidelines should be pursued. S2 Database. De-identified database of the validation cohort. (CSV) S1 File. Gastro-Intestinal Quality Improvement Consortium (GIQuIC) data-entry form. (PDF) S2 File. Transparent Reporting of a multivariable prediction model for Individual Progno- sis or Diagnosis (TRIPOD) checklist. (PDF) S1 Table. Reasons of high-risk assessment for adenoma detection and adenoma detection rate per high-risk assessment reason. (DOCX) Acknowledgments We thank Max Peters, MD, PhD, for his advice on the modeling in R. We thank Max Peters, MD, PhD, for his advice on the modeling in R. Supporting information Supporting information S1 Database. De-identified database of the derivation cohort. (CSV) S2 Database. De-identified database of the validation cohort. (CSV) S1 Data Dictionary. Description of the variables within the databases. (XLSX) S1 File. Gastro-Intestinal Quality Improvement Consortium (GIQuIC) data-entry form. (PDF) S2 File. Transparent Reporting of a multivariable prediction model for Individual Progno- sis or Diagnosis (TRIPOD) checklist. (PDF) S1 Table. Reasons of high-risk assessment for adenoma detection and adenoma detection rate per high-risk assessment reason. (DOCX) S1 Database. De-identified database of the derivation cohort. (CSV) S2 Database. De-identified database of the validation cohort. (CSV) S2 Database. De-identified database of the validation cohort. (CSV) References 1. Nishihara R, Wu K, Lochhead P, Morikawa T, Liao X, Qian ZR, et al. Long-term colorectal-cancer inci- dence and mortality after lower endoscopy. N Engl J Med. United States; 2013; 369: 1095–1105. https://doi.org/10.1056/NEJMoa1301969 PMID: 24047059 2. Zauber AG, Winawer SJ, O’Brien MJ, Lansdorp-Vogelaar I, van Ballegooijen M, Hankey BF, et al. 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Brand, Julia E. Crook, Colleen S. Thomas, Michael B. Wallace. Supervision: Michael B. Wallace. Visualization: Eelco C. Brand. Writing – original draft: Eelco C. Brand, Michael B. Wallace. Writing – review & editing: Eelco C. Brand, Julia E. Crook, Colleen S. Thomas, Peter D. S sema, Douglas K. Rex, Michael B. Wallace. Conceptualization: Eelco C. Brand, Julia E. Crook, Michael B. Wallace. Data curation: Colleen S. Thomas. Investigation: Eelco C. Brand, Peter D. Siersema, Douglas K. Rex, Michael B. Wallace. Methodology: Eelco C. Brand, Julia E. Crook, Colleen S. Thomas, Michael B. Wallace. Supervision: Michael B. Wallace. Writing – original draft: Eelco C. Brand, Michael B. Wallace. Writing – review & editing: Eelco C. Brand, Julia E. Crook, Colleen S. Thomas, Peter D. Sier- sema, Douglas K. Rex, Michael B. 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https://openalex.org/W2523202475	https://peerj.com/articles/2449.pdf	English	null	In vitroandin vivocomparison of transport media for detecting nasopharyngeal carriage ofStreptococcus pneumoniae	PeerJ	2,016	cc-by	4,805	In vitro and in vivo comparison of transport media for detecting nasopharyngeal carriage of Streptococcus pneumoniae Anneke Steens1,2, Natacha Milhano1,3, Ingeborg S. Aaberge1 and Didrik F. Vestrheim1 1 Infection Control and Environmental Health, Norwegian Institute of Public Health, Oslo, Norway 2 Faculty of Medicine, University of Oslo, Oslo, Norway 3 European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden 1 Infection Control and Environmental Health, Norwegian Institute of Public Health, Oslo, Norway 2 Faculty of Medicine, University of Oslo, Oslo, Norway 3 E P f P bli H l h Mi bi l T i i (EUPHEM) E C f Di 3 European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden Subjects Microbiology, Infectious Diseases Keywords Streptococcus pneumoniae, Nasopharyngeal carriage, Transport media, In vivo and in vitro comparison, Carriage study, Pneumococcus ABSTRACT Background. As a standard method for pneumococcal carriage studies, the World Health Organization recommends nasopharyngeal swabs be transported and stored at cool temperatures in a medium containing skim-milk, tryptone, glucose and glycerol (STGG). An enrichment broth used for transport at room temperature in three carriage studies performed in Norway may have a higher sensitivity than STGG. We therefore compared the media in vitro and in vivo. Methods. For the in vitro component, three strains (serotype 4, 19F and 3) were suspended in STGG and enrichment broth. Recovery was compared using latex agglutination, quantification of bacterial loads by real-time PCR of the lytA gene, and counting colonies from incubated plates. For the in vivo comparison, paired swabs were obtained from 100 children and transported in STGG at cool temperatures or in enrichment broth at room temperature. Carriage was identified by latex agglutination and confirmed by Quellung reaction. Results. In vitro, the cycle threshold values obtained by PCR did not differ between the two media (p = 0.853) and no clear difference in colony counts was apparent after incubation (p = 0.593). In vivo, pneumococci were recovered in 46% of swabs transported in STGG and 51% of those transported in enrichment broth (Kappa statistic 0.90, p = 0.063). Submitted 27 June 2016 Accepted 15 August 2016 Published 22 September 2016 Corresponding author Anneke Steens, anneke.steens@fhi.no Academic editor Luc Van Kaer Additional Information and Declarations can be found on page 7 DOI 10.7717/peerj.2449 Copyright 2016 Steens et al. Distributed under Creative Commons CC-BY 4.0 Submitted 27 June 2016 Accepted 15 August 2016 Published 22 September 2016 Corresponding author Anneke Steens, anneke.steens@fhi.no Academic editor Luc Van Kaer Additional Information and Declarations can be found on page 7 DOI 10.7717/peerj.2449 Copyright 2016 S l Discussion. Overall, no statistical differences in sensitivity were found between STGG and enrichment broth. Nevertheless, some serotype differences were observed and STGG appeared slightly less sensitive than enrichment broth for detection of nasopharyngeal carriage of pneumococci by culturing. We recommend the continued use of STGG for transport and storage of nasopharyngeal swabs in pneumococcal carriage studies for the benefit of comparability between studies and settings, including more resource-limited settings. Subjects Microbiology, Infectious Diseases Keywords Streptococcus pneumoniae, Nasopharyngeal carriage, Transport media, In vivo and in vitro comparison, Carriage study, Pneumococcus Distributed under Creative Commons CC-BY 4.0 How to cite this article Steens et al. How to cite this article Steens et al. (2016), In vitro and in vivo comparison of transport media for detecting nasopharyngeal carriage of Streptococcus pneumoniae. PeerJ 4:e2449; DOI 10.7717/peerj.2449 INTRODUCTION Monitoring carriage of Streptococcus pneumoniae (pneumococci) is important for determining changes after vaccine introduction in national immunisation programmes. To enable comparison of results from different studies and countries, the World Health Organization Pneumococcal Carriage Working Group published a set of standard methods for such studies measuring nasopharyngeal carriage of pneumococci (Satzke et al., 2013). A medium containing skim milk powder, tryptone soy broth, glucose and glycerol in distilled water (STGG) is recommended for transport and storage of nasopharyngeal specimens, and transport should be done at cool temperatures (O’Brien et al., 2001). Studies using STGG in developed countries have generally revealed prevalences of pneumococcal carriage in children of around 30–50% (Andrade et al., 2014; Van Hoek et al., 2014; Desai et al., 2015). In Norway, several carriage studies have been performed using enrichment broth (beef infusion enriched with 5% horse serum and 3.3% defibrinated horse blood (Kaltoft et al., 2008); Statens Serum Institute, Copenhagen, Denmark) for transport at room temperature. Carriage prevalence in those studies was around 80% before and after introduction of the 7-valent pneumococcal conjugate vaccine (PCV), and 62% two years after switching to the 13-valent PCV (Steens et al., 2015). Although different factors may contribute to this high prevalence, such as the percentage of children in day-care (>90% (Statistics Norway, 2010)) and the low use of antibiotics in Norway (Garcia-Rodriguez & Fresnadillo Martinez, 2002; Norwegian Veterinary Institute, 2014), results suggest that enrichment broth transported at room temperature may be more sensitive for detection of carriage than STGG transported at cool temperatures. We therefore compared (1) in vitro recovery from serial dilutions in STGG and enrichment broth and (2) in vivo detection of nasopharyngeal carriage of pneumococci from swabs that were transported and stored in STGG at cool temperatures or in enrichment broth at room temperature. ABSTRACT (2016), In vitro and in vivo comparison of transport media for detecting nasopharyngeal carriage of Streptococcus pneumoniae. PeerJ 4:e2449; DOI 10.7717/peerj.2449 MATERIALS AND METHODS In the in vitro component of the study, we compared recovery rates of pneumococci from serial dilutions that had been stored at different temperatures and media using (I) culturing, (II) a commercial latex agglutination kit and (III) quantitative real-time PCR (qPCR). In the in vivo component, we compared carriage using paired swabs taken from children attending day-care centres and transported in STGG or enrichment broth. In the second part, we used methods (I) and (II) for detection of pneumococci. See Fig. 1 for a schematic overview of the procedures. Note that we followed the recommended conditions for transport and storage for each medium (wet ice/cool box for STGG and freezing (in vivo only), room temperature and immediate processing for enrichment broth). Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 In vitro comparison Furthermore, 100 µL of the STGG samples was added to 3ml fresh enrichment broth. All was done in triplicate. Plates and tubes (broth sample made from the STGG samples and the initial enrichment broth samples) were incubated overnight at 35 ◦C with 5% CO2. Pneumococci were identified by latex agglutination (Pneumotest-Latex kit; Statens Serum Institut, Denmark; Slotved et al., 2004) from the incubated broths. Quantification of the bacterial loads was performed by qPCR (see below for details) and counting of the colony forming units (CFU) from the incubated plates. In vitro comparison Three strains belonging to different serotypes were used as pneumococcal samples; two reference strains (ATCC49619—serotype 19F, and TIGR4—serotype 4), and a strain belonging to serotype 3 obtained from the 2013 sample of a previous Norwegian carriage study (Steens et al., 2015). Colonies from each serotype were suspended in Todd-Hewitt Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 2/10 Vortex and freeze at -‐70°C Serial dilu6ons of pneumococcal suspensions in TH broth 100µL in 1ml STGG on wet ice 100µL in 3ml enrichment broth at room temperature TIME 3 hours Overnight Day 2 Latex agglu6na6on Plate 100µL on blood agar. Incubate at 35⁰C Count colonies Incubate in ini6al 3ml enrichment broth at 35⁰C Prepare 200µL sample for qPCR and freeze Plate 100µL on blood agar. Incubate at 35⁰C Incubate 100µL in 3ml enrichment broth at 35⁰C Latex agglu6na6on Count colonies Prepare 200µL sample for PCR and frozen In vitro In vivo Nasopharyngeal swab 2 Thaw STGG sample, and vortex Place in 3ml enrichment broth at room temperature TIME Within 4 hours Overnight Day 2 Latex agglu6na6on Plate the swab on GBA for culturing. Incubate at 35⁰C Quellung reac6on Incubate in ini6al 3ml enrichment broth at 35⁰C Plate 20µL on GBA for culturing, Incubate at 35⁰C Incubate 200µL in 3ml enrichment broth at 35⁰C Latex agglu6na6on Quellung reac6on Place in 1ml STGG in a cool box Freeze for minimal 18 hours and maximal 1 months Nasopharyngeal swab 1 qPCR Figure 1 Schematic overview of the experimental designs. (A) in vitro. (B) in vivo. TH, Todd-Hewitt; STGG, skim milk, tryptone, glucose and glycerol; GBA, gentamycin-blood-agar. Place in 3ml enrichment broth at room temperature Latex agglu6na6on Figure 1 Schematic overview of the experimental designs. (A) in vitro. (B) in vivo. TH, Todd-Hewitt; STGG, skim milk, tryptone, glucose and glycerol; GBA, gentamycin-blood-agar. (TH) broth at a concentration of 0.5 McFarland in serial 1:10 dilutions of 10−2 to 10−5 (minimal concentration for which recovery of pneumococcal DNA was possible; see Supplemental Information 3). A volume of 100 µL of serotype dilution in TH broth was added to each set of transport media (either 1 ml of STGG or 3 ml of enrichment broth) to prepare the pneumococcal samples. The samples were left for 3 h on wet ice (STGG) or room temperature (enrichment broth). Subsequently, 100 µL of the samples was plated on Columbia horse blood agar plates. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 DNA extraction and ampliﬁcation by qPCR From each sample 200 µL was boiled for 10 min and DNA was extracted by QIAamp DNA Mini QIAcube kit (Qiagen, Inc., Valencia, CA, US) according to the manufacturer’s recommendations. A qPCR assay for the detection of the autolysin-encoding gene (lytA) was then performed as described before by Carvalho et al. (2007). Briefly, 25 µL reaction volume composed of TaqMan Fast Universal PCR Master Mix 2x, 200 nM of each primer and probe, 10x Exo IPC-mix, 50x Exo IPC DNA and 2 µL of DNA was run at 50 ◦C for 2 min, denaturation at 95 ◦C for 10 min, followed by 40 amplification cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. Samples were considered negative if cycle thresholds (Ct) Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 3/10 exceeded 40. A positive (ATCC49619) and a non-template control (sterile water) were included in each run, along with extraction controls. Data analysis For the in vitro analyses, Ct values and CFU counts (after a logarithmic transformation; logCFU) from the two media were compared by linear regression. We used 11 mutually exclusive dummy variables identifying different dilution-serotype combinations, which enabled us to simultaneously run the comparison for all serotypes. Additionally, the analyses were conducted separately per serotype. Agreement in the in vivo comparison was determined using the kappa statistic (Landis & Koch, 1977) and the exact McNemar’s probability test for paired data. Data were analysed in Stata 14.0 and GraphPad Prism 5. In vivo comparison This comparison was performed as part of a larger carriage study (sample taken in 2015 (Steens et al., 2016)). The study was conducted in accordance with principles of the Decla- ration of Helsinki, and approved by the Regional Committee for Medical Research Ethics, South-Eastern Norway (reference number: 2014/2046). Parents/guardians of the children gave written informed consent before including their child in the study. The study design resembles the design used in previous Norwegian carriage studies (Steens et al., 2015). Two flocked nylon nasopharyngeal swabs (E-swabsTM, Copan, Italy) taken from the same nostril were collected from 100 children aged 1–6 years according to standard procedures. The first swab was placed in 1 ml STGG which was subsequently stored in a cool box and the second swab was stored and transported in 3 ml enrichment broth at room temperature. The specimens were processed within 4 h of sampling. The STGG samples were vortexed at high speed and frozen at −70 ◦C , following the recommendations of WHO (Satzke et al., 2013). Within one month but earliest 18 h after initial freezing, the STGG samples were processed further: after being thawed and vortexed, 200 µl was added to fresh enrichment broth and 20 µl was plated on gentamycin-blood-agar (GBA). The swabs from the enrichment broth samples were plated on GBA within 4 h of sampling and the swab was re-inserted into the enrichment broth. All broths and GBA plates were incubated overnight at 35 ◦C , with 5% CO2. Pneumococci were identified by latex agglutination from incubated broths. Confirma- tion and factor typing were performed by Quellung reaction. All morphologically different pneumococcal colonies per plate were typed. In cases where the latex agglutination was positive but no colonies were found on plates after incubation overnight, samples were re-cultured by plating one drop of the incubated broth and incubating this for another night before further analysis. In vitro results (A) serotype 19F; (B) serotype 4; (C) serotype 3. * Uncountable. The quantification of DNA by Ct value is presented in Fig. 2. The Ct values overall did not differ significantly between STGG and enrichment broth samples (p = 0.853), though for serotype 4, the Ct values were significantly lower for STGG samples compared to enrichment broth samples (p = 0.007). After culturing, no clear overall difference was found in the logCFU (p = 0.593) but significant differences were observed for serotype 4 (Fig. 3B; p = 0.008; more colonies on plates incubated with STGG samples) and serotype 3 (Fig. 3C; p = 0.001; more colonies on plates incubated with enrichment broth samples). In vitro results The latex agglutination test was positive for both media for all serotype dilutions tested, with the exception of serotype 3 at a dilution of 10−5 in STGG, where no pneumococci were detected. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 4/10 A B C * # Enrichment broth STGG Figure 2 DNA quantification (Ct values) at different dilutions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. At a dilution of 10−5, one of the triplicates had a cycle threshold (Ct) of 39.4, while the other two were above 40. This sample was therefore considered negative. # Ct above 40. A B C Enrichment broth STGG Figure 3 Quantification of bacterial load (log counts of colony forming units [CFU]) at different dilu- tions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. * Uncountable. A B C * # Enrichment broth STGG * # Figure 2 DNA quantification (Ct values) at different dilutions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. At a dilution of 10−5, one of the triplicates had a cycle threshold (Ct) of 39.4, while the other two were above 40. This sample was therefore considered negative. # Ct above 40. A B C Enrichment broth STGG Figure 3 Quantification of bacterial load (log counts of colony forming units [CFU]) at different dilu- tions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. * Uncountable. Figure 2 DNA quantification (Ct values) at different dilutions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. At a dilution of 10−5, one of the triplicates had a cycle threshold (Ct) of 39.4, while the other two were above 40. This sample was therefore considered negative. # Ct above 40. Figure 2 DNA quantification (Ct values) at different dilutions of enrichment broth and STGG. (A) serotype 19F; (B) serotype 4; (C) serotype 3. At a dilution of 10−5, one of the triplicates had a cycle threshold (Ct) of 39.4, while the other two were above 40. This sample was therefore considered negative. # Ct above 40. A B C Enrichment broth STGG Figure 3 Quantification of bacterial load (log counts of colony forming units [CFU]) at different dilu- tions of enrichment broth and STGG. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 In vivo results Forty-six percent of the swabs transported and stored in STGG and 51% of those transported in enrichment broth were positive for pneumococci. This resulted in a Kappa statistic for carriage of 0.90 for the paired swabs (Table 1), indicating a trend towards higher sensitivity after transportation in enrichment broth compared to STGG (p = 0.0625). If re-cultured samples were excluded, carriage was 44% for the samples transported and stored in STGG and 48% for those transported in enrichment broth (Kappa = 0.92; p = 0.125). For each child for whom both swabs were positive, the same serotype was obtained. In one child, carriage of two serotypes was found in the enrichment broth sample, while one serotype was found in the STGG sample. However, the plate incubated with the enrichment broth Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 5/10 Table 1 Pneumococcal carriage determined by culturing paired nasopharyngeal swabs stored in STGG or enrichment broth. Enrichment broth Positive Negative Total Positive 46 0 46 STGG Negative 5 49 54 Total 51 49 100 Notes. STGG, skim milk, tryptone, glucose and glycerol. sample had only one colony of the serotype that was missed in the STGG sample (serotype 3) and the agglutination test was negative for this serotype, indicating presence at a very low concentration. Table 1 Pneumococcal carriage determined by culturing paired nasopharyngeal swabs stored in STGG or enrichment broth. Enrichment broth Positive Negative Total Positive 46 0 46 STGG Negative 5 49 54 Total 51 49 100 Notes. STGG, skim milk, tryptone, glucose and glycerol. Table 1 Pneumococcal carriage determined by culturing paired nasopharyngeal swabs stored in STGG or enrichment broth. Enrichment broth Positive Negative Total Positive 46 0 46 STGG Negative 5 49 54 Total 51 49 100 Notes. STGG, skim milk, tryptone, glucose and glycerol. Table 1 Pneumococcal carriage determined by culturing paired nasopharyngeal swabs stored in STGG or enrichment broth. STGG, skim milk, tryptone, glucose and glycerol. sample had only one colony of the serotype that was missed in the STGG sample (serotype 3) and the agglutination test was negative for this serotype, indicating presence at a very low concentration. sample had only one colony of the serotype that was missed in the STGG sample (serotype 3) and the agglutination test was negative for this serotype, indicating presence at a very low concentration. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 DISCUSSION Overall, no statistical differences in sensitivity were found between STGG stored and transported at cool temperatures and enrichment broth transported at room temperature. Nevertheless, some serotype differences were observed as well as a trend towards higher sensitivity for detection of pneumococcal carriage after transportation in enrichment broth compared to STGG. There are several possible reasons for these differences. In vitro, a pure dilution of one serotype was used in place of a nasopharyngeal swab. In vivo the swab contained different respiratory bacteria and viruses, and was covered with mucus and cellular debris. The presence of other bacteria places pneumococci in competition for nutrients needed for growth and reproduction. These nutrients are available in higher concentration in enrich- ment broth than in STGG, which may explain the small difference in sensitivity observed between the in vitro and in vivo settings. In addition to the difference in available nutrients in STGG and enrichment broth, the fact that STGG samples were kept in a cool box during transport while the enrichment broth samples were kept at room temperature, may have caused the small non-significant difference in carriage prevalence. The original carriage study presenting the use of enrichment broth as transport medium used cool transport conditions (Kaltoft et al., 2008) but three previous Norwegian carriage studies used room temperature (Steens et al., 2015). The present study is not designed to differentiate between the effect of media and temperature, but while pneumococci are thought to thrive better at warmer temperatures, we did not find an overall difference between the methods in bacterial load or DNA quantity in vitro. Whether the relative abundance of other respiratory bacteria/the microbiome may have changed and would no longer be representative of the nasopharyngeal tract after storage at room temperature should be investigated were room temperature to be used in such studies. Still, for serotype 3, the bacterial load indicated a higher sensitivity of enrichment broth compared to STGG. The serotype 3 isolate was obtained from a previous carriage study. For serotype 19F and 4, reference strains were used. The origin of the isolates (reference strains versus carriage isolate) may have induced different bacterial growth characteristics. Further, the capsule structure differs between Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 6/10 serotypes (serotype 3 being very mucoid). The low number of serotypes tested and the difference in origin between serotypes are limitations to the in vitro part of this study. DISCUSSION The carriage prevalence found in this study is lower than observed previously in Norway (Steens et al., 2015), and more similar to what has been seen in other developed countries (Van Hoek et al., 2014; Desai et al., 2015; Andrade et al., 2014). The methods used for swab collection, transport and incubation in enrichment broth and culturing were unchanged from former studies, indicating a real difference in carriage prevalence that may have resulted from vaccination. Advantages of molecular based techniques compared to culture techniques include the fact that viable organisms are not required, the original composition of the nasopharyngeal specimen is preserved, and detailed quantification and characterization of the pneumococci within a sample are possible, depending on the methods used (Satzke et al., 2013). Furthermore, the sensitivity of molecular methods for detection of multiple co-colonising serotypes has been shown to be higher than conventional methods (Saha et al., 2015). Nevertheless, isolation of strains enables further characterization such as antimicrobial susceptibility testing and sequence typing, and should not be replaced by molecular methods alone, despite its high sensitivity. The additional incubation step and use of latex agglutination appears to be of value for identification and isolation of pneumococci from multiple carriage (Kaltoft et al., 2008). The PneuCarriage Project concluded that microarray with a culture amplification step has the highest sensitivity for determining carriage (Satzke et al., 2015). Finally, STGG is cheap, easy to make and can be stored longer than enrichment broth, thus enabling comparability between studies and settings, including more resource-limited settings. Furthermore, STGG transported and stored at cool temperatures enables studies to investigate the microbiome (Grijalva et al., 2014; Turner et al., 2011), whereas enrichment broth may selectively stimulate growth of pneumococci. Therefore, even though STGG ap- peared slightly less sensitive than enrichment broth for detection of nasopharyngeal carriage of pneumococci by culturing, we recommend the continued use of STGG for transport and storage of nasopharyngeal swabs at cool temperatures in future carriage studies. ACKNOWLEDGEMENTS We thank the children and parents for participating and day-care centres workers for their support. We also thank Anne Ramstad Alme, Gunnhild Rødal, Lene Haakensen, Anne Witsø and Martha Langedok Bjørnstad for the laboratory analyses, Ingvild Essén and Kristine Hartmark for the collection of nasopharyngeal swabs, and Richard White for statistical advice. ADDITIONAL INFORMATION AND DECLARATIONS Funding The authors received no funding for this work. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 Funding The authors received no funding for this work. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 Competing Interests The authors declare there are no competing interests. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.2449#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.2449#supplemental-information. Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): Regional Committee for Medical Research Ethics, South-Eastern Norway (REK sør-øst B) Approval number: 2014/2046. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: The raw data has been supplied as a Supplementary File. The raw data has been supplied as a Supplementary File. Steens et al. (2016), PeerJ, DOI 10.7717/peerj.2449 Author Contributions • Anneke Steens and Natacha Milhano conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Ingeborg S. Aaberge conceived and designed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper. • Didrik F. Vestrheim conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper. 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W3037027989.txt	https://inria.hal.science/hal-03283228/document	en		Trace Equivalence and Epistemic Logic to Express Security Properties	Lecture notes in computer science	2,020	cc-by	7,820	Trace Equivalence and Epistemic Logic to Express Security Properties Kiraku Minami To cite this version: Kiraku Minami. Trace Equivalence and Epistemic Logic to Express Security Properties. 40th International Conference on Formal Techniques for Distributed Objects, Components, and Systems (FORTE), Jun 2020, Valletta, Malta. pp.115-132, �10.1007/978-3-030-50086-3_7�. �hal-03283228� HAL Id: hal-03283228 https://inria.hal.science/hal-03283228 Submitted on 9 Jul 2021 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License Trace Equivalence and Epistemic Logic to Express Security Properties⋆ Kiraku Minami[0000−0002−4434−4222] Kyoto University, Kyoto 606-8502, Japan kminami@kurims.kyoto-u.ac.jp Abstract. In process algebra, we can express security properties using an equivalence on processes. However, it is not clear which equivalence is the most suitable for the purpose. Indeed, several definitions of some properties are proposed. For example, the definition of privacy is not unique. This situation means that we are not certain how to express an intuitive security notion. Namely, there is a gap between an intuitive security notion and the formulation. Proper formalization is essential for verification, and our purpose is to bridge this gap. In the case of the applied pi calculus, an outputted message is not explicitly expressed. This feature suggests that trace equivalence appropriately expresses indistinguishability for attackers in the applied pi calculus. By chasing interchanging bound names and scope extrusions, we prove that trace equivalence is a congruence. Therefore, a security property expressed using trace equivalence is preserved by application of contexts. Moreover, we construct an epistemic logic for the applied pi calculus. We show that its logical equivalence agrees with trace equivalence. It means that trace equivalence is suitable in the presence of a non-adaptive attacker. Besides, we define several security properties to use our epistemic logic. Keywords: Applied pi calculus · Trace equivalence · Epistemic logic 1 Introduction 1.1 Background In modern society, information technology is indispensable to our daily lives, and many communication protocols are developed to transmit data securely. Verification of security properties of each protocol is essential, but it is not easy. In the first place, how to formalize security notions is not clear. Various definitions of the same security property have been proposed, we will show an example later. One of our goals is to provide foundations to express these properties in a rigorous way. Besides, how to model communication and concurrency is also not clear; many such models have also been developed. In this work, we focus on process algebra because it allows us to handle parallel composition naturally. ⋆ This work was partly supported by JST ERATO Grant Number JPMJER1603, Japan. 2 K. Minami In process calculi, common confidentiality properties such as secrecy are represented to use an equivalence on processes. Many equivalences exist (cf. [18]), but which is the most suitable for expressing confidentiality is not clear. For instance, Delaune et al. [13] defined privacy in electronic voting in terms of the applied pi calculus [1] as follows. Definition 1 ( [13, Definition 9]). A voting protocol respects vote-privacy (or just privacy) if S[VA {a/v }\|VB {b/v }] ≈l S[VA {b/v }\|VB {a/v }] for all possible votes a and b. VA and VB denote the voters containing the free variable v. S is an evaluation context. S denotes other voters and authorities. Intuitively, when the protocol respects privacy, this definition states that an attacker cannot distinguish two situations where votes are swapped. Note that indistinguishability is expressed to use labeled bisimilarity ≈l in this definition. Is it the most suitable? This question is nontrivial. Indeed, Chadha et al. [7] gave another definition and claimed that trace equivalence is more suitable than bisimilarity to model privacy. We also claim that trace equivalence is more suitable to express security properties in the presence of non-adaptive attackers. Similar arguments are not abundant in previous work. In the applied pi calculus, a process can send not only names but also terms, but we do not explicitly express sent messages. We indirectly represent them to use alias variables. This feature enables us to handle cryptographic protocols naturally and suggests that trace equivalence means indistinguishability for attackers. This is because attackers whom we consider can observe only labeled transitions. We recall the syntax and semantics of the applied pi calculus in §2. Both bisimilarity and trace equivalence on labeled transition systems (LTSs) are well studied. However, trace equivalence in the applied pi calculus (and other variants of the pi calculus [26, 27]) has not drawn much attention. This is perhaps because trace equivalence is the coarsest among commonly used equivalences. However, security properties sometimes require that different processes are regarded as the same. For example, consider secrecy. We want to make two processes that send different messages indistinguishable. In the case, trace equivalence is enough, but bisimilarity is not always optimal because it is too strong. Bisimilarity requires that possible actions for each process are the same. However, a non-adaptive attacker cannot detect a difference in feasibility. Here, “non-adaptive” means that the attacker cannot control participants. Thereby, a fine equivalence such as bisimilarity is not always adequate. Bisimilarity is probably suitable for more powerful attackers. Epistemic logic is often used to express confidentiality directly (e.g. [7, 25, 32]). For example, when a message M sent by an agent a is anonymous, we might say that an adversary cannot know who sent M . In epistemic logic, we can express it with a formula such as ¬KSend(a, M ). This logical formulation is close to our intuition. Nevertheless, research into an epistemic logic for the applied pi calculus is not abundant. Trace Equivalence and Epistemic Logic to Express Security Properties 3 In this paper, we assume that attackers can observe only labeled transitions. Especially, they cannot observe what action participants’ can do. This assumption is natural because attackers in this paper are non-adaptive. We also assume that an attacker can send messages to participants. 1.2 Contributions We prove that trace equivalence for the applied pi calculus is a congruence in §3. Second, we give an epistemic logic that characterizes trace equivalence in §4. Besides, we define security properties such as role-interchangeability, secrecy [25, 32], and openness, using our epistemic logic. Moreover, we show that parallel composition does not generally preserve secrecy and openness. Whereas, trace equivalence characterizes total secrecy, so application of contexts preserves it. We omit many proofs. See [28] for details. Our results suggest that trace equivalence is more suitable to express (nonprobabilistic) security notions than bisimilarity. 2 The Applied Pi Calculus The applied pi calculus [1] is an extension of the pi-calculus [26, 27]. We can handle cryptographic protocols naturally to use it. 2.1 Syntax Let Σ be a signature equipped with an equational theory. Terms are made from names, variables, and function symbols. A term is ground when it contains no variables. We recall the syntax of the applied pi-calculus. Here, M, N... range over terms, while n on names and x on variables. P, Q ::= 0 \| M ⟨N ⟩.P \| M (x).P \| νn.P \| if M = N then P else Q \| P + Q \| P \|Q \| !P A, B ::= P \| νn.A \| νx.A \| A\|B \| {M /x} P, Q, ... are plain processes. ν is a binding operator. \| is a parallel composition operator. + is a nondeterministic choice operator. Plain processes are similar to pi-calculus processes, but they are not the same. A pi-calculus process can send only a name. On the other hand, an applied pi calculus process can send a term. Besides, a channel consists of a term. An object of an input prefix is a variable, so names do not change while the process runs. A, B, ... are extended processes. We call {M /x} an active substitution. This notion is peculiar to the applied pi calculus. An active substitution {M /x} substitutes M for x in a neighbor process. fn(A) and bn(A) denote the sets of free names and bound names of a process A, respectively. fv and bv are similar to them. If fn(A)∩bn(A) = ∅ and no bound 4 K. Minami names are restricted twice, we say that A is name-distinct. Variable-distinctness is defined similarly. n(M ) denotes the set of names that appear in a term M . v(M ) is similar to it. The domain dom(A) of an extended process A is inductively defined below. If variables in neighbor concurrently running processes are in dom(A), the process A affects those variables. If fv(A) = dom(A), we say that A is closed. dom(P ) = ∅, dom(νn.A) = dom(A), dom(νx.A) = dom(A) \ {x}, dom(A\|B) = dom(A) ∪ dom(B), dom({M /x}) = {x} 2.2 Semantics A context is an expression containing one hole. An evaluation context is a context whose hole is neither under a replication, a conditional branch, nor an action prefix. Structural equivalence ≡ is the smallest equivalence relation on extended processes that is closed under application of evaluation contexts and α-conversion, such that: A\|0 ≡ A (A\|B)\|C ≡ A\|(B\|C) A\|B ≡ B\|A (νu.A)\|B ≡ νu.(A\|B) if u ∈ / fn(B) ∪ fv(B) νu.νv.A ≡ νv.νu.A !P ≡ P \|!P P + Q ≡ Q + P νx.{M /x} ≡ 0 A\|{M /x} ≡ A[M /x]\|{M /x} {M /x} ≡ {N /x} if Σ ⊢ M = N The second from the last represents how an active substitution {M /x} acts. Definition 2. Internal reduction → is the smallest relation on extended processes closed under structural equivalence and application of evaluation contexts, such that: if M = N then P else Q → P when Σ ⊢ M = N if M = N then P else Q → Q when Σ ̸⊢ M = N P +Q→P M ⟨N ⟩.P \| M (x).Q → P \| Q[N /x], where terms M and N in the second rule are ground. The last line represents synchronous communication on a channel M . We emphasize that an environment cannot observe what is interchanged. Next, we recall labeled semantics and requisite equivalence relations. x∈ / fv(M ⟨N ⟩.P ) M (N ) M (x).P −→ P [N /x] M ⟨N ⟩.P α A −→ A′ u does not appear in α. α νu.A −→ νu.A′ νx.M ⟨x⟩ −→ P \|{N /x} Trace Equivalence and Epistemic Logic to Express Security Properties α A −→ A′ bv(α) ∩ fv(B) = ∅ α A\|B −→ A′ \|B 5 α A ≡ A′ A′ −→ B ′ B ′ ≡ B α A −→ B The second rule represents an output. Note that an active substitution {N /x} is generated. The term N does not appear in the action label νx.M ⟨x⟩. A frame is an extended process generated from 0 and active substitutions to use restriction and parallel composition. fr(A) denotes a process obtained by replacing plain processes in A with 0, and we call it a frame of A. We can consider that fr(A) is a list of outputted messages. µ is an action. We define =⇒ as the transitive reflexive closure of −→, and µ α α =⇒ as =⇒−→=⇒. =⇒ is the former when µ is silent and is the latter otherwise. def Definition 3. (M = N )φ ⇔ v(M ) ∪ v(N ) ⊆ dom(φ) and M σ = N σ where φ ≡ νe n.σ and n e ∩ n(M, N ) = ∅ for some names n e and active substitutions σ. (M = N )φ means that an attacker cannot distinguish M and N to use φ. Definition 4. The static equivalence on closed frames is given by def φ ≈s ψ ⇔ dom(φ) = dom(ψ) and ∀M, N ; (M = N )φ ⇔ (M = N )ψ for closed frames φ and ψ. The static equivalence on closed processes is given by def A ≈s B ⇔ fr(A) ≈s fr(B) for closed processes A and B. Static equivalence means that an attacker has the same information about which terms are equal. µ1 µn Definition 5. A trace tr is a finite derivation tr = A0 =⇒ ... =⇒ An such that every Ai is closed and fv(µi ) ⊆ dom(Ai−1 ) for all i. If An can perform no actions, the trace tr is said to be complete or maximal. Given a trace tr, µj µi+1 let tr[i] be its i-th process Ai , and tr[i, j] be the trace Ai =⇒ ... =⇒ Aj where 0 ≤ i ≤ j ≤ n. The length of the trace tr is denoted by \|tr\| = n. µ1 µn µ′ 1 Definition 6. Let tr be a trace A0 =⇒ ... =⇒ An and tr′ be a trace B0 =⇒ µ′ m ... =⇒ Bm . Static equivalence between tr and tr′ is defined as below: def tr ∼t tr′ ⇔ n = m and µi = µ′i and Ai ≈s Bi f or all i. An attacker cannot distinguish statically equivalent traces. tr(A) is a set of traces of A. trmax (A) is a set of maximal traces of A. Definition 7. Let A and B be closed processes. def A ⊆t B ⇔ ∀tr ∈ tr(A) ∃tr′ ∈ tr(B) s.t. tr ∼t tr′ , def A ≈t B ⇔ A ⊆t B and B ⊆t A. Let A and B be two processes. Let σ be a map that maps variables in (fv(A) \ dom(A)) ∪ (fv(B) \ dom(B)) to ground terms. When Aσ ≈t Bσ holds for every such σ, we also denote it as A ≈t B. 6 K. Minami A ⊆t B means that each trace of A is imitated by some trace of B. We later show that non-adaptive active attackers cannot distinguish trace equivalent processes. Trace equivalence is undecidable. However, if processes contain no replications and the equational theory on Σ is a subterm convergent destructor rewriting system, trace equivalence is coNEXP complete [9]. 3 Congruency of Trace Equivalence The theorem below is our main result. It holds that trace equivalence is a congruence even though trace equivalence for the pi-calculus is not a congruence. This is ascribed to the difference between the pi-calculus and the applied pi calculus, namely, to the fact that names and variables are distinguished in the applied pi calculus. This is why adding an input prefix does not break trace equivalence. Besides, a scheme of late instantiation for an input transition is used in pi-calculus [26, 27], so parallel composition may break trace equivalence. On the other hand, a scheme of early instantiation is used in the applied pi calculus. This scheme enables us to decompose a trace of a parallel composed process into traces of component processes. Example 1. We consider pi-calculus. We put P = z(z ′ )\|yy ′ .ww′ , Q = z(z ′ ).yy ′ .ww′ + yy ′ .z(z ′ ).ww′ + yy ′ .ww′ .z(z ′ ). Then, x(z).P and x(z).Q are trace equivalent, but xy\|x(z).P and xy\|x(z).Q are not trace equivalent. On the other hand, x(z).P and x(z).Q are not trace equivalent in the applied pi calculus because instantiation is early. Abadi et al. [1] defined partial normal forms to prove that labeled bisimilarity is closed by application of closing evaluation contexts. They gave an operational semantics on partial normal forms. They classified transitions between ordinal processes into six cases to use partial normal forms. To prove the next theorem, we use partial normal forms and define concurrent normal forms of traces. Transitions in a concurrent normal trace have to be particular forms. Abadi et al. [1] studied decomposition and composition of reductions on partial normal forms. We study decomposition and composition of concurrent normal traces. Theorem 1. ≈t is a congruence. The proof is very long and complicated, so we only present an outline of our proof for the proposition below. Other cases are easy. The proof is given in [28]. Proposition 1. A ≈t B ⇒ A\|C ≈t B\|C. Proof outline. First, we define concurrent normal forms. A concurrent normal form is a particular form of a trace of a parallel composed process. A concurrent Trace Equivalence and Epistemic Logic to Express Security Properties 7 normal trace captures changes of scopes of bound names. Each process in a concurrent normal trace is of the form νe rse.(νe x.(σ\|P )ρ \| ν ye.(ρ\|Q)σ), where σ and ρ are (active) substitutions. Terms sent by the left process are accumulated in σ. Bound names sent by the left process P are accumulated in se. Symmetric cases are similar. Second, for any trace t of A\|C, we prove that there exists a concurrent normal trace t′ of A\|C such that t ∼t t′ . Third, given a concurrent normal trace tr of A\|C, we prove that we can construct traces of A and C which each process in them is of the form νe s.(σ\|P )ρ or νe r.(ρ\|Q)σ. Finally, we take a trace tr′ of B which is statically equivalent to the extracted trace of A as the above, combine it with tr′ , and prove that the result is statically equivalent to the given trace tr. 2 Example 2. Let h be a unary function symbol which cannot be inverted. νm.a(x).x⟨m⟩ ≈t νm.a(x).x⟨h(m)⟩ holds. Then, νm.a(x).x⟨m⟩ \| νn.a⟨n⟩.n(y).b⟨y⟩ ≈t νm.a(x).x⟨h(m)⟩ \| νn.a⟨n⟩.n(y).b⟨y⟩ is shown as follows. We arbitrarily take a trace tr of the left hand side. We consider tr :νm.a(x).x⟨m⟩ \| νn.a⟨n⟩.n(y).b⟨y⟩ νz.a⟨z⟩ −→ νm.a(x).x⟨m⟩ \| νn.(n(y).b⟨y⟩ \| {n/z }) a(z) −→νmn.(n⟨m⟩ \| n(y).b⟨y⟩ \| {n/z }) −→νmn.(b⟨m⟩ \| {n/z }) νw.b⟨w⟩ −→ νmn.{n/z , m/w} as an example. We transform it into a concurrent normal form. tr′ :νm.a(x).x⟨m⟩ \| νn.a⟨n⟩.n(y).b⟨y⟩ νz.a⟨z⟩ −→ νn.((νm.a(x).x⟨m⟩)[n/z ] \| n(y).b⟨y⟩ \| {n/z }) a(z) −→νn.((νm.z⟨m⟩)[n/z ] \| n(y).b⟨y⟩ \| {n/z }) −→νnm.((νv.{m/v })[n/z ] \| (b⟨v⟩ \| {n/z })[m/v ]) νw.b⟨w⟩ −→ νnm.((νv.{m/v })[n/z , v /w] \| {n/z , v /w}[m/v ]) Next, we decompose it into traces of component processes. tr1 :νm.a(x).x⟨m⟩ tr2 :νn.a⟨n⟩.n(y).b⟨y⟩ a(n) νz.a⟨z⟩ νv.n⟨v⟩ z(m) −→(νm.z⟨m⟩)[n/z ] −→ (νm.{m/v })[n/z ] −→ νn.(n(y).b⟨y⟩ \| {n/z }) −→νn.(b⟨v⟩ \| {n/z })[m/v ] νw.b⟨w⟩ −→ νn.{n/z , v /w}[m/v ] 8 K. Minami Since νm.a(x).x⟨m⟩ ≈t νm.a(x).x⟨h(m)⟩ holds, we can take a trace of νm.a(x).x⟨h(m)⟩ which is statically equivalent to the former. tr3 :νm.a(x).x⟨h(m)⟩ a(n) −→(νm.z⟨h(m)⟩)[n/z ] νv.n⟨v⟩ −→ (νm.{h(m)/v })[n/z ] Finally, we compose tr2 and tr3 and obtain a desired trace tr4 . tr4 :νm.a(x).x⟨h(m)⟩ \| νn.a⟨n⟩.n(y).b⟨y⟩ νz.a⟨z⟩ −→ νn.((νm.a(x).x⟨h(m)⟩)[n/z ] \| n(y).b⟨y⟩ \| {n/z }) a(z) −→νn.((νm.z⟨h(m)⟩)[n/z ] \| n(y).b⟨y⟩ \| {n/z }) −→νnm.((νv.{h(m)/v })[n/z ] \| (b⟨v⟩ \| {n/z })[h(m)/v ]) νw.b⟨w⟩ −→ νnm.((νv.{h(m)/v })[n/z , v /w] \| {n/z , v /w}[h(m)/v ]) Cheval et al. [10] established congruence property of trace equivalence for image-finite processes. They proved that trace equivalence is equivalent to maytesting equivalence for image-finite processes. On the other hand, taking all processes into account, may-testing equivalence does not imply trace equivalence. They gave a concrete counterexample. Thus, we cannot use the same technique. 4 An Epistemic Logic for the Applied Pi Calculus 4.1 Syntax We propose an epistemic logic for the applied pi calculus. It was inspired by [7], but our logic is a bit different. We give syntax of formulas. δ ::=⊤\|M1 = M2 \|M ∈ dom\|δ1 ∨ δ2 \|¬δ φ ::=δ\|φ1 ∨ φ2 \|¬φ\|⟨µ⟩− φ\|F φ\|Kφ where M1 , M2 and M are terms, and µ is an action. We call δ a static formula and φ a modal formula. A static formula δ mentions equality of terms. A modal formula φ mentions traces. ⟨µ⟩− φ states that the previous action is µ, and φ holds just before observing µ. F φ states that φ holds some time or other. The operator K expresses an attacker’s knowledge, i.e., Kφ means an attacker knows that φ holds. 4.2 Semantics Our logic is an LTL-like logic with an epistemic operator. Let A be a namevariable-distinct process that fv(A) \ dom(A) = x e, ρ be an assignment which Trace Equivalence and Epistemic Logic to Express Security Properties 9 maps x e to ground terms, tr ∈ tr(Aρ), 0 ≤ i ≤ \|tr\|, and M1 and M2 be terms. Please remember that fr(A) is a frame of A. We suppose that δ and φ contain no variables other than x e ∪ dom(tr[i]). We omit semantics of logical operators. They are defined as expected. A, ρ, tr, i \|= M1 = M2 iff (M1 ρ = M2 ρ)fr(tr[i]) A, ρ, tr, i \|= M ∈ dom iff M is a variable x, and x ∈ dom(tr[i]) µ A, ρ, tr, i \|= ⟨µ⟩− φ iff tr[i − 1] =⇒ tr[i] in tr and A, ρ, tr, i − 1 \|= φ A, ρ, tr, i \|= F φ iff ∃j ≥ i s.t. A, ρ, tr, j \|= φ A, ρ, tr, i \|= Kφ iff ∀ρ′ ∀tr′ ∈ tr(Aρ′ ); tr[0, i] ∼t tr′ [0, i] ⇒ A, ρ′ , tr′ , i \|= φ We suppose that an attacker does not know what terms are assigned to free variables before the process runs. Hence, the definition of K contains a quantifier over assignments ∀ρ′ . Recall that an attacker can observe only labeled transitions, so accessibility is defined based on static equivalence on traces. We also define satisfiability of formulas containing free variables. We put ye = dom(tr[i]). We suppose that φ contains no variables other than x e, ye, and ze. f; A, ρ, tr, i \|= φ(e f), A, ρ, tr, i \|= φ(e x, ye, ze) iff ∀M x, ye, M f is a sequence of ground terms. where M From now, we suppose that all processes are name-variable-distinct. We often omit restriction of a domain of definition. D(ρ) is a domain of definition of ρ. When a formula φ is satisfied over all possible runs of a process A, we say that A satisfies φ. def Definition 8. A \|= φ ⇔ ∀ρ ∀tr ∈ tr(Aρ); A, ρ, tr, 0 \|= φ, where D(ρ) = fv(A) \ dom(A). def Definition 9. A ⊑L B ⇔ ∀ρ ∀tr ∈ tr(Aρ) ∃tr′ ∈ tr(Bρ) s.t. ∀i ∀φ; [A, ρ, tr, i \|= φ ⇔ B, ρ, tr′ , i \|= φ], where D(ρ) = (fv(A) \ dom(A)) ∪ (fv(B) \ dom(B)). def A ≡L B ⇔ A ⊑L B and B ⊑L A. 4.3 Correspondence with trace equivalence We prove that trace equivalent processes satisfy the same formulas. Theorem 2. 1. A ≈t B ⇒ A ⊑L B; 2. A ⊑L B ⇒ A ⊆t B Proof. Let x e = (fv(A) \ dom(A)) ∪ (fv(B) \ dom(B)). 1) We prove ∀ρ ∀tr ∈ tr(Aρ), tr′ ∈ tr(Bρ); tr ∼t tr′ ⇒ ∀i ∀φ; [A, ρ, tr, i \|= φ ⇔ B, ρ, tr′ , i \|= φ], 10 K. Minami where D(ρ) = x e, by induction on the syntax of formulas. 2) We arbitrarily take an assignment ρ and tr ∈ tr(Aρ). By A ⊑L B, ∃tr′ ∈ tr(Bρ) s.t.∀i ∀φ; [A, ρ, tr, i \|= φ ⇔ B, ρ, tr′ , i \|= φ]. Then, we can prove tr ∼t tr′ . By arbitrariness of tr, it immediately follows that A ⊑L B ⇒ A ⊆t B. Theorem 3. A ≈t B ⇔ A ≡L B. This theorem suggests that trace equivalence is suitable to define security properties. We give Proposition 2 as an example in the next subsection. 4.4 Applications In this subsection, we often use abbreviations. Notably, P expresses ¬K¬, and G expresses ¬F ¬. P φ means that an attacker does not know φ does not hold. In other words, the attacker thinks that the possibility that φ holds remains. We define minimal secrecy. We regard it as generalized minimal anonymity [25]. Definition 10. A variable x is minimally secret with respect to δ in A iff A \|= G(δ(x) → P (¬δ(x))). This definition means that attackers cannot know that δ(x) holds. This property is very weak. For instance, although x is minimally secret with respect to a nontrivial formula δ, x is not always minimally secret with respect to ¬δ. Hereafter, we often omit objects. Example 3. We put δ(z) : z ̸= a ∧ z ̸= b. We consider a process if x = a then c else d. Then x is minimally secret with respect to δ, but not secret with respect to ¬δ. Moreover, ∨ does not preserve minimally secret. However, ∧ preserves it. Although x is minimally secret in A, x is not always secret in A\|A. Besides, restriction does not always preserve minimal secrecy. Example 4. We put δ(z) : z = a. We put P = if x = a then (a⟨s⟩ + b⟨s⟩) else a⟨s⟩, Q = if x = b then b⟨s⟩ else c⟨s⟩. Then x is minimally secret with respect to δ in P + Q, but not secret in (P + Q)\|(P + Q). Example 5. We put δ(z) : z = a. Then, x is minimally secret with respect to δ in x + a, but not secret in νa.(x + a). Here, we omitted objects. We define total secrecy. We can also regard it as generalized total anonymity [25]. Total secrecy states attackers can obtain no information about x. Trace Equivalence and Epistemic Logic to Express Security Properties 11 Definition 11. x is totally secret in A(x, ye) iff ∀δ(z, ze, w); e A(x, ye) \|= G(δ(x, ye, w) e → P (¬δ(x, ye, w))) e where δ contains no variables other than ones in {z} ∪ ze ∪ w e and satisfies that e ∀ψ∃M : ground s.t. ψ \|= ¬δ(M, N e , w). ∀N e Besides, \|e y \| = \|e z \| and w e ∩ ({x} ∪ ye) = ∅. Proposition 2. x is totally secret in A(x, ye) iff A(x, ye) ≈t A(x′ , ye). Proof. ⇒) We suppose for the sake of contradiction that A(x, ye) ̸≈t A(x′ , ye). e that are ground such that A(M1 , N e ) ̸≈t A(M2 , N e ). There exist M1 , M2 and N e e e )) We suppose that A(M1 , N ) ̸⊆t A(M2 , N ). Then, there exists tr ∈ tr(A(M1 , N e such that any trace of A(M2 , N ) is not statically equivalent to tr. e . Then We put δ(z, ze) : z ̸= M2 ∨ ze ̸= N e ), tr, \|tr\| \|= Kδ(x, ye). A(x, ye), (x 7→ M1 , ye 7→ N This contradicts total secrecy. ⇐) We arbitrarily take δ, ρ, tr and i, where δ meets the demand of Definition 11 and D(ρ) = {x} ∪ ye. We suppose that A(x, ye), ρ, tr, i \|= δ(x, ye, w). e We take M such that fr(tr[i]) \|= ¬δ(M, ρ(e y ), w). e Let ρ′ be { M (y = x) ′ ρ (y) = ρ(y) (otherwise). By assumption, A(ρ(x), ρ(e y )) ≈t A(M, ρ(e y )). Hence, there exists tr′ ∈ tr(A(M, ρ(e y ))) such that tr ∼t tr′ . Then, A(x, ye), ρ′ , tr′ , i \|= ¬δ(M, ρ(e y ), w). e Therefore, A(x, ye), ρ, tr, i \|= P (¬δ(x, ρ(e y ), w)). e Then, A(x, ye) \|= G(δ(x, ye, w) e → P (¬δ(x, ρ(e y ), w))). e Theorem 4. If x is totally secret in A(x, ye), then x is also totally secret in E[A(x, ye)] for every context E[_] which does not contain x. Our framework can handle role interchangeability [25]. When xi satisfies a property δk and xl satisfies a property δj , an attacker thinks that it is possible that xl satisfies a property δk and xi satisfies a property δj . Definition 12. We put fv(A) \ dom(A) = {x1 , ..., xp }, J = {1, ..., q}, and I = {1, ..., p}. (xi , δk ) is role interchangeable regarding {δj (zj , yej )}j∈J in A iff A(x1 , ..., xp ) \|= G(δk (xi , yek ) → ∧∧ (δj (xl , yej ) → P (δk (xl , yek ) ∧ δj (xi , yej )))) l∈I j∈J where yej ∩ {x1 , ..., xp } = ∅ for all j ∈ J. 12 K. Minami Proposition 3. f ∀i ∀tr ∈ tr(A(M1 , ..., Mp )) ∀M e ∃tr′ ∈ tr(A(Mi , N2 , ..., Ni−1 , M1 , Ni+1 , ..., Np )) s.t. tr ∼t tr′ ∃N ⇔ (x1 , δk ) is role interchangeable with respect to {δj } in A for all {δj } and k. Corollary 1. ∀l ∈ I \ {i}; A(x1 , ..., xi , ..., xl , ..., xp ) ≈t A(x1 , ..., xl , ..., xi , ..., xp ) ⇒ (xi , δk ) is role interchangeable with respect to {δj } in A for all {δj } and k. The converse holds only when p = 2. We give a counterexample for p = 3. Example 6. We put A(x, y, z) = if x = y then x + z else if x = z then x + y else y + z. Then, (x, δk ) is role interchangeable regarding {δj } in A for all {δj }j∈J and k, but A(a, b, a) ̸≈t A(b, a, a). Thus, A(x, y, z) ̸≈t A(y, x, z). We can also consider role permutativity. Mano [24] showed that it is strictly stronger than role interchangeability. Role permutativity states that even if p values are swapped, an attacker cannot notice it. Definition 13. We put fv(A) \ dom(A) = {x1 , ..., xp }, J = {1, ..., q}, and I = {1, ..., p}. {δj }j∈J is role permutable in A iff ∧ ∧ ∀n ≤ p ∀ψ ∈ Sp ; A(x1 , ..., xp ) \|= G( δik (xik , yek ) → P ( δik (xiψ(k) , yek ))) k≤n k≤n where yej ∩ {x1 , ..., xp } = ∅ for all j and each ik differs. Proposition 4. ∀ψ ∈ Sp ; A(x1 , ..., xp ) ≈t A(xψ(1) , ..., xψ(p) ) ⇔ {δj }j∈J is role permutable in A for all {δj }j∈J . We define openness. We regard it as generalized identity [32]. Parallel composition does not preserve openness. Definition 14. x is open in A under ∆(x) iff ∀ρ ∀tr ∈ trmax (Aρ); A, ρ, tr, \|tr\| \|= ∆(x) → K(∆(x) → (x = xρ)). Example 7. We put ∆(z) : z = r ∨ z = s. We put P = if x = r then a⟨n⟩ else b⟨n⟩, Q = if x = r then b⟨n⟩ else a⟨n⟩. Then x is open in P and Q under ∆(x), but x is not open in P \|Q under ∆(x). Problem 1. Input: An extended process A, an assignment ρ, a trace tr ∈ tr(A), an integer 0 ≤ i ≤ \|tr\|, and a formula φ. Question: Does A, ρ, tr, i \|= φ hold? Proposition 5. Even if the word problem in Σ is decidable, Problem 1 can be undecidable. Trace Equivalence and Epistemic Logic to Express Security Properties 13 Abadi and Cortier [2] proved that static equivalence can be undecidable even if the word problem in Σ is decidable. Proposition 5 follows from it. We change semantics. We repeat the definition of satisfaction. A \|= φ iff ∀ρ ∀tr ∈ tr(Aρ); A, ρ, tr, 0 \|= φ Now, we restrict ρ to be an assignment which maps free variables to only names and restrict inputted messages in tr to be only variables. That is, we assume that an attacker cannot tamper with a message. In other words, the attacker can only transfer messages without any change. Notably, the attacker cannot make a tuple of messages. Problem 2. Input: An extended process A and a formula φ. Question: Does A \|= φ hold? A convergent subterm theory is an equational theory defined by finite equations whose each right-hand side is a proper subterm of the left-hand side. Proposition 6. If the equational theory on Σ is a convergent subterm theory and A contains no replications, Problem 2 is decidable. 4.5 Comparison with the Work of Chadha et al. Chadha et al. developed the definition of privacy in e-voting as follows. They considered protocol instances in which two voters Alice and Bob participate, and voting options are 0 and 1. Definition 15 ( [7, Definition 9]). The voting process V respects privacy if V \|= Aprivacy ∧ Bprivacy where def - Aprivacy = ∧v∈{0,1} 2(K(Avote(v)) → Bvote(v)), and def - Bprivacy = ∧v∈{0,1} 2(K(Bvote(v)) → Avote(v)). Avote(v) means that Alice voted v, and Bvote(v) is similar. Minimal secrecy of a vote never holds because an attacker trivially knows votes when all votes agree. We consider protocol instances in which m voters participate and n voting options exist. Let vi be a vote of i. We consider the property below: ∨j,k vj ̸= vk → ∧i ∧v G(K(vi = v) → v1 = v∧...∧vi−1 = v∧vi+1 = v∧...∧vm = v) The consequence in G(...) implies that vi ̸= v due to the antecedent condition, so we can rewrite the property. ∨j,k vj ̸= vk → ∧i ∧v G(K(vi = v) → vi ̸= v) Moreover, we take the contraposition in G. ∨j,k vj ̸= vk → ∧i ∧v G(vi = v → P (vi ̸= v)) 14 K. Minami This consequence is exactly minimal secrecy. Besides, minimal secrecy of voting implies privacy, so privacy and minimal secrecy of voting agree under the disagreement condition ∨j,k vj ̸= vk . It was shown that V (0, 1) ≈t V (1, 0) implies that V respects privacy, and the partial converse was given in [7]. We give several properties of minimal secrecy. Proposition 7. We assume that a voting process V is equivalent for aborts, and minimal secrecy of each vote in V (v1 , ..., vm ) holds under the disagreement condition ∨j,k vj ̸= vk . 1. m = 2 ⇒ V (v1 , v2 ) ≈t V (v2 , v1 ). 2. m = 3 and n = 2 ⇒ V (v1 , v2 , v3 ) ≈t V (v2 , v1 , v3 ). 3. Otherwise, V (v1 , ..., vi , ..., vj , ..., vm ) ≈t V (v1 , ..., vj , ..., vi , ..., vm ) does not always hold. 5 Related Work Logics about behavior of labeled transition systems originate from HennessyMilner logic [20] that is a modal logic characterizing observational congruence. That is, observational equivalent systems satisfy the same modal formulas when these systems are image-finite. Process algebra is a special LTS. The spi calculus [3] is an extension of the pi-calculus. It enables us to handle symmetric key encryption based on the DolevYao model [14]. In the spi calculus, two ciphertexts obtained by encrypting two different plaintexts are indistinguishable unless an observer gets a secret key. Abadi and Gordon formalized security properties to use testing equivalence. We focused on the applied pi calculus [1] because it is more powerful than the spi calculus. That is, we intend to handle more various security notions. In the calculus, a process can send not only names but also terms via alias variables. Due to this feature, we can handle not only secrecy but also stricter properties. The authors proved that observational equivalence and labeled bisimilarity correspond. Chadha et al. [7] already developed an epistemic logic for the applied pi d Has directly represents attackcalculus. They defined formulas Has and evt. d means that a particular event had occurred. Temporal ers’ knowledge, and evt modalities were also used, but they do neither mention the just previous nor next action. The epistemic operator K was defined based on static equivalence on traces. Authors suggested that trace equivalence is more suitable than labeled bisimilarity when we consider privacy. However, a correspondent relation between logic and behavior of processes was not provided. As a matter of fact, α-equivalent processes do not always satisfy the same formulas in their framework because secret values are expressed as bound names or through events. In our framework, trace equivalent processes satisfy the same formulas. Horne [21] introduced quasi-open bisimilarity, and he proved that it coincides open bisimilarity. Moreover, intuitionistic modal logic FM characterizes quasiopen bisimilarity. The law of excluded middle does not hold in the logic because processes containing a free variable are also considered. Trace Equivalence and Epistemic Logic to Express Security Properties 15 Knight et al. [22] defined an epistemic logic for an LTS. This framework is based on Hennessy-Milner logic, and it handles multiple agents’ knowledge. They also proved weak completeness. However, compositionality was not discussed. Process algebra is one of nominal transition systems. Parrow et al. [29] developed modal logic characterizing bisimilarity for a nominal transition system. Toninho and Caires [31] proposed a dynamic spatial epistemic logic, which reasons what information a process can obtain. The epistemic operator means not only an attacker’s knowledge but also a participant’s knowledge, so, for example, the logic can reason a correspondence assertion. Tsukada et al. [32] studied sequential and parallel compositionality of security notions to use an epistemic logic for a multiagent system. They proved that neither anonymity nor privacy is generally preserved by composition. They also gave a sufficient condition for preservation. However, this word “parallel” merely means that the same agent acts two actions in the paper. That is, concurrency was not considered. Fiore and Abadi [16] developed symbolic models of processes. They gave a procedure to decide whether an environment can derive a message M . Their technique can be used for verification. However, equivalences on processes were not studied in the paper. Clarkson and Schneider [12] generalized trace properties to hyperproperties, and Clarkson et al. [11] developed hyperLTL and hyperCTL* for hyperproperties. Hyperproperties can express security properties which cannot be expressed by trace properties. The authors regarded systems as sets of traces, so hyperproperties are properties about systems. Our security properties are also proper hyperproperties. The advantage of our work over these works is to relate trace equivalence to attackers’ knowledge. In [11, 12], the relation between the equivalence and knowledge is not clear. Goubault-Larrecq et al. [19] proposed the probabilistic applied pi calculus. In this case, Theorem 1 no longer holds. It is known that trace distribution preorder [30] is not a congruence. On the other hand, it is shown in [5] that probabilistic trace equivalence for nondeterministic and probabilistic LTS is a congruence with respect to parallel composition. Probabilistic trace equivalence is coarser than trace distribution equivalence. Canetti et al. [6] defined implementation for task-PIOAs. According to their definition, T1 implements T2 iff the set of behaviors of T1 composed with E is included in the set of behaviors of T2 composed with E for every environment E. Here, behavior is the set of trace distributions. The implementation relation is preserved by parallel composition. Giro and D’Argenio [17] pointed out that ordinary schedulers may give rise to unnatural behavior. A scheduler may leak information if it can look at the whole of the system. To solve this problem, they provided several reasonable subclasses of schedulers. The problem of the scheduler in the formalization of security properties was also pointed out in [4, 8], which proposed other approaches. Eisentraut et al. [15] also studied subclasses of schedulers for probabilistic automata. They defined late distribution bisimulation and proved that late dis- 16 K. Minami tribution bisimulation with respect to distributed schedulers is compositional. We may need to specify subclasses of schedulers to state a probabilistic variant of Theorem 1. Knight et al. [23] developed spatial and epistemic process calculus. Their study is for concurrent constraint programming, so their processes can add constraints. They proved that observational equivalence is a congruence. Their processes do not have labeled actions, so observational equivalence means that equivalent processes provide the same results. On the other hand, in the applied pi calculus, trace equivalent processes provide equivalent traces and indistinguishable information. In this paper, we characterized trace equivalence in terms of our epistemic logic. That is, we showed that a non-adaptive active intruder cannot distinguish trace equivalent processes. We also focused on how composition of systems affects security properties. We proved that any composition preserves total secrecy and role permutativity. This is because trace equivalence is a congruence. 6 Conclusion 6.1 Summary In this paper, we provided an epistemic logic for the applied pi calculus. This logic is an LTL-like logic, so we can describe security notions. We formulated secrecy, role-interchangeability, and openness. These are generalized security properties regarding multiagent systems. Moreover, we associated trace equivalence with total secrecy. Application of context does not preserve minimal secrecy, but total secrecy is preserved because trace equivalence is a congruence. We also give a necessary and sufficient condition for role-interchangeability. We conclude that trace equivalence is suitable to express non-probabilistic indistinguishability in the view of security in the presence of a non-adaptive active adversary. 6.2 Future Work First, our epistemic logic states an adversary’s knowledge. We intend to construct a logic for a process’s knowledge. It will bridge a gap between multiagent systems and process calculi. Second, formalizations of other security properties such as non-malleability are also the next topics. Finally, what logic is suitable for security in the presence of an adaptive attacker is still open. Acknowledgments The author thanks Prof. M. Hasegawa and Prof. N. Yoshida for discussions. He also thanks the anonymous reviewers for helpful comments and suggestions. Trace Equivalence and Epistemic Logic to Express Security Properties 17 References 1. Abadi, M., Blanchet, B., Fournet, C.: The applied pi calculus: Mobile values, new names, and secure communication. J. 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https://openalex.org/W2789620726	http://www.journalijar.com/uploads/946_IJAR-21511.pdf	English	null	VERRUCIFORM EPIDERMODYSPLASIA (STUDY OF 2 CASES) IN CHU MOHAMED V MARRAKECH MORROCCO.	International journal of advanced research	2,018	cc-by	2,482	Introduction:- Verruciform Epidermodysplasia (EV) was described in 1922 by Lewandowski-Lutz[1]. It is a rare genodermatosis, with autosomal recessive inheritance, characterized by a facilitation of cutaneous infection by certain types of human papilloma virus (HPV) associated with a deficiency of cellular immunity [2,3]. We report two cases of EV associated with malignant transformation, specifying the clinical and evolutionary features of this condition through our observations and a review of the literature. Patients and observations:- Our study is retrospective about 2 cases collected from the plastic surgery; restorative and burns department, in Mohamed VI Hospital of Marrakech in Morocco, carriers of epidermodysplasue verruciform transformed into malignant tumors. Ouafaa Dhaidah, Moulay Driss Elamrani, Yassine Benchamkha and Salwa Ettalbi. Ouafaa Dhaidah, Moulay Driss Elamrani, Yassine Benchamkha and Salwa Ettalbi. Ouafaa Dhaidah, Moulay Driss Elamrani, Yassine Benchamkha and Salwa Ettalb Department of Plastic and Reconstructive Surgery in CHU Mohamed VI, Marrakech, Mor , y , rtment of Plastic and Reconstructive Surgery in CHU Mohamed VI, Marrakech, Morocco. ………………………………… Manuscript Info ……………………. Manuscript History Received: 10 November 2017 Final Accepted: 12 December 2017 Published: January 2018 Key words:- Verruciform epidermodysplasia, dermatology, oncology, plastic Surgery. ……………………………………………………………… Verruciform epidermodysplasia (VE) or Lutz-Lewandowski disease is a rare genodermatosis, most often autosomal recessive, characterized by abnormal susceptibility to infection by certain types of HPV. We report 2 cases with malignant transformation in squamous cell and basocellular carcinomas. We study 2 cases of patients aged of 50 and 34 years old that suffer from this illness and have been received in plastic restorative and burns Department in CHU Mohamed V Marrakech Morocco. In our study we try to focus on the origin, factors and the diagnostic of the Verruciform epidermodysplasia and compare them to those evocated by other authors and the treatment that has been followed. Verruciform epidermodysplasia is a rare disease that requires early diagnosis to avoid the high potential for transformation for these patients. Copy Right, IJAR, 2018,. All rights reserved. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 6(1), 632-635 Patient N: 1 J.J is a woman aged 50 years, resulting from the first siblings cousins parents without pathological antecedents. The beginning of the symptomatology was at the age of 15 marked by the spontaneous appearance of the flat verrucous lesions disseminated all over the face; the trunk and limbs and generalized after all over the body with healthy skin intervals. The evolution was marked by the rapid appearance and increase of multiple tumors in 2 years (when she was 48 years old). The diagnosis of a verruciform epidermodysplasia was posed during her hospitalization in the dermatology department then sent to ours for additional care. On examination: achromatic warts and macules at the level of the face and the upper and lower limbs, with ulcero-budding tumors at the forehead, 8 cm long axis, another 632 Corresponding Author:- Ouafaa Dhaidah. Address:- Department of Plastic and Reconstructive Surgery in CHU Mohamed VI, Marrakech, Morocco. 632 Corresponding Author:- Ouafaa Dhaidah. 632 Corresponding Author:- Ouafaa Dhaidah. Address:- Department of Plastic and Reconstructive Surgery in CHU Mohamed VI, Marrakech, Morocco. Int. J. Adv. Res. 6(1), 632-635 Int. J. Adv. Res. 6(1), 632-635 ISSN: 2320-5407 at the level of the internal cantus with a size of 2 cm and the third one was at the level of the external face of the leg with a size of 6 cm.Biopsies were made for these 3 tumors under local anesthesia.The histological study has shown that it is a moderately differentiated squamous cell carcinoma. A cervicofacial CT is done as part of a local extension assessment showed a cutaneous process at the forehead, and opposite the root of the nose without invasive bone opposite. Tumor excision was made with margins of one centimeter for the tumor of the leg and the forehead and bone cut at the level of the latter for the tumor of the internal cantus of the eye and the septum was respected. The histological study showed epidermoid carcinoma at the three sites, with tumor limits at the level of the internal cantus (the patient refuses the ocular exenteration). A locoregional extension assessment was negative, the coverage made by a thin skin graft at the level of the loss of substance of the forehead and the leg was performed and for the internal cantus a cicatrization directed by Vaseline tulles and local topicals. The patient was sent to the oncology department for additional care. Patient N: 1 They decided that radiotherapy is not recommended on the eye. Due to the ocular recurrence of the tumor; the patient accepted the ocular exenteration and readressed back to the oncology department for care. Patient 2:- O.Z patient, 34 years old woman with no pathological history; born of a consanguineous marriage, admitted to plastic, restorative and burns surgery department for a frontal tumor. The examination shows few warts on the face and limbs with an ulcero-budding tumor of 7 centimeters. The patient was operated several times; the pathological anatomy showed epidermoid carcinoma with a deep incomplete excision. Cervico-facial CT showed a tumor process of the left fronto-palpebral scalp without intracranial bone extension with infra-centimetric peripheral ganglia. A large surgical revision with ocular exenteration is done with coverage by a flap of the scalp. The patient was referred to the radiotherapy department where she did the sessions. Recurrence occurred after 6 months and cerebral CT showed cerebral parenchymal infiltration. A multidisciplinary staff (plastic surgery, dermatology, oncology and neurosurgery) decided on a tumor excision intraparechymate and covered by a free flap but due to the general deterioration, the treatment was palliative. Discussion:- Adv. Res. 6(1), 632-635 ISSN: 2320-5407 ubiquitous without racial or geographic limitation, although the condition has rarely been described in black subjects. ubiquitous without racial or geographic limitation, although the condition has rarely been described in black subjects. The skin lesions are the cause of hospitalization for both patients. We generally find the same lesional aspects described in the literature. It is :  Maculopapular lesions with warts. We note that these lesions are disseminated throughout the integument with healthy skin interval, small sizes with regular hypochromic contours or even non painful and itchy achromic.  Maculopapular lesions with warts. We note that these lesions are disseminated throughout the integument with healthy skin interval, small sizes with regular hypochromic contours or even non painful and itchy achromic.  erythematous macular lesions of pityriasis versicolor almost are generalized to the entire hyperchromic integument and sometimes hypochromic. Diffuse and very squamous with bending folds.  erythematous macular lesions of pityriasis versicolor almost are generalized to the entire hyperchromic integument and sometimes hypochromic. Diffuse and very squamous with bending folds.  hyperpigmented palmoplantar keratotic lesions often found in the OS literature. The histological examination was performed from biopsies or tumor excisions of cutaneous tissue fragment and allowed to find the characteristic lesions described by LEDERMAN which associates four signs in the histology of verruciform epidermodysplasia called hyperkeratosis, hyperacanthosis, hypergranulosis and intense vacuolar alteration in the center point which is the cytopathic effect of HPV viruses. This examination confirmed the diagnosis in all our patients who presented for the most part the characteristic signs of the histology of verruciform epidermodysplasia [10, 11]. None of our two patients was able to benefit from genetic and biochemical studies or research of viral DNA. This would have been important. This is to know what type of HPV virus has infected each patient. The small number of our observations does not allow us to draw a definitive conclusion on the average age of onset of the disease. All authors agree to locate the age of onset in childhoodIn the literature, we find this cancerization in 20 to 25% of cases - which is a very low rate compared to our patients who present 100% of malignant transformation found in basal cell carcinoma and squamous cell carcinoma that make all the seriousness of the disease [12]. The transformation into spino-cellular epithelioma after a rather long delay. Discussion:- Invasive cancer appears after 15 years, sometimes beyond 25 years, or even 40 years according to Lutzner. It is the possibility of this cancerous transformation that makes all of LEWANDOWSKY-LUTZ's disease. The age of malignant transformation for our population is after 30 years that is almost the same result found by other authors[13]. The prognosis of EV is related to the oncogenic potential of some HPV inducing carcinomas on the photo-exposed areas in 30 to 60% of cases [14]. We note the notion of recurrence with the appearance of other tumors despite an oncology surgical treatment associated with radiotherapy which makes the fatal prognosis of this pathology. Patients must protect themselves from UV radiation at an early age. The retinoids, by their antiproliferative and inductive actions of the cellular differentiation, make it possible to prevent and to delay the occurrence of cutaneous cancers. They have only a partial effect on warts since keratinocytes are always infested with HPV. Their long-term use exposes them to the risk of side effects. Some tests based on interferon, vitamin D analogs and the combination of retinoic acid with interferon, give hope for a better therapeutic management of this ailment [15, 16]. Discussion:- Discussion:- LEWANDOWSKY-LUTZ disease is a rare condition. The evaluation of its frequency in the general population has rarely been done in the literature. LEWANDOWSKY-LUTZ disease is a rare condition. The evaluation of its frequency in the general population has rarely been done in the literature. The small number of our observations does not allow us to draw a definitive conclusion on the average age of onset of the disease. All authors agree on the age of onset is in childhood, the age of malignant transformation for our population is after 30 years [4]. In the literature we do not note a predisposition of sex in disease, while in our population we find a female predominance [4]. The familial nature of the disease was recognized since S. HIDAKA (1924) who called it "verrucas dyskeratoticas congenitalis", It is a multifactorial disease involving genetic factors extrinsic (ultraviolet) and presumably immunological [ 5]. We find the hereditary character net in our cases. Indeed, Our patient N 2 had a deceased brother that had the same lesions as her. But at no time, none of our patients has reported a dermatitis concept to his parents. Note that in all our observations; we find a parental consanguinity [6,7]. We retain in our observations 50% of family cases. This is a high percentage compared to the literature which finds that vary between 10 and 20% [8,9]. We retain in our observations 50% of family cases. This is a high percentage compared to the literature which finds that vary between 10 and 20% [8,9]. The parental consanguinity of these cases is consistent with the literature that says that the mode of transmission is autosomal recessive and rarely the mode of transmission is recessive X-linked because in all our cases we do not find the same lesions among the ascendants. The parental consanguinity of these cases is consistent with the literature that says that the mode of transmission is autosomal recessive and rarely the mode of transmission is recessive X-linked because in all our cases we do not find the same lesions among the ascendants. The incidence of race seems relative . Our two patients belong to the brown race, recorded in North Africa zone (south of Morocco).Most cases of LEWANDOWSKY-LUTZ disease are white. SORYA ORGA MALIKI's study shows that there were five black patients in his study in Senegal. According to ANGO-PADONOU[9], the disease is 633 Int. J. Conclusion:- EV is a rare genodermatosis. Its relationship with certain genetic diseases is well established. Our study of EV associated with a malignant transformation into cutaneous tumors that change the prognosis of this pathology. 634 Int. J. Adv. Res. 6(1), 632-635 ISSN: 2320-5407 Bibliography:- g p y 1. Ortak T, Uysal C A, AlagosMS,Orbay H, Sensoz O. Epidermodysplasia Verruciformis : An Unusual Presentation. 2 l S 2006 32 302 6 2. Dermatol Surg 2006 ; 32 : 302-6 2. Dermatol Surg 2006 ; 32 : 302-6 3. Micalli G, Nasca MR, Dall’oglio F, Musumeci ML. Cimetine therapy for epidermodysplasia verruciformis. J Am Acad 4 Dermatol 2003 ; 48 : 9-10 4. Dermatol 2003 ; 48 : 9-10. 5. 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J Am Acad.Dermatol 2001 ; 45 : 296-9 22. Junko Hi, Chihiro M, Tsuyoshi M, Makoto K,, Masaaki M.Treatment of localized ep verruciformis witht acalcitol ointment. Internat J Dermatology 2002 ; 41 : 817–20. 635
https://openalex.org/W1990934473	https://zenodo.org/records/1535753/files/article.pdf	English	null	Sixty-Eight Cities of the United States in 1920	Journal of geography	1,921	public-domain	2,644	Journal of Geography Journal of Geography ISSN: 0022-1341 (Print) 1752-6868 (Online) Journal homepage: http://www.tandfonline.com/loi/rjog20 Date: 21 June 2016, At: 15:42 This is the first of a series of articles by Professor Ridgley based on the census reports of 1920. The next article will deal with population by states. Editor. Sixty-Eight Cities of the United States in 1920 Douglas C. Ridgley To cite this article: Douglas C. Ridgley (1921) Sixty-Eight Cities of the United States in 1920, Journal of Geography, 20:2, 75-79, DOI: 10.1080/00221342108984922 To link to this article: http://dx.doi.org/10.1080/00221342108984922 Published online: 22 Feb 2008. Submit your article to this journal Article views: 4 View related articles Published online: 22 Feb 2008. Download by: [Universitaetsbibliothek Heidelberg] Download by: [Universitaetsbibliothek Heidelberg] SIXTY-EIGHT CITIES OF THE UNITED STATES FEB., 1921 75 SIXTY-EIGHT CITIES OF THE UNITED STATES IN 1920' DOUGLAS c. RIDGLEY State Normal University, Normal, Illinois State Normal University, Normal, Illinois State Normal University, Normal, Illinois The United States Census is taken every ten years. This includes exhaustive reports concerning population, agriculture, manufactures, forestry and forest products, mines and quarries. Preparation for taking the census of 1920 began in July, 1919, and 70,000 enumerators started their work on January 2, 1920, as January 1 fell on Sunday. Their task involved a call at every home in the land during the month of January. Inquiry was made and facts recorded concerning all the people of the United States. Detailed reports were also made concerning agriculture, manufactures, and other subjects. Downloaded by [Universitaetsbibliothek Heidelberg] at 15:42 21 June 2016 In the Bureau of the Census of the Department of Commerce at Washington, D. C., hundreds of persons are engaged in studying, classifying, tabulating, and publishing the census reports. Publication of all reports will not be completed until 1922. In order to give infor- mation concerning the census returns, the Bureau of the Census issues reports from day to day to the newspapers and others who are inter- ested. The earlier reports are concerning population. Since February, 1920, readers have found census population facts almost daily in the newspapers. A recent report on which this article is based gives the population of cities having 25,000 inhabitants or more. Some of the leading facts of this report are here summarized. Populations are given in round numbers. CLASSES OF CITIES, NUMBER, AND TOTAL POPULATION OF EACH CLASS CLASSES OF CITIES, NUMBER, AND TOTAL POPULATION OF EACH CLAS Increase per cent Group 1. 200,000 inhabitants or more 33 22,000,000 24.8 Group 2. 100,000 to 200,000 inhabitants. . . . . . . . . . . . 36 4,700,000 28.8 Group 3. 50,000 to 100,000 inhabitants. . . . . . . . . . . . 70 5,200,000 33.2 Group 4. 25,000 to 50,000 inhabitants. . . . . . . . . . . . 144 5,100,000 34.1 14.9 Cities having Number Population 1910-1920 Total 25,000 inhabitants or more.. . . . . . . . . . . . . 288 37,000,000 Total population ConCinental United States.. . . . . . . . . .105,000,000 The 33 larger cities have about 20 per cent of the population of the United States, and the 288 cities included in the summary have 35 THE JOURNAL OF GEOGRAPHY 76 VOL. CITIES OF THE 100,000 CLASS Downloaded by [Universitaetsbibliothek Heidelberg] at 15:42 21 June 2016 Groups 1 and 2 of the above classification are usually referred to as cities of the 100,000 class, and this article deals with these 68 cities as a single group. The material is presented in such form as to be made the basis of a few geography lessons with classes now studying the United States or who hare studied the United States previously. Names of states are not given after the names of cities in the lists below. Pupils should always be required to give the name of the state after the name of the cit>y. The cities should be located by use of wall map, maps of United States and groups of states in textbook, and in atlases. In case a name indicates well-known cities in two or more states, the state is indicated. Since each decennial census stands for ten years as the only accurate count of population, it is of real importance to get promptly from the official census significant facts, classify them, and study their larger relationships as a sure foundation for accurate comparison during the next decade. Downloaded by [Universitaetsbibliothek Heidelberg] at 15:42 21 June 2 In 1910, there were 50 cities of the 100,000 class. The 1920 list contains these 50 and 18 additional cities. During the decade every one of the 68 cities grew in population, the percentage varying from less than one-tenth of one per cent for Spokane, Washington, to 201 per cent for Akron, Ohio, and 113 per cent for Detroit, Michigan. Los Angeles, California made the next largest percentage gain with 80 per cent. The increases in number of inhabitants varied from 35 for Spokane, Washington, to 850,000 for New York City. Detroit made the next largest gain, 520,000, and Chicago next with 510,000. Among the 50 cities of the 100,000 class in 1910, 7 retain the same relative rank as in 1920. New York, Chicago, and Philadelphia still rank Nos. 1, 2, and 3 respectively. 3an Francisco retains rank as No. 11, Minneapolisas No. 18, Birmingham as 36, and Dayton as 43. De- troit moved from No. 9 to No.4; LOS Angeles from No. 17 to No. 10. No other of the 50 cities were advanced in rank more than 4. SIXTY-EIGHT CITIES OF THE UNITED STATES IN 1920' DOUGLAS c. RIDGLEY State Normal University, Normal, Illinois 20 per cent of the entire population. It is interesting to note that the first two groups of the above summary have about the same number of cities, the third group is about double the second group in number with about the same population as the second group, The,fourth group is about double the third group in number with approximately the same population as the third group. Exactly one-half of the 288 cities included in the summary are in the fourth group. THE SIXTY-EIGHT CITIES WITH POPULATIONS IN ROUND NUMBER THE SIXTY-EIGHT CITIES WITH POPULATIONS IN ROUND NUMBER THE SIXTY-EIGHT CITIES WITH POPULATIONS IN ROUND NUMBER In the following list, the cities are arranged in order of population, the numbers indi- cating the rank of the city in 1920. In the following list, the cities are arranged in order of population, the numbers indi- cating the rank of the city in 1920. 1. New York City, 5,600,000. 3. Philadelphia, 1,800,000. 4. Detroit, 993,000. 5. Celveland, 796,000. 6. St. Louis, 772,000. 7. Boston, 748,000. 8. Baltimore, 733,000. 9. Pittsburgh, 588,000. 10. Los Angeles, 576,000. 11. San Francisco, 508,000. 12. Buffalo, 506,000. 13. hfilwaukee, 457,000. 14. Washington, 437,000. 15. Newark, 414,000. 16. Cincinnati, 401,000. 17. New Orleans, 387,000. 18. Minneapolis, 380,000. 19. Kansas City (Mo.), 324,000. 20. Seattle, 315,000. 21. Indianapolis, 314,000. 22. Jersey City, 297,000. 23. Rochester, 295,000. 24. Port- land (Oregon), 258,000. 25. Denver, 256,000. 26. Toledo, 243,000. 27. Providence, 237,000. 28. Columbus, 237,000. 29. Louisville, 234,000. 30, St. Paul, 234,000. 31. Oakland, 210,000. 32. Akron, 208,000. 33. ,4tlanta, 200,000. 34. Omaha, 191,000. 35. Worcester, 179,000. 36. Birmingham, 178,000. 37. Syracuse, 171,000. 38. Rich- mond, 171,000. 39. New Haven, 162,000. 40. Memphis, 162,000. 41. San Antonio, 161,000. 42. Dallas, 158,000. 43. Dayton, 152,000. 44. Bridgeport, 143,000. 45. Houston, 138,000. 46. Hartford, 138,000. 47. Scranton, 137,000. 48. Grand Rapids, 137,000. 49. Paterson, 135,000. 50. Youngstown, 132,000. 51. Springfield (Mass.), 129,000. 52. Des Moines, 126,000. 53. New Bedford, 121,000. 54. Fall River, 120,000. 55. Trenton, 119,000. 56. Nashville, 118,000. 57. Salt Lake City, 118,000. 58. Cam- den, 116,000. 59. Norfolk, 115,000. 60. Albany, 113,000. 61. Lowell, 112,000. 62. Wilmington, 110,000. 63. Cambridge, 109,000. 64. Reading, 107,000. 65. Fort Worth, 106,000. 66. Spokane, 104,000. Kansas City (Kans.), 101,000. 68. Yonkers, 100,000. 2. Chicago, 2,700,000. Downloaded by [Universitaetsbibliothek Heidelberg] at 15:42 21 June 2 CITIES OF THE 100,000 CLASS Spokane moved downward in rank from 48 to 66, Cambridge from 47 to 63, four others mol-ed don~mard in rank 10 or more. Of these 50 cities, SIXTY-EIGHT CITIES OF THE UNITED STATES FEB., 1921 FEB., 1921 77 7 retain the same rank, 16 advance in rank and 27 rank lower, but all have gained in population. 7 retain the same rank, 16 advance in rank and 27 rank lower, but all have gained in population. CLASSIFICATION OF THE SIXTY-EIGHT CITIES 1. Open your geography textbook at the political map of the United States. Find on the map the cities of the above list in order of rank, pronounce the name of each city and the name of the state in which it is located. If the city is not given on the map of the United States, find it on the larger map of the group of states in which it is found . 2. On an outline map of the United States, place a cross for each city, so that the two lines of the cross shall intersect at the exact loca- tion of the city. Refer to the map in your geography to make sure of the location. Near the cross place the number indicating the rank of the city. y 3. With only your outline map before you, see whether you can name all the cities promptly, giving the name of the state after the name of each city. 4. Make a table to show the following facts: 4. Make a table to show the following facts: a. Number of states having 7 cities of the 100,000 class Write the names of states follomed by the names of the cities for each state. a. Number of states having 7 cities of the 100,000 class a. Number of states having 7 cities of the 100,000 class Write the names of states follomed by the names of the cities for each state. Write the names of states follomed by the names of the cities for each state. THE JOURXXL OF GEOGRAPHY VOL. 20 VOL. 20 78 b. Number of states having 6 cities of the 100,000 class Write the list as under "a" in each case. b. Number of states having 6 cities of the 100,000 class i h li d " " i h b. Number of states having 6 cities of the 100,000 class Write the list as under "a" in each case. c. Number of states having 5 cities of the 100,000 class d. Number of states having 4 cities of the 100,000 class e. Number of states having 3 cities of the 100,000 class f. Number of states having 2 cities of the 100,000 class g. Number of states having 1 city of the 100,000 class h. CLASSIFICATION OF THE SIXTY-EIGHT CITIES Number of states having no city of the 100,000 class In listing the states as above, the District of Columbia is to be considered among the list of states. The complete list therefore will consist of the names of 48 states and 1 federal district. 5 . Make a list of the groups of states as shown on the various maps of your textbook. After the name of each state-group, write the num- ber indicating the number of cities in the group. Xdd your numbers and see whether the sum is 68. Xame orally the cities in each group of states, note on the map the exact position of the city, and give reasons for its location. Read in the textbook what is given about each city. 5 . Make a list of the groups of states as shown on the various maps of your textbook. After the name of each state-group, write the num- ber indicating the number of cities in the group. Xdd your numbers and see whether the sum is 68. Xame orally the cities in each group of states, note on the map the exact position of the city, and give reasons for its location. Read in the textbook what is given about each city. g y 6. On a map of the United States trace the 100th meridian. This meridian divides the land surface of Continental United States into two almost equal parts. How many of the 68 cities lie west of the 100th meridian? How many east? Give reasons for this difference. 7. Name the cities of the 100,000 class located on the Mississippi River; on the Ohio River; on the Missouri River; on the Great Lakes; on the Atlantic coast; on the Gulf coast; on the Pacific coast. What proportion of the 68 cities is included in this list? p p 8. Which occupations-agriculture. mining, manufacturing, com- merce-tend to develop large cities? Give examples and explain. 9. How many of the 68 cities are in the 11 Western States? How many in the 8 Plateau States? In the 3 Pacific Coast States? Give reasons. 10. Select 10 cities in which you are most interested and learn all you can about their advantages of location and the reasons for their development. Use textbook, commercial geographies, encyclopedias, and other reference books. FOR THE GEOGRAPHY CLASS OR THE ARITHMETIC CLASS The large facts of the United States Census are of sufficient impor- t~ance, significance, and permanence to warrant a careful study. The facts concerning 68 cities have great geographical significance. The real meaning of the census reports is emphasized and crystallized by SIXTY-EIGHT CITIES OF THE UNITED STATES 79 FEB., 1921 FEB., 1921 having pupils solve arithmetical problems based on census figures. These problems may be solved as a geography lesson or an arith- metic lesson. The total population of Continental United States is 105,000,000; of these 68 cities, 27,000,000; of the 33 larger cities, each having 200,000 inhabitants or more, 22,000,000 ; of the 35 cities having between 100,000 and 200,000 inhabitants approximately 5,000,000. The following arithmetical problems are suggestive of helpful arithme- tic work based on census reports. The per cents may be expressed as a whole number and one decimal place. bliothek Heidelberg] at 15:42 21 June 2016 1. What per cent of the population of Continental United States live : 1. What per cent of the population of Continental United States live : a. In the 68 cities of the 100,000 class? b. In the 33 cities of the 200,000 class? c. In the 10 larger cities? d. In New York City? e. In Chicago? e. In Chicago? 2. New York state has a population of 10,000,000. What per cent of this population live in New York City? 3. Illinois has a population of 6,400,000. What per cent of this population live in Chicago? 4. Ohio has a population of 7,500,000. What per cent live in the 7 cities of the 100,000 class? 5. Massachusetts has a population of 3,800,000. What per cent live in its cities of the 100,000 group? 6. California has a population of 3,400,000. What per cent live in cities of over 100,000? 7. Akron, Ohio, had a population in 1910 of 69,000. Find its in- crease in population and its percentage of increase. 8. New York City had a population in 1910 of 4,700,000. Find its increase in population and its percentage of increase. Explain clearly why the percentage of increase for Akron and New York City are so widely different. 9. Rhode Island has a population OF 600,000. What per cent live in Providence? 10. District of Columbia has a population of 437,000. What per cent live in the city of Washington? y g 11. FOR THE GEOGRAPHY CLASS OR THE ARITHMETIC CLASS Study the population figures of this article and make additional problems to be solved by members of your class. Look for large and interesting comparisons. Make problems that will help the student to understand geography as well as arithmetic.
https://openalex.org/W4390664015	https://www.e3s-conferences.org/articles/e3sconf/pdf/2024/04/e3sconf_icite2023_01066.pdf	English	null	Calculation and manufacture of multilayer composite structures	E3S web of conferences	2,024	cc-by	3,738	ΎCorresponding author: yakubov@tyuiu.ru Calculation and manufacture of multilayer composite structures Yu. Е. Yakubovskiy, V. I. Kolosov, A. G. Kuzyaev, and S. O. Kruglov Industrial University of Tyumen, Tyumen, Russia Yu. Е. Yakubovskiy, V. I. Kolosov, A. G. Kuzyaev, and S. O. Kruglov Industrial University of Tyumen, Tyumen, Russia Abstract. A brief overview of the work done to solve the problems of bending of composite plates and shells is presented. A mathematical model has been developed that takes into account the physically nonlinear properties of the layer`s material. The seams between the layers have finite longitudinal stiffness. Seams can be continuous or discrete (anchors). The proposed mathematical model makes it possible to take into account the creep properties of the aging material. The reliability of calculations is substantiated by comparison with experimental data and known calculations. Taking into account the temperature mode made it possible to develop a technology for manufacturing multilayer structures with predicted properties of the material of the layers. Based on the results presented, methods for calculating multilayer structures and technologies for their manufacture have been developed. It is proposed to create products from cement-based composites with unique planned characteristics, oriented to the intended purpose of the manufactured structures. The intended purpose of products can be achieved by using a material with special characteristics in each layer. To protect against rodents and microorganisms, light antiseptics can be used in the process of making the composite. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems. The results of the creation of composites are presented. © 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 474, 01066 (2024) ICITE 2023 E3S Web of Conferences 474, 01066 (2024) ICITE 2023 https://doi.org/10.1051/e3sconf/202447401066 2 Main points Further development of the theory of composite plates led to the decision to physically nonlinear problems of bending considered designs. To implement the mathematical model, a system of resolving differential equations was built taking into account the nonlinear properties of the material of each layer. The layers are connected by flexible shear links, which are described by nonlinear relationships, since the stiffness coefficient of the seams depends on the magnitude of the shear stresses. Comparison of the results of solving the problem with experimental data (in the inelastic area) confirmed the effectiveness of the proposed method [3]. The tasks were considered taking into account the isotropy and anisotropy of the properties of the materials of the structural layers and shear seams. Kirchhoff - Love hypothesis is fulfilled for a separate layer, but not for the package as a whole. The layers are interconnected by elastic - yielding shear links, which allow one layer to shift with respect to another. Cross-links are absolutely rigid. The load normal to the surface of the package is distributed according to an arbitrary principle. The total number of layers is n + 1, and the seams are n. Nonlinear equations are derived for active elastic-plastic deformations. To record the relationship between stresses and strains, the dependences of the deformation theory are used. The material of the layers is compressed, the coefficient of transverse deformation is variable. The influence of the stiffness of the anchorage of the plate layers on the behavior of the composite package was also investigated [2]. As a result, a system of nonlinear differential equations of equilibrium, continuity and seam work has been developed. The total number of equations is 2 (n + 1). 1 Introduction Recently, a large number of materials have been created that meet increased requirements for the reliability of structures, including strength, thermal conductivity, resistance to aggressive environment, etc. It is possible to achieve the desired combination of the required properties by selecting and alternating layers in composite shells. Structures of two or more monolithic elements have been known for a long time and have found very wide application in all areas of activity. These are all kinds of load-bearing and enclosing structures, multilayer elements in buildings and mechanisms, steel-concrete pipes with high tightness and strength, and much more. If the work of the seams is taken into account when calculating multilayer structures, then such systems are classified as composite [1, 2]. y p Two-layer composite beams, plates and shells are often made from a concrete layer, reinforced on the side of the tension zone with a steel sheet that only takes membrane forces. E3S Web of Conferences 474, 01066 (2024) ICITE 2023 https://doi.org/10.1051/e3sconf/202447401066 Three-layer and multi-layer beams and slabs are arranged in such a way that layers of a more rigid material strengthen the stretched area of the structure. Such systems are used in the construction of foundations and bases of industrial construction projects with increased requirements for thermal insulation. They are also used in foundations for residential buildings erected on permafrost. 2 Main points 𝜕𝜑𝜕𝜑𝐵𝜕𝜑𝜕𝐵𝜕𝜑 y Continuity equations for the middle surface of the i-th layer are presented in the form [4]: 𝐵𝑖𝜕𝜑𝑖𝜕𝑥𝜕𝜑𝑖𝜕𝑦𝐵𝑖𝐵𝑖𝜕𝜑𝑖𝜕𝑥𝜕𝑦𝜕𝐵𝑖𝜕𝜕𝜕𝜑𝑖𝜕𝑥 𝐵𝐵11 ∗𝑖𝑖(𝜕𝜕4𝜑𝜑𝑖𝑖 𝜕𝜕𝑥𝑥4 + 𝜕𝜕4𝜑𝜑𝑖𝑖 𝜕𝜕𝑦𝑦4 ) + 2 (𝐵𝐵33 ∗𝑖𝑖 2 −𝐵𝐵12 ∗𝑖𝑖) 𝜕𝜕4𝜑𝜑𝑖𝑖 𝜕𝜕𝑥𝑥2𝜕𝜕𝑦𝑦2 + 2[𝜕𝜕𝐵𝐵11 ∗𝑖𝑖 𝜕𝜕𝜕𝜕 𝜕𝜕3𝜑𝜑𝑖𝑖 𝜕𝜕𝑥𝑥3 + 2 2 https://doi.org/10.1051/e3sconf/202447401066 E3S Web of Conferences 474, 01066 (2024) E3S Web of Conferences 474, 01066 (2024) ICITE 2023 ICITE 2023 + 𝜕𝜕 𝜕𝜕𝜕𝜕(𝐵𝐵33 ∗𝑖𝑖 2 −𝐵𝐵12 ∗𝑖𝑖) 𝜕𝜕3𝜑𝜑𝑖𝑖 𝜕𝜕𝜕𝜕𝜕𝜕𝑦𝑦2 + 𝜕𝜕𝐵𝐵11 ∗𝑖𝑖 𝜕𝜕𝜕𝜕 𝜕𝜕3𝜑𝜑𝑖𝑖 𝜕𝜕𝑦𝑦3 + 𝜕𝜕 𝜕𝜕𝜕𝜕(𝐵𝐵33 ∗𝑖𝑖 2 −𝐵𝐵12 ∗𝑖𝑖) 𝜕𝜕3𝜑𝜑𝑖𝑖 𝜕𝜕𝑥𝑥2𝜕𝜕𝜕𝜕] + + (𝜕𝜕2𝐵𝐵11 ∗𝑖𝑖 𝜕𝜕𝑦𝑦2 −𝜕𝜕2𝐵𝐵12 ∗𝑖𝑖 𝜕𝜕𝑥𝑥2 ) 𝜕𝜕2𝜑𝜑𝑖𝑖 𝜕𝜕𝑦𝑦2 + (𝜕𝜕2𝐵𝐵11 ∗𝑖𝑖 𝜕𝜕𝑥𝑥2 −𝜕𝜕2𝐵𝐵12 ∗𝑖𝑖 𝜕𝜕𝑦𝑦2 ) 𝜕𝜕2𝜑𝜑𝑖𝑖 𝜕𝜕𝑥𝑥2 + 𝜕𝜕2𝐵𝐵33 ∗𝑖𝑖 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕 𝜕𝜕2𝜑𝜑𝑖𝑖 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕− −𝜕𝜕2 𝜕𝜕𝑦𝑦2 [(𝐵𝐵11 ∗𝑖𝑖−𝐵𝐵12 ∗𝑖𝑖)(𝑇𝑇𝑖𝑖−𝑇𝑇𝑖𝑖−1)] −𝜕𝜕2 𝜕𝜕𝑥𝑥2 [(𝐵𝐵11 ∗𝑖𝑖−𝐵𝐵12 ∗𝑖𝑖)(𝑇𝑇𝑖𝑖−𝑇𝑇𝑖𝑖−1)] = = 𝜕𝜕2𝜀𝜀1𝑖𝑖 𝜕𝜕𝑦𝑦2 + 𝜕𝜕2𝜀𝜀2𝑖𝑖 𝜕𝜕𝑥𝑥2 + 2 𝜕𝜕2𝛾𝛾12 𝑖𝑖 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕(2) To describe the joint operation of layers on both sides of the i-th seam, it was taken into account that the characteristics of the shear stiffness of the seam are considered isotropic in the seam plane. Cross-links are absolutely rigid: 𝜕𝜕𝜕𝜕𝑇𝑖𝜂𝜕𝜕𝜕𝜕𝜕𝜕𝑇𝑖𝜂𝜕𝜕𝜂𝜕𝑇𝑖𝜕𝑥𝜕𝑇𝑖𝜕𝑦𝑐𝑖𝜕𝑊𝜕𝑥𝜕𝑊𝜕𝑦 𝜕𝜕 𝜕𝜕𝜕𝜕 𝜕𝜕𝑇𝑇𝑖𝑖 𝜂𝜂𝑖𝑖𝜕𝜕𝜕𝜕+ 𝜕𝜕 𝜕𝜕𝜕𝜕 𝜕𝜕𝑇𝑇𝑖𝑖 𝜂𝜂𝑖𝑖𝜕𝜕𝜕𝜕+ 1 𝜂𝜂𝑖𝑖(𝜕𝜕2𝑇𝑇𝑖𝑖 𝜕𝜕𝑥𝑥2 + 𝜕𝜕2𝑇𝑇𝑖𝑖 𝜕𝜕𝑦𝑦2 ) = 𝑐𝑐𝑖𝑖(𝜕𝜕2𝑊𝑊 𝜕𝜕𝑥𝑥2 + 𝜕𝜕2𝑊𝑊 𝜕𝜕𝑦𝑦2 ) + +𝜀𝜀𝑥𝑥 𝑖𝑖+1 −𝜀𝜀𝑥𝑥 𝑖𝑖+ 𝜀𝜀𝑦𝑦 𝑖𝑖+1 −𝜀𝜀𝑦𝑦 𝑖𝑖(3) The obtained equations of the mathematical model were transformed into algebraic ones by the asymptotic expansion of the determined functions into sinusoidal series. In this case, the procedure for orthogonalization of the Bubnov - Galerkin method is implemented. The system of algebraic equations was solved by the iterative method according to the Seidel scheme [3]. The method of variable parameters of elasticity in the form of I.A. Birger was used in the calculations. This allowed the solution of a nonlinear problem to be reduced to a sequence of solutions to linear problems. At the same time, in the process of calculations, variable coefficients of the stiffness of the material of the layers of the composite structure were calculated. The calculation was reduced to fixing variable stiffness coefficients at each stage. At the beginning of the calculation, elastic values of mechanical characteristics were introduced 𝐄𝐄𝐜𝐜𝐜𝐜 𝐢𝐢 = 𝐄𝐄𝟎𝟎 𝐢𝐢, 𝛎𝛎𝐜𝐜𝐜𝐜 𝐢𝐢 = 𝛎𝛎𝟎𝟎 𝐢𝐢 . The system of linear equations (1), (2), (3) was solved by the Bubnov - Galerkin method and the Seidel iterative method to determine the stress - strain state of the structure. 2 Main points The number of the sought functions is also 2 (n + 1): the deflection function W (x, y), n + 1 functions of efforts 𝜑𝜑𝑖𝑖(𝑥𝑥, 𝑦𝑦) and n shear functions 𝑇𝑇𝑖𝑖(𝑥𝑥, 𝑦𝑦): 𝐷𝜕𝑊𝜕𝑥𝜕𝑊𝜕𝑦𝐷𝐷𝜕𝑊𝜕𝑥𝜕𝑦𝜕𝐷𝜕𝜕𝜕𝑊𝜕𝑥 𝐷𝐷11 ∗(𝜕𝜕4𝑊𝑊 𝜕𝜕𝑥𝑥4 + 𝜕𝜕4𝑊𝑊 𝜕𝜕𝑦𝑦4 ) + 2(𝐷𝐷12 ∗+ 2𝐷𝐷33 ∗) 𝜕𝜕4𝑊𝑊 𝜕𝜕𝑥𝑥2𝜕𝜕𝑦𝑦2 + 2[𝜕𝜕𝐷𝐷11 ∗ 𝜕𝜕𝜕𝜕 𝜕𝜕3𝑊𝑊 𝜕𝜕𝑥𝑥3 + + 𝜕𝜕 𝜕𝜕𝜕𝜕(𝐷𝐷12 ∗+ 2𝐷𝐷33 ∗) 𝜕𝜕3𝑊𝑊 𝜕𝜕𝜕𝜕𝜕𝜕𝑦𝑦2 + 𝜕𝜕𝐷𝐷11 ∗ 𝜕𝜕𝜕𝜕 𝜕𝜕3𝑊𝑊 𝜕𝜕𝑦𝑦3 + 𝜕𝜕 𝜕𝜕𝜕𝜕(𝐷𝐷12 ∗+ 2𝐷𝐷33 ∗) 𝜕𝜕3𝑊𝑊 𝜕𝜕𝑥𝑥2𝜕𝜕𝜕𝜕] + + (𝜕𝜕2𝐷𝐷11 ∗ 𝜕𝜕𝑥𝑥2 + 𝜕𝜕2𝐷𝐷12 ∗ 𝜕𝜕𝑦𝑦2 ) 𝜕𝜕2𝑊𝑊 𝜕𝜕𝑥𝑥2 + (𝜕𝜕2𝐷𝐷11 ∗ 𝜕𝜕𝑦𝑦2 + 𝜕𝜕2𝐷𝐷12 ∗ 𝜕𝜕𝑥𝑥2 ) 𝜕𝜕2𝑊𝑊 𝜕𝜕𝑦𝑦2 + 4 𝜕𝜕2𝐷𝐷33 ∗ 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕 𝜕𝜕2𝑊𝑊 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕= = 𝑞𝑞+ ∑ с𝑖𝑖( 𝜕𝜕2𝑇𝑇𝑖𝑖 𝜕𝜕𝑥𝑥2 + 𝜕𝜕2𝑇𝑇𝑖𝑖 𝜕𝜕𝑦𝑦2 ) + ∑ ( 𝜕𝜕2𝑀𝑀1 𝑖𝑖 𝜕𝜕𝑥𝑥2 + 2 𝜕𝜕2𝑀𝑀12 𝑖𝑖 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕+ 𝜕𝜕2𝑀𝑀2 𝑖𝑖 𝜕𝜕𝑦𝑦2 ) 𝑛𝑛+1 𝑖𝑖=1 𝑛𝑛 𝑖𝑖=1 (1) (1) Here 𝐷𝐷𝑚𝑚𝑚𝑚 ∗ - is the single cylindrical stiffness of the layer package; ; 𝑀𝑀𝑚𝑚𝑚𝑚 𝑖𝑖 - bending moments in the middle of the i-th layer; с𝑖𝑖= 𝑎𝑎𝑖𝑖+1 + 𝑏𝑏𝑖𝑖 - the distance between the middle surfaces of the layers on both sides of the i-th seam. 𝜕𝜑𝜕𝜑𝐵𝜕𝜑𝜕𝐵𝜕𝜑 Here 𝐷𝐷𝑚𝑚𝑚𝑚 ∗ - is the single cylindrical stiffness of the layer package; ; 𝑀𝑀𝑚𝑚𝑚𝑚 𝑖𝑖 - bending moments in the middle of the i-th layer; с𝑖𝑖= 𝑎𝑎𝑖𝑖+1 + 𝑏𝑏𝑖𝑖 - the distance between the middle surfaces of the layers on both sides of the i-th seam. 2 Main points The stresses in the i-th layer and the i-th seam were determined by the expressions [4]. The intensity of normal stresses was calculated and, taking into account the properties of the material of the layers of the plate, the secant modulus was determined 𝐄𝐄𝐜𝐜𝐜𝐜 𝐢𝐢(𝐱𝐱, 𝐲𝐲, 𝐳𝐳) and the lateral deformation coefficient 𝛎𝛎𝐜𝐜𝐜𝐜 𝐢𝐢(𝐱𝐱, 𝐲𝐲, 𝐳𝐳). Next, a new linear bending problem was solved and the deformed state of the plate was determined. After determining 𝛔𝛔𝐢𝐢𝐢𝐢𝐢𝐢 𝐢𝐢 (𝐱𝐱, 𝐲𝐲, 𝐳𝐳) by dependence 𝛔𝛔𝐢𝐢𝐢𝐢 𝐢𝐢−𝛆𝛆𝐢𝐢𝐢𝐢 𝐢𝐢 were calculated 𝐄𝐄𝐜𝐜𝐜𝐜 𝐢𝐢(𝐱𝐱, 𝐲𝐲, 𝐳𝐳) etc. The calculation was carried out up to the specified difference between the subsequent and previous values: 𝝈𝒊𝒊𝒊𝒊𝝈𝒊𝒊𝒊𝟏𝒊 𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊 −𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊−𝟏𝟏 𝒊𝒊 𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊 ≤∆, 𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊 −𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊−𝟏𝟏 𝒊𝒊 𝝈𝝈𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊 ≤∆, after which the problem was considered solved. after which the problem was considered solved. To assess the reliability of solving the problem of bending of the considered structure, taking into account the physically nonlinear properties of the material of the middle layer, a comparison with experiment was carried out. Calculations revealed a change in stresses up to 25% due to the physical nonlinearity of the properties of the layer materials. The dependence of the strength of the composite shell 3 E3S Web of Conferences 474, 01066 (2024) ICITE 2023 https://doi.org/10.1051/e3sconf/202447401066 revealed on the stiffness of the connecting seams, and not on their type (using glue or anchors) [2] revealed on the stiffness of the connecting seams, and not on their type (using glue or anchors) [2]. As a further development of the mathematical modeling of the bending of composite structures, the account of plastic deformations and creep was introduced. In a viscoelastic material under a constant load, deformations increase (accumulate) over time. This leads to a qualitative and significant quantitative change in the stress-strain state: a decrease in stiffness, a redistribution of forces between structural elements, etc. To describe these phenomena, it was proposed to record the relationship between stresses and deformations for an aging material. On the basis of the theory of an elastic-creeping body, the possibility of an analytical description of fast-flowing creep is shown. Using the technique of N.Kh. Arutyunyan and A.A. Zevin, new creep kernels were constructed in relation to the aging material [5]: 𝐾𝑡𝜏𝑄𝑡𝜏𝐵𝑡𝜏𝑄𝑠𝐵𝑠𝜏𝑑𝑑𝑡 𝐾𝐾1(𝑡𝑡, 𝜏𝜏) = 𝑄𝑄(𝑡𝑡, 𝜏𝜏) + 𝐵𝐵(𝑡𝑡−𝜏𝜏) + ∫𝑄𝑄(𝑠𝑠)𝐵𝐵(𝑠𝑠−𝜏𝜏)𝑑𝑑𝑑𝑑 𝑡𝑡 𝜏𝜏 (4) 𝐾𝐾2(𝑡𝑡, 𝜏𝜏) = 𝑄𝑄(𝑡𝑡, 𝜏𝜏) + 𝐵𝐵(𝑡𝑡−𝜏𝜏) + 𝑄𝑄(𝜏𝜏) ∫𝐵𝐵(𝑡𝑡−𝑠𝑠)𝑑𝑑𝑑𝑑 𝑡𝑡 𝜏𝜏 . 2 Main points 4 4 https://doi.org/10.1051/e3sconf/202447401066 E3S Web of Conferences 474, 01066 (2024) ICITE 2023 3 Conclusion The formation of a new creep core by adding the regular and difference cores has led to the possibility of describing the rapidly flowing creep of an aging material. The use of a weakly singular expression in the difference part as applied to an aging body made it possible to simplify the numerical implementation without involving special techniques. The possibility of using the new relationships in real practice was proved by comparison with experimental creep curves of an aging material (Ross data) [5]. The solution of the problem of deformation in time of the composite plate showed that the displacements and stresses in the composite plate change, depending on the age of loading, by 50% or more. The results obtained were used as a theoretical basis for the creation of structures from composite plates and shells. 2 Main points 𝒕𝝉𝑲𝟐𝒕𝝉𝑸𝒕𝝉𝑩𝒕𝝉 𝐾𝐾1(𝑡𝑡, 𝜏𝜏) = 𝑄𝑄(𝑡𝑡, 𝜏𝜏) + 𝐵𝐵(𝑡𝑡−𝜏𝜏) + ∫𝑄𝑄(𝑠𝑠)𝐵𝐵(𝑠𝑠−𝜏𝜏)𝑑𝑑𝑑𝑑 𝑡𝑡 𝜏𝜏 (4) 𝐾𝑡𝜏𝑄𝑡𝜏𝐵𝑡𝜏𝑄𝜏𝐵𝑡𝑠𝑑𝑑𝑡 𝐾𝐾2(𝑡𝑡, 𝜏𝜏) = 𝑄𝑄(𝑡𝑡, 𝜏𝜏) + 𝐵𝐵(𝑡𝑡−𝜏𝜏) + 𝑄𝑄(𝜏𝜏) ∫𝐵𝐵(𝑡𝑡−𝑠𝑠)𝑑𝑑𝑑𝑑𝑡𝜏 . 𝑲𝟏𝒕𝝉𝑲𝟐𝒕𝝉𝑸𝑩𝒕𝝉 𝜏𝜏 Here 𝑲𝑲𝟏𝟏(𝒕𝒕, 𝝉𝝉), 𝑲𝑲𝟐𝟐(𝒕𝒕, 𝝉𝝉) – are the creep kernels of the aging material; 𝑸𝑸(𝒕𝒕, 𝝉𝝉) – is the regular part of the creep kernel; 𝑩𝑩(𝒕𝒕−𝝉𝝉) – is the difference part of the creep kernel; 𝒕𝒕−𝝉𝝉 – is the difference argument; 𝒕𝒕 – considered moment in time; 𝝉𝝉 – is an intermediate moment in time; 𝒔𝒔 – is an intermediate argument. A weakly singular kernel satisfying all the necessary requirements was used as the difference component: 𝑩𝒕𝝉𝝌𝒆𝝆𝒕𝝉𝜶𝒕𝝉𝝑𝟏𝟏 𝑩𝑩(𝒕𝒕−𝝉𝝉) = 𝝌𝝌 𝒆𝒆−𝝆𝝆(𝒕𝒕−𝝉𝝉)𝜶𝜶 (𝒕𝒕−𝝉𝝉)𝝑𝝑−𝟏𝟏 𝟏𝟏 Г(𝝑𝝑) (5) 𝜗𝜗𝑡𝜏 where 𝐵𝐵(𝑡𝑡−𝜏𝜏) – is the difference part of the creep kernel; Г(𝜗𝜗) – gamma function; χ, ρ, 𝜗𝜗 – core parameters [χ > 0; ρ > 0; 𝜗𝜗 ϵ (0;1); α ϵ (0;1)]; 𝑡𝑡−𝜏𝜏 – is the difference argument; 𝑡𝑡 – considered moment in time; 𝜏𝜏 – intermediate time. The core was used as a regular component, the basis of which - the creep measure had the form: 𝑸𝒕𝝉𝝏𝑪𝟎𝑨𝒆𝜷𝜷𝟏𝒆𝜸𝒕𝝉𝒂𝒌𝒆𝜸𝒌𝒕𝒆𝜶𝒌𝝉𝟑𝒌𝟏 𝑸𝑸(𝒕𝒕, 𝝉𝝉) = − 𝝏𝝏 𝝏𝝏𝝏𝝏 [𝑪𝑪𝟎𝟎+ 𝑨𝑨𝒆𝒆−𝜷𝜷𝜷𝜷][𝟏𝟏− 𝒆𝒆−𝜸𝜸(𝒕𝒕−𝝉𝝉)] = ∑ 𝒂𝒂𝒌𝒌𝒆𝒆−𝜸𝜸𝒌𝒌𝒕𝒕𝒆𝒆𝜶𝜶𝒌𝒌𝝉𝝉 𝟑𝟑 𝒌𝒌=𝟏𝟏 (6) 𝑎𝛽𝛽𝑎𝐶𝛾𝑎𝐴𝛾𝛽𝛼𝛽𝛼𝛾𝛼𝛾𝛽𝛾𝛾𝛾𝛾 (6) 𝑎𝑎1 = 𝛽𝛽𝛽𝛽, 𝑎𝑎2 = 𝐶𝐶0𝛾𝛾, 𝑎𝑎3 = 𝐴𝐴(𝛾𝛾− 𝛽𝛽), 𝛼𝛽𝛼𝛾𝛼𝛾𝛽𝛾𝛾𝛾𝑡𝜏 𝑎𝑎1 = 𝛽𝛽𝛽𝛽, 𝑎𝑎2 = 𝐶𝐶0𝛾𝛾, 𝑎𝑎3 = 𝐴𝐴(𝛾𝛾− 𝛽𝛽), 𝑎𝑎1 = 𝛽𝛽𝛽𝛽, 𝑎𝑎2 = 𝐶𝐶0𝛾𝛾, 𝑎𝑎3 = 𝐴𝐴(𝛾𝛾− 𝛽𝛽), 𝛼𝛼1 = −𝛽𝛽, 𝛼𝛼2 = 𝛾𝛾, 𝛼𝛼3 = 𝛾𝛾−𝛽𝛽, 𝛾𝛾1 = 0, 𝛾𝛾2 = 𝛾𝛾3 = 𝛾𝛾. 𝑄𝑡𝜏𝑡𝜏𝜏 𝑎1 𝛽𝛽𝛽𝑎 2𝐶 0𝛾𝛾𝑎 3𝐴 (𝛾𝛾 𝛽𝛽) 𝛼𝛼1 = −𝛽𝛽, 𝛼𝛼2 = 𝛾𝛾, 𝛼𝛼3 = 𝛾𝛾−𝛽𝛽, 𝛾𝛾1 = 0, 𝛾𝛾2 = 𝛾𝛾3 = 𝛾𝛾. 𝑄𝑡𝜏𝑡𝜏𝜏 Here 𝑄𝑄(𝑡𝑡, 𝜏𝜏) – regular part of the creep core; 𝑡𝑡−𝜏𝜏 – is the difference argument; 𝑡𝑡 – considered moment in time; 𝜏𝜏 – intermediate time. After transformations, a formula was obtained that was used to calculate the bending of a composite structure, taking into account the visco-elastic properties of the aging material: 𝑲𝒓𝒕𝝉𝑸𝒕𝝉𝑩𝒕𝝉𝝌𝒂𝒌𝟑𝒌𝟏𝒆𝜶𝒌𝝉𝜸𝒌𝒕𝒆𝜼𝒓𝒌𝒛𝝆𝒛𝜶𝒕𝝉𝒛𝝑𝟏𝒅𝒅 𝑲𝑲𝒓𝒓(𝒕𝒕, 𝝉𝝉) = 𝑸𝑸(𝒕𝒕, 𝝉𝝉) + 𝑩𝑩(𝒕𝒕−𝝉𝝉) + 𝝌𝝌 Г(𝝑𝝑) ∑ [𝒂𝒂𝒌𝒌 𝟑𝟑 𝒌𝒌=𝟏𝟏 𝒆𝒆𝜶𝜶𝒌𝒌𝝉𝝉−𝜸𝜸𝒌𝒌𝒕𝒕∫ 𝒆𝒆𝜼𝜼𝒓𝒓𝒌𝒌𝒛𝒛−𝝆𝝆𝒛𝒛𝜶𝜶 𝒕𝒕−𝝉𝝉 𝟎𝟎 𝒛𝒛𝝑𝝑−𝟏𝟏𝒅𝒅𝒅𝒅 (7) where r = 1, 2; 𝜂𝜂1𝑘𝑘= 𝛼𝛼𝑘𝑘; 𝜂𝜂2𝑘𝑘= 𝛾𝛾𝑘𝑘, 𝑎𝑎𝑘𝑘, 𝜒𝜒 – required parameters. where r = 1, 2; 𝜂𝜂1𝑘𝑘= 𝛼𝛼𝑘𝑘; 𝜂𝜂2𝑘𝑘= 𝛾𝛾𝑘𝑘, 𝑎𝑎𝑘𝑘, 𝜒𝜒 – required parameters For numerical implementation, the exponential functions in the integrand were expanded into a Maclaurin power series. 4 Discussion The work carried out made it possible to proceed to the development of materials and structures with predictable properties. As a result of the use of nanomaterials, a more precise adjustment of such structural properties as strength, deformability, durability, rest of it. Development of the technology for manufacturing products is based on the study of the effect of temperature on the formation of the properties of composites [1]. Composite materials are planned with the specified parameters, oriented to the purpose (target application). Depending on the purpose of the product, a specially selected composition and manufacturing technology will provide the necessary characteristics: strength, chemical resistance, plasticity, etc. The solution of manufacturing issues is associated with the creation of new and modernization of existing installations, the selection of the composition of the composite, ensuring the specified quality of products at a lower cost compared to the existing analogues, which will ensure the commercialization of the project. The proposed technology makes it possible to manufacture and carry out thermal insulation of production sites of oil and gas fields in permafrost conditions. Samples of composite boards are shown in the figure 1. Thermal insulation materials are often used in the construction of various types of roads. The latter include railways, motorways and other roads, runways, pedestrian zones, etc. Protection against freezing of road surfaces is one of the important issues. The material for thermal insulation will protect the road from the destructive effects of frost heaving of the soil. a) b) a) b) b) a) a) 5 https://doi.org/10.1051/e3sconf/202447401066 E3S Web of Conferences 474, 01066 (2024) ICITE 2023 c) d) Fig. 1. Samples of composite boards. a) – perlite, b) – expanded polystyrene, c) – expanded clay, d) – multilayer slab. d) c) d) c) Fig. 1. Samples of composite boards. Fig. 1. Samples of composite boards. – perlite, b) – expanded polystyrene, c) – expanded clay, d) – multilayer slab. It is proposed to use composite plates based on cement-polymer materials with fillers for the manufacture of heat-shielding insulation elements for roadbeds for various purposes. The influence of the composition and size of the composite on the properties and characteristics of products are summarized in the table 1. characteristics of products are summarized in the table 1. Table 1. Composition and sizes of composite ingredient. 4 Discussion Material Indicators Specific gravity (kg/m3) Coefficient of thermal conductivity (watt/(mdegree)) Particle size (mm) Cost (rubles) Cement М 500 1350 0,25 5-50 10-3 7000 per ton Sand (10% of humidity) 1500 0,97 0,1-1,5 1000 per ton Perlite М76 76 0,05 0,16-1,25 2500 per m3 Expanded polystyrene granules 15 0,037 2-2,5 2000 per m3 Expanded clay 0-5 mm 450 0,2 0-5 1500 per m3 Expanded clay 10-20 mm 300 0,16 10-20 1300 per m3 The composite consists of particles of materials of various sizes: nanometers, microns, millimeters and centimeters. Nano-technologies implemented. This allows to obtain a material with the required characteristics of strength, water resistance and thermal insulation. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems. The intended purpose of products can be achieved by using a material with special characteristics in each layer. Depending on the purpose, the characteristics of the layers can provide gas tightness, strength etc. Table 1. Composition and sizes of composite ingredient. Material Indicators Specific gravity (kg/m3) Coefficient of thermal conductivity (watt/(mdegree)) Particle size (mm) Cost (rubles) Cement М 500 1350 0,25 5-50 10-3 7000 per ton Sand (10% of humidity) 1500 0,97 0,1-1,5 1000 per ton Perlite М76 76 0,05 0,16-1,25 2500 per m3 Expanded polystyrene granules 15 0,037 2-2,5 2000 per m3 Expanded clay 0-5 mm 450 0,2 0-5 1500 per m3 Expanded clay 10-20 mm 300 0,16 10-20 1300 per m3 Table 1. Composition and sizes of composite ingredient. The composite consists of particles of materials of various sizes: nanometers, microns, millimeters and centimeters. Nano-technologies implemented. This allows to obtain a material with the required characteristics of strength, water resistance and thermal insulation. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems The intended purpose of products The composite consists of particles of materials of various sizes: nanometers, microns, millimeters and centimeters. Nano-technologies implemented. This allows to obtain a material with the required characteristics of strength, water resistance and thermal insulation. The composite consists of particles of materials of various sizes: nanometers, microns, millimeters and centimeters. Nano-technologies implemented. This allows to obtain a material with the required characteristics of strength, water resistance and thermal insulation. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems. References 1. Yu. E. Yakubovskiy, V. S. Goltsov, V. I. Kolosov, Procedia Engineering 165, 1238–45 (2016) 2. Yu. E. Yakubovskiy, V. I. Kolosov, B. A. Gulyaev, V. S. Goltsov, Procedia Engineering 165, 1246–53 (2016) 3. Yu. E. Yakubovskiy, V. I. Kolosov, “Physically Nonlinear Bending of Composite Plates”, in Proceedings of Energy, Environmental and Construction Engineering 2019 (2020), pp. 307-318 4. Yu. E. Yakubovskiy, B. A. Gulyaev, V. I. Kolosov, N. A. Krivchun, S. V. Yakubovskaya, Bending of composite plates and thin shells (Tyumen Industrial University Press, Tyumen, 2016) 5. Yu. E. Yakubovskiy, V. I. Kolosov, I. A. Donkova, S. O. Kruglov, Physical and mathematical modeling. Oil, gas, energy 4, 181-191 (2018) 6. D. Ding, L. Lv, G. Xiao, J. Luo, C. Lei, Y. Ren, X. Hou, International Journal of Applied Ceramic Technology 17(2), 645-656 (2020) 7. Y. Gao, W. Xiao, H. Zhu, European Journal of Mechanics, A/Solids 82, 103993 (2020). 8. Z. Han, J. Ren, J. Zhou, S. Zhang, Z. Zhang, L. Yang, C. Yin, International Journal of Hydrogen Energy 45(11), 7223-7233 (2020) 9. N. Jiang, Y. Liu, X. Yu, H. Zhang, M. Wang, International Journal of Electrochemical Science 15, 5520-5528 (2020) 10. C. Li, Z. Li, S. Li, Y. Zhang, B. Sun, Y. Yu, W. Yue, Optics Express 28(5), 6071-6083 (2020) 11. M. Li, Z. Zhang, H. Gao, Y. Wang, J. Liang, D. Shu, B. Sun, Materials Characterization 159, 110018 (2020) 12. W. Peng, K. Sun, Mechanics of Materials 141, 103270 (2020) 13. Y. Shang, G. Yang, F. Su, Y. Feng, Y. Ji, D. Liu, C. Shen, Composites Communications 19, 147-153 (2020) 14. A. Sheng, W. Ren, Y. Yang, D. Yan, H. Duan, G. Zhao, Z. Li, Composites Part A: Applied Science and Manufacturing 129, 105692 (2020) 15. S. Tajmiri, E. Azimi, M. R. Hosseini, Y. Azimi, Environmental Research 182, 108997 (2020) 16. L. Wang, R. Luo, G. Cui, Z. Chen, Corrosion Science 167, 108522 (2020) 17. Y. Wang, W. Wang, X. Ding, D. Yu, Chemical Engineering Journal 380, 122553 (2020) 18. L. Xu, H. Wang, Z. Chu, L. Cai, H. Shi, C. Zhu, Y. Lei, ACS Applied Polymer Materials 2(2), 741-750 (2020) 19. X. Xu, H. F. Lu, K. Y. Luo, J. H. Yao, L. Z. Xu, J. Z. Lu, Y. F. Lu, Journal of Materials Processing Technology 284, 116736 (2020) 20. Y. Zhang, H. Yang, Z. Dang, S. Zhan, C. Sun, G. Hu, Q. 4 Discussion The intended purpose of products can be achieved by using a material with special characteristics in each layer. Depending on the purpose, the characteristics of the layers can provide gas tightness, strength etc. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems. The intended purpose of products can be achieved by using a material with special characteristics in each layer. Depending on the purpose, the characteristics of the layers can provide gas tightness, strength etc. The solution of these issues in thin-walled structures is associated with the development of technologies for creating multilayer composite systems. The intended purpose of products can be achieved by using a material with special characteristics in each layer. Depending on the purpose, the characteristics of the layers can provide gas tightness, strength etc. 6 6 6 https://doi.org/10.1051/e3sconf/202447401066 E3S Web of Conferences 474, 01066 (2024) ICITE 2023 References Yuan, ACS Applied Materials and Interfaces 12(19), 22137-22145 (2020) 7 7
https://openalex.org/W4290981402	https://www.nature.com/articles/s41467-022-31891-3.pdf	English	null	N-Heterocyclic carbene-based C-centered Au(I)-Ag(I) clusters with intense phosphorescence and organelle-selective translocation in cells	Nature communications	2,022	cc-by	8,804	ARTICLE 1 Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Research Center for Computational Science, Institute for Molecular Science and SOKENDAI, Myodaiji, Okazaki, Aichi 444-8585, Japan. 3 Department of Life Science and Technology, Tokyo Institute of Technology, 2-12-1-M6-7 Ookayama, Meguro-ku, Tokyo 152-8550, Japan. 4Present address: Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan. 5These authors contributed equally: Zhen Lei, Mizuki Endo. ✉email: ehara@ims.ac.jp; ozawa@chem.s.u-tokyo.ac.jp; shionoya@chem.s.u-tokyo.ac.jp ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Fig. 1 Schematic diagram of this study. Carbon(C)-centered AuI-AgI clusters with N-heterocyclic carbene (NHC) ligands with intense phosphorescence and their ligand-speciﬁc, organelle-selective translocation in cells. S ub-nanoscale gold clusters with atomic precision are pro- mising miniature nanoscale materials. Gold clusters often exhibit photoluminescence properties such as phosphores- cence, in addition to the unique molecular structures and aurophilicity1,2. To date, several excellent protocols have been developed to improve the photoluminescence performance of gold clusters. Examples include alloying by metal kernels, supramolecular networking by self-assembly of clusters, surface hardening by additives, chemical modiﬁcation by capping ligands with electron-donating/withdrawing groups, and so on3–10. The structure, electronic state, and reactivity of the gold cluster moiety can be greatly affected by the protective ligands and different metals that additionally bind to the gold atoms. y g The octahedral hexagold(I) cluster with a hyper-coordinated carbon center (CAuI6), ﬁrst developed by Schmidbaur et al. using phosphine ligands, is one of the most classical models of AuI clusters11,12. These clusters emit bright yellowish-green light in the solid-state, but not in solution. Wang et al. reported a het- eronuclear metal cluster capable of emitting light even in solution by the pyridyl-phosphine bidentate ligand13,14. This method has made it possible to construct a series of heterometallic AuI clusters that exhibit strong red phosphorescence in solution15,16. This luminescence is thought to be due to the formation of a solid sphere in which the surface of the cluster is completely protected17. It has also been reported that such cluster complexes are a promising group of compounds that emit light at speciﬁc locations in the cell18. Fig. 1 Schematic diagram of this study. Carbon(C)-centered AuI-AgI clusters with N-heterocyclic carbene (NHC) ligands with intense phosphorescence and their ligand-speciﬁc, organelle-selective translocation in cells. can improve the phosphorescence emission efﬁciency of C-centered AuI clusters, but also guide the development of functions for metal cluster-based functional molecules in che- mical synthesis approaches. More recently, N-heterocyclic carbene (NHC) ligands have been developed as one of the most promising organic ligands for Au0 clusters with high designability19–22. Carbene ligands have strong electron-donating properties and are known to enhance the stability of metal surfaces, metal nanoparticles, and metal nanorods23–25. Therefore, NHC ligands have been used in the synthesis of Au0 clusters26–30, and their interfacial structure, stability, and catalytic activity have been elucidated. Results Synthesis and characterization. To further polynucleate the AuI cluster with different metals, a nitrogen donor was introduced into the wing-tip portion of each NHC ligand (N-isopropyl-N’−2-(5- methylpyridyl)benzimidazolylidene (1a), N-isopropyl-N’−2-pyr- idylbenzimidazolylidene (1b), N-isopropyl-N’−2-(5-methylpyridyl) imidazolylidene (1c), N-isopropyl-N’−2-pyridylimidazolylidene (1d); Supplementary Figs. 1–4). Speciﬁcally, CAuI6 complexes [(C) (AuI-L)6](BF4)2 (2a–d, L = 1a–d) in which only the carbene ligand is coordinated to AuI, were synthesized from unsymmetrical bidentate ligands 1a–d. The isolation yields of 2a–d were 8–52% based on the amount of AuI used according to previously reported literatures11–18, and their molecular structures in the solid-state were determined by single-crystal X-ray diffraction (ScXRD) (Fig. 2, Supplementary Figs. 5, 6, and Supplementary Table 8). It was found by NMR spectroscopy (Supplementary Figs. 8–27) and mass spectrometry (Supplementary Fig. 28) that the solid-state structure was maintained in solution. The heterometallic CAuI6AgI2 clusters, [(C)(AuI-L)6AgI2](BF4)4 (3a–d, L = 1a–d) were synthesized from CAuI6 clusters 2a–d with pyridyl pendants-introduced ligands 1a–d. The processes of complexation of CAuI6 clusters 2a–d with AgBF4 were investi- gated by UV-vis absorption, phosphorescence spectroscopy, mass spectrometry, and NMR spectroscopy (Supplementary Figs. 34–46). CAuI6AgI2 clusters, 3a–d, were isolated as single crystals by layering dry Et2O on a solution of each reaction mixture in CH2Cl2/CH3OH. As shown in Fig. 3a, the molecular structures of CAuI6AgI2 clusters, [(C)(AuI-L)6AgI2](BF4)4 (3a–d, L = 1a–d), were determined by ScXRD, and all clusters were found to have a bicapped octahedral core. The two AgI ions in each cluster are located on two opposite sides of the octahedron, each anchored by three pyridyl groups and interacting with three neighboring AuI atoms. These CAuI6AgI2 clusters 3a–d showed strong intramolecular C −H ∙∙∙Au interactions34–36, with the shortest H-Au distances being 2.690, 2.818, 2.765, and 2.730 Å, respectively (Supplementary Fig. 47). The structures are similar to In this study, we design and synthesize bidentate ligands consisting of an NHC ligand linked to a pyridyl ligand, and clarify the detailed structure and photochemical properties of hetero- nuclear clusters of AuI and AgI, which exhibit very high phos- phorescence quantum yields even in solution. Furthermore, the strong phosphorescence emission of these clusters is used to elucidate the cellular uptake and organelle-selective translocation pathways of the cluster complexes (Fig. 1). Interestingly, the NHC ligand-protected heterometallic clusters are translocated to a speciﬁc intracellular organelle in a ligand-speciﬁc manner. This is in sharp contrast to phosphine-protected clusters with the same metal core structure, which are non-selectively dispersed in the cytosol. ARTICLE In this con- text, we successfully applied the NHC ligand to the CAuI6 cluster in 201831–33. We found that when an imidazolylidene carbene ligand was attached to each gold atom of the C-centered CAuI6 cluster, the phosphorescence emission was red-shifted in the solid-state compared to clusters protected by phosphine ligands31. On the other hand, when a benzimidazolylidene ligand with one benzene ring fused to the imidazole ring was used, the phosphorescence emission showed a large blue shift of about 60 nm32,33. These two examples indicate that such a simple chemical modiﬁcation can signiﬁcantly change the photochemical properties of the clusters. Phosphorescent metal cluster com- plexes have the potential to precisely control the structure and electronic state of the metal cluster part by ligand design, and are expected to contribute not only to the creation of structure- speciﬁc photochemical functions but also to live-cell imaging and elucidation of intracellular molecular behaviors. NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications N-Heterocyclic carbene-based C-centered Au(I)- Ag(I) clusters with intense phosphorescence and organelle-selective translocation in cells Zhen Lei 1,5, Mizuki Endo 1,5, Hitoshi Ube 1, Takafumi Shiraogawa2, Pei Zhao 2, Koichi Nagata 1,4, Xiao-Li Pei1, Tomoya Eguchi3, Toshiaki Kamachi 3, Masahiro Ehara 2✉, Takeaki Ozawa 1✉& Mitsuhiko Shionoya 1✉ Zhen Lei 1,5, Mizuki Endo 1,5, Hitoshi Ube 1, Takafumi Shiraogawa2, Pei Zhao 2, Koichi Nagata 1,4, Xiao-Li Pei1, Tomoya Eguchi3, Toshiaki Kamachi 3, Masahiro Ehara 2✉, Takeaki Ozawa 1✉& Mitsuhiko Shionoya 1✉ Photoluminescent gold clusters are functionally variable chemical modules by ligand design. Chemical modiﬁcation of protective ligands and introduction of different metals into the gold clusters lead to discover unique chemical and physical properties based on their signiﬁcantly perturbed electronic structures. Here we report the synthesis of carbon-centered Au(I)-Ag(I) clusters with high phosphorescence quantum yields using N-heterocyclic carbene ligands. Speciﬁcally, a heterometallic cluster [(C)(AuI-L)6AgI2]4+, where L denotes benzimidazolylidene- based carbene ligands featuring N-pyridyl substituents, shows a signiﬁcantly high phosphor- escence quantum yield (Φ = 0.88). Theoretical calculations suggest that the carbene ligands accelerate the radiative decay by affecting the spin-orbit coupling, and the benzimidazolylidene ligands further suppress the non-radiative pathway. Furthermore, these clusters with carbene ligands are taken up into cells, emit phosphorescence and translocate to a particular organelle. Such well-deﬁned, highly phosphorescent C-centered Au(I)-Ag(I) clusters will enable ligand- speciﬁc, organelle-selective phosphorescence imaging and dynamic analysis of molecular dis- tribution and translocation pathways in cells. 1 Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Research Center for Computational Science, Institute for Molecular Science and SOKENDAI, Myodaiji, Okazaki, Aichi 444-8585, Japan. 3 Department of Life Science and Technology, Tokyo Institute of Technology, 2-12-1-M6-7 Ookayama, Meguro-ku, Tokyo 152-8550, Japan. 4Present address: Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan. 5These authors contributed equally: Zhen Lei, Mizuki Endo. ✉email: ehara@ims.ac.jp; ozawa@chem.s.u-tokyo.ac.jp; shionoya@chem.s.u-tokyo.ac.jp 1 NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 ARTICLE Fig. 2 Heteronuclear CAuI6AgI2 clusters 3a–d were used in this study. a Synthetic route for CAuI6AgI2 clusters 3a–d. b Chemical structures of ligands 1a–d. c Molecular structure of [(C)(AuI−1a)6](BF4)2 (2a, 25% probability) with the anions BF4- simpliﬁed. Fig. 2 Heteronuclear CAuI6AgI2 clusters 3a–d were used in this study. a Synthetic route for CAuI6AgI2 clusters 3a–d. b Chemical structures of ligands 1a–d. c Molecular structure of [(C)(AuI−1a)6](BF4)2 (2a, 25% probability) with the anions BF4- simpliﬁed. those of the previously reported [(C)(AuI-dppy)6AgI2](BF4)4 (4, dppy = 2-pyridyldiphenylphosphine)14, but differ in the following two points. First, because the C −AuI bond distances in 3a–d with NHC ligands are shorter than those of the P −AuI bonds in 4 with phosphine ligands, and also because the AuI-AgI distances are shorter in 3a–d, the overall structures of the CAuI6AgI2 parts of 3a–d are signiﬁcantly smaller than that of 4 (Fig. 3b). The AuI- AgI distances in 3a–d are in a very similar range (Supplementary Table 13). Second, there are no intramolecular C −H ∙∙∙Au interactions in 4. consistent with those observed when AgBF4 was added to the solutions of 2a–d. Their molar absorbance coefﬁcients of 3a and 3b, protected with benzimidazolylidene ligands, were signiﬁcantly higher than those of 3c and 3d, protected with imidazolylidene ligands. g Importantly, the use of the bidentate NHC ligands and the coordination of AgI ions have a synergistic effect, and the heterogeneous metal clusters emit very strong phosphorescence in solution (Fig. 3e and Supplementary Fig. 66). In comparison, the metal clusters 3a–d with NHC ligands emit yellow light in solution, whereas 4 with phosphine ligands emit red phosphor- escence, with a difference in wavelength of about 90 nm. The phosphorescence quantum yields of 3a and 3b were determined to be 0.88 and 0.86 in CH2Cl2 (λemmax = 562 nm), respectively, the highest values among the reported AuI clusters (Fig. 3g). In contrast, single crystals of 3c and 3d with imidazolylidene ligands showed similar yellow luminescence, but the phosphor- escence quantum yields in CH2Cl2 and CH2Cl2/CH3OH were remarkably low, 0.14 and 0.01, respectively. In addition, the phosphorescence lifetimes of 3a and 3b protected with benzimidazolylidene ligands were 1.85 and 1.66 μs, respectively, which were signiﬁcantly longer than those of 3c and 3d protected with imidazolylidene ligands, 0.32 and 0.16 μs, respectively (Fig. 3f). The radiative (kr) and non-radiative rate constants (knr) shown in Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 3g indicate that clusters with NHC ligands have signiﬁcantly higher radiative emission rates than clusters with phosphine ligands. Furthermore, the benzimidazolylidene ligand could dramatically improve the quantum yield and microsecond-level lifetime of the phosphorescence of the CAuI6AgI2 clusters, 3a and 3b, by signiﬁcantly suppressing the non-radiative relaxation pathways. Upon photoexcitation, 3a–d showed strong yellow lumines- cence in the solid-state with λemmax lying in the range from 559 to 578 nm, which signiﬁcantly red-shifted as compared to 2a–d (from 482 to 490 nm), and is similar to 4 (553 nm, Fig. 3c and Supplementary Figs. 29 and 48). The two AgI atoms do not simply bind to the CAuI6 core but may change the electronic structure of the whole clusters13–17. The CAuI6AgI2 clusters 3a–d were further characterized by solution-phase NMR spectroscopy (Supplementary Figs. 49–64). Two interesting features were observed in the 1H NMR spectra. First, in the 1H NMR spectra of 3a–d, the septet signals of the secondary hydrogen atoms of the isopropyl groups were shifted to the high ﬁeld by about 0.5 ppm compared to 2a–d, which is a change similar to that observed when AgBF4 was added to 2a–d (Supplementary Figs. 42–45). This result conﬁrmed the existence of C −H ∙∙∙Au interactions in 3a–d even in solution36. The second feature is that split signals of methyl of isopropyl groups were observed in the both 1H and 13C NMR spectra of 3a–d. For example, the methyl groups of 2b showed only one set of signals in the 1H (1.49 ppm) and 13C NMR (21.9 ppm) spectra, whereas two sets of signals were observed in the 1H NMR (1.44 and 1.33 ppm) and 13C NMR (22.6 and 22.1 ppm) spectra for 3b. It was inferred from the molecular structures of 3a–d that this signal change was caused by the helical arrangement of the ligands in the bicapped octahedral CAuI6AgI2 and the two opposing CAuI3AgI moieties showing different helical directions14,15. Heterometallic species, [(C)(AuI-L)4AuIAgI](BF4)+ and [(C) (AuI-L)6AgI](BF4)2+ (L = 1a–d), were also observed in the MS spectra (Supplementary Fig. 65). These results strongly suggest that all of 3a–d maintain the bicapped octahedral structures even in solution. Theoretical calculations of CAuI6AgI2 clusters. In order to mechanistically clarify how the photochemical properties change depending on the type of carbene ligands, the absorption and phosphorescence properties of 3b with benzimidazolylidene ligands and 3d with imidazolylidene ligands were theoretically calculated and comparatively analyzed (Fig. 4). Results Thus, the control of intracellular translocation of C-centered AuI clusters is achieved by a slight modiﬁcation of organic ligands. These results not only show that NHC ligands 2 NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 reproduced. The distributions of SOMO and SOMO −1, which characterize phosphorescence, are similar to those of LUMO and HOMO respectively (Fig 4c d) The imidazolylidene and ben close agreement with the experimental values of 2.21 and 2.17 eV, respectively, within error37 (Supplementary Table 21). It is worth noting that the molecular structure of the clusters Fig. 3 Molecular structures and photochemical properties of CAuI6AgI2 clusters 3a–d and 4. a Molecular structures of 3a–d (50% probability for 3a, 3c, and 3d; 25% probability for 3b), with the anions BF4- simpliﬁed. b Comparison of the key structure parameters of 3a–d and 4. c Emission spectra of 3a–d and 4 in the solid-state, with λemmax being 559, 573, 578, 570, and 553 nm, respectively, and corresponding photos at room temperature. d UV-vis absorption spectra of 3a (ε336 = 8.4 × 104 M−1 cm−1), 3b (ε336 = 8.3 × 104 M−1 cm−1), 3c (ε333 = 3.4 × 104 M−1 cm−1), 3d (ε330 = 3.3 × 104 M−1 cm−1), and 4 (ε321 = 1.8 × 104 M−1 cm−1) in CH2Cl2 (293 K). e Emission spectra of 3a–d and 4 in CH2Cl2 at 293 K, with λemmax of 562, 562, 564, 571, and 650 nm, respectively, and the corresponding photos taken at room temperature. f Emission decay of 3a–d and 4 in CH2Cl2 at room temperature, with τ of 1.85, 1.66, 0.32, 0.16, and 3.74 µs, respectively. g Quantum yields, radiative rate constants (kr) and non-radiative rate constants (knr) of 3a–d and 4 in CH2Cl2 at room temperature. Note that a mixed solvent CH2Cl2/CH3OH (9:1, v:v) was used for 3d42. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Fig. 3 Molecular structures and photochemical properties of CAuI6AgI2 clusters 3a–d and 4. a Molecular structures of 3a–d (50% probability for 3a, 3c, and 3d; 25% probability for 3b), with the anions BF4- simpliﬁed. b Comparison of the key structure parameters of 3a–d and 4. c Emission spectra of 3a–d and 4 in the solid-state, with λemmax being 559, 573, 578, 570, and 553 nm, respectively, and corresponding photos at room temperature. d UV-vis absorption spectra of 3a (ε336 = 8.4 × 104 M−1 cm−1), 3b (ε336 = 8.3 × 104 M−1 cm−1), 3c (ε333 = 3.4 × 104 M−1 cm−1), 3d (ε330 = 3.3 × 104 M−1 cm−1), and 4 (ε321 = 1.8 × 104 M−1 cm−1) in CH2Cl2 (293 K). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 As a result, the calculated absorption spectra agreed well with the experimental observations in the calculated energy range. The trend of the energy difference was reproduced; the ﬁrst peaks for 3b and 3d were calculated to be 387 and 388 nm, respectively. The order of the molar absorbance coefﬁcients (3b > 3d) was also well Solutions of CAuI6AgI2 clusters 3a–d in CH2Cl2 or CH2Cl2/ CH3OH showed multiple optical absorption bands in the range of 300–450 nm (Fig. 3d). These optical absorption modes were 3 ARTICLE NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Finally, in order to evaluate knr, the minimum energy crossing point (MECP) between S0 and T1 states was calculated using the Harvey method43,44. The results showed that the core of CAuI6AgI2 is signiﬁcantly deformed at the MECP. For example, the shape of CAuI6AgI2 of 3d is signiﬁcantly changed compared to the shape of the T1 state (Supplementary Fig. 72). The energy barrier from the minimum of T1 state to MECP were calculated as 11.6 and 10.8 kcal/mol for 3b and 3d, respectively, which qualitatively agrees with the knr of these complexes (Supplemen- tary Table 29). However, the energy difference of the MECP for 3b and 3d is small, so there may be another cause. Overall, although NHC ligands are less involved in the electronic structure of the clusters than phosphine ligands, they can accelerate radiative decay by enhancing the spin–orbit coupling of the low- lying spin–orbit states. On the other hand, when the benzimi- dazolylidene ligand was used, non-radiative decays did not occur preferentially, resulting in 3a and 3b having very high quantum yields. We then directly investigated the phosphorescence lifetimes and the radiative rate constants of 3b and 3d using the ZORA method with spin–orbit interaction in a perturbative way40 implemented in the ADF program package41. The results are compared to the experimental values, which approximate the calculated kr as kr = 1/τ (Supplementary Table 27). The phosphorescence is attributed to the three lowest, nearly degenerate spin–orbit states, which are about 0.1 eV lower than other states, with T1 being the dominant contribution. Although the order of the absolute values of τ differs from the experimental values, the trend of kr (3b > 3d) agrees with each other. Importantly, the kr values of imidazolylidene- and benzimidazolylidene-protected clusters were almost quantita- tively reproduced in the calculations. Note that the low kr of 3d with imidazolylidene ligands is due to the solvent effect of CH3OH42. In addition, we analyzed the wavefunction and spin–orbit coupling of the low-lying spin–orbit states (Supple- mentary Tables 27 and 28). These states are described mainly by the T1, T2, and T3 components, with small contributions from the S1 and S3 components. It can be seen that the NHC ligand signiﬁcantly changed the main component of each state and the coupling of the spin–orbit states. This is thought to be the origin of the different kr values of these compounds. Ligand-speciﬁc translocation in cells. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Fig. 4 TD-DFT calculations for CAuI6AgI2 clusters 3b and 3d. a, b Calculated and experimental absorption spectra of 3b (calc. ε388 = 3.0 × 104 M−1 cm−1, exp. ε390 = 3.7 × 104 M−1 cm−1) and 3d (calc. ε389 = 2.0 × 104 M−1 cm−1, exp. ε385 = 1.6 × 104 M−1 cm−1). The excitation energies, oscillator strengths, and transition characters for the absorption spectra are summarized in Supplementary Tables 18, 19, and the associated molecular orbitals (MOs) are shown in Supplementary Figs. 68, 69. The lowest peaks observed around 400 nm for 3b and 3d were considered to be transitions between MOs consisting mainly of AuI, AgI and the central carbon. The strong absorption peaks around 330 nm for 3b and 3d are due to the MLCT transition from AuI to ligands37. The absorption in the high-energy region (<300 nm) is mainly due to the ππ* transition of the ligands. c, d Calculated MOs (lowest unoccupied molecular orbital, LUMO; highest occupied molecular orbital, HOMO; singly occupied molecular orbital, SOMO, in the triplet state), comparisons of calculated and experimental excitation energies, and analysis of orbital composition by Mulliken partition of ligands in 3b and 3d. Fig. 4 TD-DFT calculations for CAuI6AgI2 clusters 3b and 3d. a, b Calculated and experimental absorption spectra of 3b (calc. ε388 = 3.0 × 104 M−1 cm−1, exp. ε390 = 3.7 × 104 M−1 cm−1) and 3d (calc. ε389 = 2.0 × 104 M−1 cm−1, exp. ε385 = 1.6 × 104 M−1 cm−1). The excitation energies, oscillator strengths, and transition characters for the absorption spectra are summarized in Supplementary Tables 18, 19, and the associated molecular orbitals (MOs) are shown in Supplementary Figs. 68, 69. The lowest peaks observed around 400 nm for 3b and 3d were considered to be transitions between MOs consisting mainly of AuI, AgI and the central carbon. The strong absorption peaks around 330 nm for 3b and 3d are due to the MLCT transition from AuI to ligands37. The absorption in the high-energy region (<300 nm) is mainly due to the ππ* transition of the ligands. c, d Calculated MOs (lowest unoccupied molecular orbital, LUMO; highest occupied molecular orbital, HOMO; singly occupied molecular orbital, SOMO, in the triplet state), comparisons of calculated and experimental excitation energies, and analysis of orbital composition by Mulliken partition of ligands in 3b and 3d. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 e Emission spectra of 3a–d and 4 in CH2Cl2 at 293 K, with λemmax of 562, 562, 564, 571, and 650 nm, respectively, and the corresponding photos taken at room temperature. f Emission decay of 3a–d and 4 in CH2Cl2 at room temperature, with τ of 1.85, 1.66, 0.32, 0.16, and 3.74 µs, respectively. g Quantum yields, radiative rate constants (kr) and non-radiative rate constants (knr) of 3a–d and 4 in CH2Cl2 at room temperature. Note that a mixed solvent CH2Cl2/CH3OH (9:1, v:v) was used for 3d42. close agreement with the experimental values of 2.21 and 2.17 eV, respectively, within error37 (Supplementary Table 21). reproduced. The distributions of SOMO and SOMO −1, which characterize phosphorescence, are similar to those of LUMO and HOMO, respectively (Fig. 4c, d). The imidazolylidene and ben- zimidazolylidene ligand moieties are involved in the SOMO −1 orbitals. The calculated phosphorescence energies of 3b and 3d are 2.09 and 2.08 eV (592 and 596 nm, respectively), which are in p y pp y It is worth noting that the molecular structure of the clusters in the singlet state is very different from that in the triplet state8,9,38,39 (Supplementary Fig. 71 and Supplementary Table 23). For example, the AuI-AuI distances of 3b in the 4 NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 At a concentration of 5.0 μM, similar localization was observed by extending the incubation time (30 and 60 min), but at the same time, the cells changed to a round shape and the nuclear envelope showed the characteristics of cells under stress45. Similar nucleoli spots and morphological changes were also observed in the cells labeled with 3a-c at a concentration of 5.0 or 10 μM. HEK293T cells and COS7 cells also showed a similar trend (Supplementary Figs. 75–81). It is noteworthy that CAuI6AgI2 clusters protected by the NHC or phosphine ligands showed different distributions in the cells, even though they have almost the same core structure. To clarify the cellular uptake pathway of 3a and 4, cells were incubated with 3a and 4 at a lower temperature (4 °C), where endocytosis was expected to be non-speciﬁcally inhibited. As shown in Fig. 6a, the cells labeled under the condition did not show any luminescence signals. Then, route-speciﬁc inhibitors such as wortmannin, sucrose, and genistein were used at 37 °C to speciﬁcally inhibit macropinocytosis, clathrin- and caveolin- dependent endocytosis, respectively. For cluster 3a with benzimidazolylidene ligands, the genistein treatment effectively inhibited the ER accumulation. These results suggest that the uptake of cluster 3a is due to a caveolin-dependent endocytosis process. In contrast, the accumulation of cluster 4 with phosphine ligands in the cytosol was blocked by all inhibitors. Thus cluster 4 was taken up into the cells and dispersed in a nonspeciﬁc manner. Based on these results, we proposed a possible route of cellular uptake and intracellular translocation of NHC- and phosphine-protected CAuI6AgI2 clusters (Fig. 6b). These clusters ﬁrst accumulate on the surface of the cell membrane and are then taken up into the cells by endocytosis. Clusters 3a and 3b were suggested to accumulate in speciﬁc organelles, whereas cluster 4 was uniformly distributed in the cytosol. HEK293T and COS7 cells labeled with 3a or 3b showed a similar distribution pattern. To identify the accumulated struc- tures, the cells labeled with 3a or 3b were further labeled with several organelle markers. Confocal microscope analysis showed that 3a and 3b were selectively located in the ER. The time-lapse imaging revealed that clusters 3a and 3b were transported into the cells within 5 min after accumulation on the surface. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Fig. 5 Optimized bioimaging results of CAuI6AgI2 clusters 3a, 3b, and 4. a Confocal luminescence and differential interference contrast (DIC) images of 3a and 3b in HeLa, HEK293T, and COS7 cells. Scale bar, 50 μm. b Confocal luminescence and DIC images of 4 in HeLa cells. c Co-localization images of 3a and 3b with ER-tracker Red in HeLa cells. d Time-lapse images of 3a in Hela cells. White arrows indicate nuclear accumulation. e PLIM images and lifetime plot of 3a in HeLa cells. Scale bar, 20 μm. Each experiment was independently repeated at least three times with similar results. As previously reported18, strong luminescence spots were observed in the nucleoli of the HeLa cells labeled with 4 at a concentration of 10 μM for 10 min. Time-lapse monitoring also conﬁrmed that cellular uptake and nucleoli accumulation were fast under these conditions. At a concentration of 5.0 μM, similar localization was observed by extending the incubation time (30 and 60 min), but at the same time, the cells changed to a round shape and the nuclear envelope showed the characteristics of cells under stress45. Similar nucleoli spots and morphological changes were also observed in the cells labeled with 3a-c at a concentration of 5.0 or 10 μM. HEK293T cells and COS7 cells also showed a similar trend (Supplementary Figs. 75–81). Fig. 5 Optimized bioimaging results of CAuI6AgI2 clusters 3a, 3b, and 4. a Confocal luminescence and differential interference contrast (DIC) images of 3a and 3b in HeLa, HEK293T, and COS7 cells. Scale bar, 50 μm. b Confocal luminescence and DIC images of 4 in HeLa cells. c Co-localization images of 3a and 3b with ER-tracker Red in HeLa cells. d Time-lapse images of 3a in Hela cells. White arrows indicate nuclear accumulation. e PLIM images and lifetime plot of 3a in HeLa cells. Scale bar, 20 μm. Each experiment was independently repeated at least three times with similar results. As previously reported18, strong luminescence spots were observed in the nucleoli of the HeLa cells labeled with 4 at a concentration of 10 μM for 10 min. Time-lapse monitoring also conﬁrmed that cellular uptake and nucleoli accumulation were fast under these conditions. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Luminescent metal com- plexes have been used as promising bioimaging reagents for the past two decades45–47. Li and Wang et al. have reported on the nucleolus-selective labeling behavior of phosphine-protected CAuI6AgI2 cluster 418. Since carbene-protected CAuI6AgI2 clus- ters 3a and 3b emit phosphorescence sufﬁcient for bioimaging in DMSO/PBS (1:1000, v:v) (Supplementary Figs. 73 and 74, Sup- plementary Table 30), we further examined their behavior in living cells to determine if there are differences in subcellular distribution and cell renewal pathway depending on the organic ligands. Figure 5 shows the optimized bioimaging results for 3a, 3b, and 4. At the concentration of 1.0 or 2.0 μM, where no cytotoxicity was observed by confocal microscopy analysis (Supplementary Figs. 75–82), 3a, 3b, and 4 entered HeLa cells within 10 min. 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 3a or 3b had accumulated in the ER after 10 min, and the longer the incubation time, the more it accumulated in the nucleus region (Fig. 5d and Supplementary Figs. 78, 80). The accumulation of 3a in the ER, induced by a 10-min incubation, was maintained for 36 h (Supplementary Fig. 84). Thus, clusters with the NHC ligands can be selectively translocated to an intracellular organelle in a manner speciﬁc to the ligand structure. Furthermore, since these clusters have long emission lifetimes on the order of micro- seconds, phosphorescence lifetime imaging (PLIM) was con- ducted. The calculated lifetime of 3a was 0.15 μs, which was in good agreement with the in vitro result. Thus, 3a can be used as an ER-selective targeting reagent, and its phosphorescence can be well distinguishable from autoﬂuorescence. NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications 6 ARTICLE Methods For phosphine-protected 4, the uptake is an energy-dependent, nonspeciﬁc process that is associated with macropinocytosis, clathrin-mediated, and/or caveolin-dependent endocytosis. In the case of NHC-protected 3a–d, caveolin-dependent endocy- tosis is the main pathway for cellular uptake. NHC-based clusters 3a and 3b were sequentially translocated to ER after intracellular uptake, but phosphine-based cluster 4 was released from intracellular compartments to cytosol within 10 min. Prolonged incubation allowed the nuclear accumulation of the clusters 3a, 3b, and 4, which in turn increased the permeability of their membranes. In addition, high concentration of clusters acceler- ated these processes. Synthesis of 1a·HI and 1b·HI. Under a nitrogen atmosphere, a Schlenk tube was charged with benzimidazole (5.00 g, 42.3 mmol), K2CO3 (3.90 g, 28.2 mmol), and the corresponding bromopyridine derivatives: 2-bromo-5-methylpyridine (2.43 g, 14.1 mmol) for 1a·HI and 2-bromopyridine (1.37 mL, 14.1 mmol) for 1b·HI. The reaction mixture was heated at 200 °C for 12 h and then allowed to cool to room temperature. After it was diluted with water (50 mL) and extracted with CH2Cl2 (50 mL × 3), the combined organic phase was washed with sat. Na2CO3 aqueous solution (50 mL × 3), and brine (50 mL), and then dried over anhydrous MgSO4 before ﬁltration. Concentration under reduced pressure gave a light red oil. This intermediate was then transferred into a Schlenk ﬂask and dissolved in CH3CN (50 mL) under a nitrogen atmosphere. 2-Iodopropane (1.51 mL, 15.2 mmol) was added to the solution, and the reaction mixture was heated at reﬂux for 12 h. After cooling to room temperature, the mixture was concentrated to ca. 5 mL under reduced pressure. When diethyl ether (50 mL) was added to this residue, a pale yellow precipitate was obtained as a crude product. Colorless crystals 1a·HI and 1b·HI were obtained by layering diethyl ether on CH2Cl2/CH3CN (9:1, v:v) solu- tion containing the crude product. Yields: 2.30 g (43%, based on 2-bromo-5- methylpyridine) for 1a·HI; 1.90 g (37%, based on 2-bromopyridine) for 1b·HI. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 Fig. 6 Mechanisms and proposed routes of cellular uptake of carbene- and phosphine-protected CAuI6AgI2 clusters. a Confocal luminescence and DIC images of clusters 3a and 4 in HeLa cells at 37 °C without pretreatment of inhibitor (control), at 4 °C without pretreatment of inhibitor, or at 37 °C with pretreatment of endocytosis inhibitors wortmannin, sucrose or genistein. Scale bars, 50 μm. Each experiment was independently repeated at least three times with similar results. b Schematic representation of the proposed cellular uptake routes for carbene-protected 3a–d and phosphine-protected 4. Fig. 6 Mechanisms and proposed routes of cellular uptake of carbene- and phosphine Fig. 6 Mechanisms and proposed routes of cellular uptake of carbene- and phosphine-protected CAuI6AgI2 clusters. a Confocal luminescence and DIC images of clusters 3a and 4 in HeLa cells at 37 °C without pretreatment of inhibitor (control), at 4 °C without pretreatment of inhibitor, or at 37 °C with pretreatment of endocytosis inhibitors wortmannin, sucrose or genistein. Scale bars, 50 μm. Each experiment was independently repeated at least three times with similar results. b Schematic representation of the proposed cellular uptake routes for carbene-protected 3a–d and phosphine-protected 4. NATURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunicati ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 (s, 18H, methyl), 1.19 (d, 18H, isopropyl), 0.94 (d, 18H, isopropyl; 13C NMR (126 MHz, CD2Cl2, δ, ppm): 178.6, 150.8, 149.6, 143.1, 136.6, 123.0, 121.8, 119.7, 54.7, 23.0, 22.4, 18.8. ESI-MS (CH2Cl2): 2681.5 ([(C)(Au−1c)6Ag](BF4)2+). (s, 18H, methyl), 1.19 (d, 18H, isopropyl), 0.94 (d, 18H, isopropyl; 13C NMR (126 MHz, CD2Cl2, δ, ppm): 178.6, 150.8, 149.6, 143.1, 136.6, 123.0, 121.8, 119.7, 54.7, 23.0, 22.4, 18.8. ESI-MS (CH2Cl2): 2681.5 ([(C)(Au−1c)6Ag](BF4)2+). CH3CN (50 mL) under a nitrogen atmosphere. 2-Iodopropane (1.70 g, 10.0 mmol) was added to the solution, and the reaction mixture was heated at reﬂux for 12 h. After cooling to room temperature, the mixture was concentrated to ca. 5 mL under reduced pressure. When diethyl ether (50 mL) was added to this residue, a pale yellow precipitate was obtained as a crude product. Colorless crystals of 1c·HI were obtained by layering diethyl ether on CH2Cl2/CH3CN (9:1, v:v) solution containing the crude product. Yield: 1.71 g (52%, based on 2-bromo-5- methylpyridine). CH3CN (50 mL) under a nitrogen atmosphere. 2-Iodopropane (1.70 g, 10.0 mmol) was added to the solution, and the reaction mixture was heated at reﬂux for 12 h. After cooling to room temperature, the mixture was concentrated to ca. 5 mL ( 2 2) ( ( )( )6 g ( 4)2 ) For 3d: 1H NMR (500 MHz, CD2Cl2/CD3OD = 3:1 (v:v), δ, ppm): 8.12 (t, 6H), 7.78 (d, 6H), 7.62 (d, 12H), 7.45 (d, 6H), 7.31 (t, 6H), 4.64 (sept, 6H, isopropyl), 1.16 (d, 18H, isopropyl), 0.96 (d, 18H, isopropyl). ESI-MS (CH2Cl2/CH3OH = 9:1 (v:v)): 1939.2 ([(C)(Au−1d)4AuAg](BF4)+). 1H NMR (500 MHz, CDCl3, δ, ppm): 11.37 (s, 1H, imidazolyl), 8.54 (d, 1H, pyridyl), 8.32 (d, 1H, pyridyl), 8.26 (t, 1H, imidazolyl), 7.86 (dd, 1.8 Hz, 1H, pyridyl), 7.42 (t, 1H, imidazolyl), 5.27 (sept, 1H, isopropyl), 2.43 (s, 3H, methyl), 1.73 (d, 6H, isopropyl). X-ray crystallography. Intensity data of compounds 1a–c·HI, 2a–d, and 3a–d were collected on a Rigaku XtaLAB Synergy-DW system (CuKα) at 93 K. The structures were solved by direct methods, and non-hydrogen atoms except for the disordered BF4- and CH2Cl2 in 2b were reﬁned anisotropically by the least squares on F2 using the SHELXTL program. The hydrogen atoms of organic ligands were generated geometrically; no attempt was made to locate the hydrogen atoms of disordered dichloromethane molecules in 2b and water molecules in 2d. Synthesis of 2a–d. References Cluster linker approach: preparation of a luminescent porous framework with NbO topology by linking silver ions with gold(I) clusters. Angew. Chem. Int. Ed. 53, 12771–12775 (2014). 7. Ryo, K. et al. Ultrabright luminescence from gold nanoclusters: rigidifying the Au(I)-thiolate shell. J. Am. Chem. Soc. 137, 8244–8250 (2015). Synthesis of 3a–d. CAuI6 cluster (2a (29.6 mg, 10.0 μmol) for 3a; 2b (28.8 mg, 10.0 μmol) for 3b; 2c (25.8 mg, 10.0 μmol) for 3c; 2d (24.9 mg, 10.0 μmol) for 3d) was dissolved in dry CH2Cl2 (3 mL). AgBF4 (6.0 mg, 30.0 μmol) in dry CH3OH (1 mL) was added to the solution under stirring. The mixture was then ﬁltered and the ﬁltrate was layered with dry Et2O. Yellow crystals can be obtained within 1 week. Yield: 30.3 mg (90%, based on 2a) for 3a; 30.2 mg (93%, based on 2b) for 3b; 26.3 mg (89%, based on 2c) for 3c; 18.3 mg (64%, based on 2d) for 3d. 8. Li, Q. et al. A mono-cuboctahedral series of gold nanocluster: photoluminescence origin, large enhancement, wide tunability, and structure −property correlation. J. Am. Chem. Soc. 141, 5314–5325 (2019). y 9. Weerawardene, K. L. D. M. et al. Luminescence and electron dynamics in atomically precise nanoclusters with eight superatomic electrons. J. Am. Chem. Soc. 141, 18715–18726 (2019). 10. Yao, C. et al. Giant emission enhancement of solid-state gold nanoclusters by surface engineering. Angew. Chem. Int. Ed. 59, 8270–8276 (2020). g g For 3a: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 7.91 (d, 6H), 7.81 (d, 6H), 7.74 (s, 6H), 7.64 (dt, 12H), 7.59 (t, 6H), 7.44 (d, 6H), 5.34 (sept, 6H, isopropyl), 1.41 (d, 18H, isopropyl), 1.34 (s, 18H, isopropyl), 1.12 (s, 18H, methyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 183.6, 151.8, 147.4, 143.1, 137.2, 134.5, 131.9, 127.6, 127.1, 123.5, 114.1, 113.4, 54.1, 22.3, 22.1, 16.4. ESI-MS (CH2Cl2): 2389.2 ([(C)(Au- 1a)4AgAg2](BF4)2+). 11. Scherbaum, F., Grohmann, A., Huber, B., Krüger, C. & Schmidbaur, H. “Aurophilicity” as a consequence of relativistic effects: the hexakis(triphenylphosphaneaurio)methane dication [(Ph3PAu)6C]2+. Angew. Chem. Int. Ed. Engl. 27, 1544–1546 (1988). References For 2b: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.43 (s, 6H, pyridyl), 7.86 (br, 6H, pyridyl), 7.75 (m, 12H, benzimidazolylidene), 7.53 (m, 12H, benzimidazolylidene), 7.30 (br, 6H, pyridyl), 6.45 (br, 6H, pyridyl), 5.71 (br, 6H, isopropyl), 1.49 (br, 36H, isopropyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 186.6, 151.0, 149.4, 139.1, 134.1, 132.2, 125.7, 125.7, 123.7, 121.3, 115.0, 113.3, 53.9, 21.9. ESI-MS (CH2Cl2): 1308.3 ([(C)(Au-1b)6]2+). 1. Schmidbaur, H. & Schier, A. A brieﬁng on aurophilicity. Chem. Soc. Rev. 37, 1931–1951 (2008). 2. Schmidbaur, H. & Schier, A. Aurophilic interactions as a subject of current research: an up-date. Chem. Soc. Rev. 41, 370–412 (2012). research: an up-date. Chem. Soc. Rev. 41, 370–412 (2012). 3. Kang, X. & Zhu, M. Tailoring the photoluminescence of atomically preci nanoclusters. Chem. Soc. Rev. 48, 2422–2457 (2019). 3. Kang, X. & Zhu, M. Tailoring the photoluminescence o nanoclusters. Chem. Soc. Rev. 48, 2422–2457 (2019). For 2c: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.41 (d, 6H, pyridyl), 8.19 (s, 6H, pyridyl), 7.87 (s, 6H, imidazolylidene), 7.21 (s, 6H, imidazolylidene), 6.58 (d, 6H, pyridyl), 5.22 (sept, 6H, isopropyl), 2.08 (s, 18H, methyl), 1.15 (d, 36H, isopropyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 179.8, 149.1, 148.7, 138.6, 133.9, 120.1, 117.2, 116.9, 53.6, 22.8, 18.1. ESI-MS (CH2Cl2): 1200.2 ([(C)(Au-1c)6]2+). 4. Santiago-Gonzalez, B. et al. Permanent excimer superstructures by supramolecular networking of metal quantum clusters. Science 353, 571–575 (2016). 5. Huang, R.-W. et al. Hypersensitive dual-function luminescence switching of a silver chalcogenolate cluster-based metal-organic framework. Nat. Chem. 9, 689–697 (2017). For 2d: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.58 (d, 6H, pyridyl), 8.38 (d, 6H, pyridyl), 7.91 (s, 6H, imidazolylidene), 7.21 (s, 6H, imidazolylidene), 7.06 (dd, 6H, pyridyl), 6.85 (t, 6H, pyridyl), 5.20 (sept, 6H, isopropyl), 1.15 (d, 36H, isopropyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 179.9, 150.9, 149.2, 138.2, 123.8, 120.3, 117.5, 117.5, 53.8, 23.0. ESI-MS (CH2Cl2): 1158.2 ([(C)(Au-1d)6]2+). For 2d: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.58 (d, 6H, pyridyl), 8.38 (d, 6H, pyridyl), 7.91 (s, 6H, imidazolylidene), 7.21 (s, 6H, imidazolylidene), 7.06 (dd, 6H, pyridyl), 6.85 (t, 6H, pyridyl), 5.20 (sept, 6H, isopropyl), 1.15 (d, 36H, isopropyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 179.9, 150.9, 149.2, 138.2, 123.8, 120.3, 117.5, 117.5, 53.8, 23.0. ESI-MS (CH2Cl2): 1158.2 ([(C)(Au-1d)6]2+). 6. Lei, Z., Pei, X.-L., Jiang, Z.-G. & Wang, Q.-M. Data availability The data that support the ﬁndings of this study are available within the manuscript and its supplementary information and from the corresponding author upon request. The X-ray crystallographic coordinates for structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 2103969 (1a ∙HI), CCDC 2103970 (1b ∙HI), CCDC 2103971 (1c ∙HI), CCDC 2103972 (2a), CCDC 2103973 (2b), CCDC 2103974 (2c), CCDC 2103975 (2d), CCDC 2103976 (3a), CCDC 2103977 (3b), CCDC 2103978 (3c), and CCDC 2103979 (3d). These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Received: 27 November 2021; Accepted: 8 July 2022; For 2a: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.40–8.15 (br, 6H, pyridyl), 7.91–7.38 (br, 30H, pyridyl + benzimidazolylidene), 7.22–6.72 (br, 6H, pyridyl), 5.75 (br, 6H, isopropyl), 1.45 (br, 54H, isopropyl + methyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 187.9, 150.3, 148.5, 140.0, 135.6, 134.4, 132.4, 125.9, 125.7, 120.9, 113.8, 113.2, 54.3, 22.4, 18.3. ESI-MS (CH2Cl2): 1350.2 ([(C)(Au-1a)6]2+). For 2a: 1H NMR (500 MHz, CD2Cl2, δ, ppm): 8.40–8.15 (br, 6H, pyridyl), 7.91–7.38 (br, 30H, pyridyl + benzimidazolylidene), 7.22–6.72 (br, 6H, pyridyl), 5.75 (br, 6H, isopropyl), 1.45 (br, 54H, isopropyl + methyl). 13C NMR (126 MHz, CD2Cl2, δ, ppm): 187.9, 150.3, 148.5, 140.0, 135.6, 134.4, 132.4, 125.9, 125.7, 120.9, 113.8, 113.2, 54.3, 22.4, 18.3. ESI-MS (CH2Cl2): 1350.2 ([(C)(Au-1a)6]2+). ARTICLE Imidazolium/benzimidazolium halide (1a·HI (114 mg, 0.30 mmol) for 2a; 1b·HI (110 mg, 0.30 mmol) for 2b; 1c·HI (98.7 mg, 0.30 mmol) for 2c; 1d·HBr (80.4 mg, 0.30 mmol) or 1d·HI (94.5 mg, 0.30 mmol) for 2d) was dissolved in dry CH2Cl2 (10 mL). Tht-AuCl (96.0 mg, 0.30 mmol) was added and then the solution was stirred for 5 min, which was followed by adding K2CO3 (828 mg, 6.00 mmol). After the mixture was stirred for 12 h in the dark, it was ﬁltered through a thin layer of Celite. The solvent was then removed under reduced pressure using a rotary evaporator. After adding NaBF4 (165 mg, 1.50 mmol) and CH3OH (10 mL), the suspension was stirred for 5 min. CH2Cl2 (5 mL), a solution of KOH (28.0 mg, 0.50 mmol) in CH3OH (3 mL), a solution of AgBF4 (58.5 mg, 0.30 mmol) in CH3OH (1 mL), and H2O (50 μL) were sequen- tially dropwise added into the mixture under stirring, which leads to a brown suspension. After another 5 min stirring, the suspension was again ﬁltered through Celite and evaporated to dryness. The solid was then transferred to a Schlenk ﬂask with a nitrogen atmosphere, and dry CH2Cl2 (5 mL), Et3N (30.0 μL, 0.20 mmol), and a 2.0 M solution of Me3SiCHN2 in hexanes (48.0 μL, 0.10 mmol) were added. The resulting mixture was stirred for another 1 h. After ﬁltration into a tube, a layer of dry Et2O was added to the CH2Cl2 solution, which gave the products colorless block-like crystals within 2 weeks. Yields: 59.5 mg (40%, based on tht- AuCl) for 2a; 74.3 mg (52%, based on tht-AuCl) for 2b; 31.3 mg (24%, based on tht-AuCl) for 2c; 11.6 mg (9%, based on tht-AuCl (1d·HBr)) for 2d; 10.0 mg (8%, based on tht-AuCl (1d·HI)) for 2d. g g y p y g disordered dichloromethane molecules in 2b and water molecules in 2d. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Discussion h d In this study, we have established a rational design and synthesis method for a series of phosphorescent AuI-AgI clusters, and revealed that the organic ligands bound to the surface of the metal clusters are important for their photochemical property and subcellar distribution. The NHC ligands were found to remark- ably shift the emission wavelength of the CAuI6AgI2 clusters, and signiﬁcantly affecting the phosphorescence quantum yield and lifetime, compared to the same cluster protected by phosphine ligands. More importantly, the CAuI6AgI2 clusters were taken up into the living cells and showed structure-speciﬁc translocation pathways and distribution. This suggests that molecular design may be able to control molecular behaviors in the cell. Therefore, this study is expected to provide a strategy to create metal clusters with tunable functionalities, and also to lead to clear guidelines for designing active and applicable metallodrugs. y py g py For 1a·HI: 1H NMR (500 MHz, CDCl3, δ, ppm): 11.41 (s, 1H, benzimidazolyl), 8.77 (d, 1H, pyridyl), 8.61–8.59 (m, 1H, benzimidazolyl), 8.46 (d, 1H, pyridyl), 7.95 (dd, 1H, pyridyl), 7.84–7.82 (m, 1H, benzimidazolyl), 7.71 (td, 2H, benzimidazolyl), 5.39 (sept, 1H, isopropyl), 2.48 (s, 3H, methyl), 1.99 (d, 6H, isopropyl). For 1b·HI: 1H NMR (500 MHz, CDCl3, δ, ppm): 11.46 (s, 1H, benzimidazolyl), 8.94 (d, 1H, pyridyl), 8.67 (m, 2H, benzimidazolyl + pyridyl), 8.16 (td, 1H, pyridyl), 7.86–7.84 (m, 1H, benzimidazolyl), 7.73 (t, 2H, benzimidazolyl), 7.54 (dd, 1H, pyridyl), 5.42 (sept, 1H, isopropyl), 2.01 (d, 6H, isopropyl). Synthesis of 1c·HI. Under a nitrogen atmosphere, a Schlenk tube was charged with imidazole (2.04 g, 30.0 mmol), K2CO3 (2.76 g, 20.0 mmol), and 2-bromo-5- methylpyridine (1.72 g, 10.0 mmol). The reaction mixture was heated at 190 °C for 12 h and then allowed to cool to room temperature. After it was diluted with water (50 mL) and extracted with CHCl3 (50 mL × 3), the combined organic phases were washed with sat. Na2CO3 aqueous solution (50 mL × 3), and then dried over anhydrous MgSO4 before ﬁltration. Concentration under reduced pressure gave a colorless oil, which was then transferred into a Schlenk ﬂask and dissolved in 7 TURE COMMUNICATIONS \| (2022) 13:4288 \| https://doi.org/10.1038/s41467-022-31891-3 \| www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-31891-3 15. Yang, Y., Pei, X.-L. & Wang, Q.-M. Postclustering dynamic covalent modiﬁcation for chirality control and chiral sensing. J. Am. Chem. Soc. 135, 16184–16191 (2013). 42. 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Carbene ligands in surface chemistry: from stabilization of discrete elemental allotropes to modiﬁcation of nanoscale and bulk substrates. Chem. Rev. 115, 11503–11532 (2015). Acknowledgements This research was supported by JSPS KAKENHI Grants No. JP16H06509 (Coordination Asymmetry) and No. JP21H00453 to M.S., JSPS KAKENHI Grant No. JP19K05538 to M.E., and CREST (JPMJCR1752) from Japan Science and Technology (JST) to T.O. Part of the computation was performed using Research Center for Computational Science, Okazaki, Japan (Project: 20-IMS-C100). 22. Zhong, R., Lindhorst, A. C., Groche, F. J. & Kühn, F. E. Immobilization of N‐ heterocyclic carbene compounds: a synthetic perspective. Chem. Rev. 117, 1970–2058 (2017). 23. Crudden, C. M. et al. Ultra stable self-assembled monolayers of N-heterocyclic carbenes on gold. Nat. Chem. 6, 409–414 (2014). 24. Wang, G. et al. Ballbot-type motion of N-heterocyclic carbenes on gold surfaces. Nat. Chem. 9, 152–156 (2017). Competing interests 28. Narouz, M. R. et al. N-heterocyclic carbene-functionalized magic-number gold nanoclusters. Nat. Chem. 11, 419–425 (2019). The authors declare no competing interests. 29. Narouz, M. R. et al. Robust, highly luminescent Au13 superatoms protected by N‐heterocyclic carbenes. J. Am. Chem. Soc. 141, 14997–15002 (2019). , , g y 13 p p y N‐heterocyclic carbenes. J. Am. Chem. Soc. 141, 14997–15002 (2019). 30. Shen, H. et al. Highly robust but surface-active: an N-heterocyclic carbene- stabilized Au25 nanocluster. Angew. Chem., Int. Ed. 58, 17731–17735 (2019). Author contributions 25. MacLeod, M. J. et al. Robust gold nanorods stabilized by bidentate N- heterocyclic-carbene–thiolate ligands. Nat. Chem. 11, 57–63 (2019). Z.L. designed this work, performed synthetic experiments, analyzed data, and prepared the manuscript. M.Endo performed bioimaging experiments, analyzed data, and pre- pared the manuscript. H.U. designed this work and directed the research. T.S., P.Z., K.N., and M.Ehara performed theoretical calculations. X.-L.P. assisted in the synthetic experiments. T.E. performed PLIM experiments. T.K., and T.O. directed experiments. M.S. proposed and supervised this study and prepared the manuscript. 26. Polgar, A. M., Weigend, F., Zhang, A., Stillman, M. J. & Corrigan, J. F. A N‐ heterocyclic carbene-stabilized coinage metal-chalcogenide framework with tunable optical properties. J. Am. Chem. Soc. 139, 14045–14048 (2017). p p p 27. Jin, L. et al. Trinuclear gold clusters supported by cyclic (alkyl)(amino)carbene ligands: mimics for gold heterogeneous catalysts. Angew. Chem., Int. Ed. 53, 9059–9063 (2014). 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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/. 36. Bakar, M. A., Sugiuchi, M., Iwasaki, M., Shichibu, Y. & Konishi, K. Hydrogen bonds to Au atoms in coordinated gold clusters. Nat. Commun. 8, 576 (2017). Additional information bonds to Au atoms in coordinated gold clusters. Nat. Commun. 8, 576 (2017). 37. Greisch, J.-F. et al. Gas-phase photoluminescence and photodissociation of silver-capped hexagold clusters. J. Phys. Chem. A 122, 5799–5810 (2018). 38. Aikens, C. M. Electronic and geometric structure, optical properties, and excited state behavior in atomically precise thiolate-stabilized noble metal g ( ) 37. Greisch, J.-F. et al. Gas-phase photoluminescence and photodissociation of silver-capped hexagold clusters. 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https://openalex.org/W3005646976	https://www.scielo.br/j/brjp/a/pZQKDfvBhwkZxkF5mGXT43c/?lang=en&format=pdf	English	null	Effects of duloxetine, fluoxetine and pregabalin on fentanyl-induced hyperalgesia in rattus novergicus	BrJP	2,020	cc-by	5,375	RESUMO JUSTIFICATIVA E OBJETIVOS: Opioides são fármacos uti- lizados para o alívio da dor, porém, podem causar aumento da sensibilidade dolorosa, denominada hiperalgesia induzida por opioides, que afeta negativamente o tratamento da dor. O ob- jetivo deste estudo foi avaliar se o fentanil, opioide amplamente utilizado na prática clínica, produz hiperalgesia que pode ser ate- nuada pela duloxetina, fluoxetina e pregabalina. dely used in the clinical practice, produces hyperalgesia that can be attenuated by duloxetine, fluoxetine and pregabalin. METHODS: Thirty male Wistar rats were divided into six groups. The animals in group 1 received 1mL of 0.9% saline solution in- traperitoneally (IP) and gavage; group 2 received fentanyl at a dose of 100µg.kg-1 IP and 0.9% saline solution per gavage; groups 3, 4 and 5 received fentanyl at the dose of 100µg.kg-1 IP, and gavage with duloxetine, 40mg.kg-1, fluoxetine, 40mg.kg-1 and pregabalin, 40mg.kg-1, respectively. Under general anesthesia with isoflurane, all animals were submitted to plantar surgical incision. The appli- cation of Von Frey filaments assessed hyperalgesia at the second hour, one, three, five and seven days after treatment. RESULTS: Two hours after the procedure, no differences were observed between G1 and G2, although G3, G4, and G5 sho- wed less hyperalgesia. On day one and day three, a greater hype- ralgesic effect was observed in G2 when compared to G1, G3, G4 and G5. On day five, there was a hyperalgesic effect on G2, and on day seven, there were no differences among the groups. CONCLUSION: The results suggest that fentanyl induces hype- ralgesia and the efficacy of duloxetine, fluoxetine, and pregabalin in reducing it. METHODS: Thirty male Wistar rats were divided into six groups. The animals in group 1 received 1mL of 0.9% saline solution in- traperitoneally (IP) and gavage; group 2 received fentanyl at a dose of 100µg.kg-1 IP and 0.9% saline solution per gavage; groups 3, 4 and 5 received fentanyl at the dose of 100µg.kg-1 IP, and gavage with duloxetine, 40mg.kg-1, fluoxetine, 40mg.kg-1 and pregabalin, 40mg.kg-1, respectively. Under general anesthesia with isoflurane, all animals were submitted to plantar surgical incision. The appli- cation of Von Frey filaments assessed hyperalgesia at the second hour, one, three, five and seven days after treatment. l MÉTODOS: Trinta ratos Wistar machos, foram divididos em 6 grupos. RESUMO No 1º e 3º dias foi obser- vado maior efeito hiperalgésico em G2 quando comparado com G1, G3, G4 e G5. No 5º dia foi observado efeito hiperalgésico no G2, e no 7º dia não houve diferenças entre os grupos. CONCLUSÃO: Os resultados sugerem que o fentanil induz hi- peralgesia e eficácia da duloxetina, fluoxetina e pregabalina na sua redução. RESULTADOS: Na 2ª hora pós-procedimento não foram ob- servadas diferenças entre G1 e G2, entretanto, G3, G4 e G5 se mostraram com menor hiperalgesia. No 1º e 3º dias foi obser- vado maior efeito hiperalgésico em G2 quando comparado com G1, G3, G4 e G5. No 5º dia foi observado efeito hiperalgésico no G2, e no 7º dia não houve diferenças entre os grupos. RESULTADOS: Na 2ª hora pós-procedimento não foram ob- servadas diferenças entre G1 e G2, entretanto, G3, G4 e G5 se mostraram com menor hiperalgesia. No 1º e 3º dias foi obser- vado maior efeito hiperalgésico em G2 quando comparado com G1, G3, G4 e G5. No 5º dia foi observado efeito hiperalgésico no G2, e no 7º dia não houve diferenças entre os grupos. CONCLUSÃO: Os resultados sugerem que o fentanil induz hi- peralgesia e eficácia da duloxetina, fluoxetina e pregabalina na sua redução. Oscar César Pires – https://orcid.org/0000-0002-7033-0764; Maria Luiza Dalcim – https://orcid.org/0000-0002-5942-9691; Ana Luiza Montes Pigozzi – https://orcid.org/0000-0001-5623-6096; Fabiana Mara Scarpelli de Lima Alvarenga Caldeira – https://orcid.org/0000-0001-9084- 9822; Marta Helena Rovani Pires – https://orcid.org/0000-0003-0592-5304; Lafayette de Almeida Neto – https://orcid.org/0000-0002-5449-3344; Irimar de Paula Posso – http://orcid.org/0000-0003-0337-2531. 1. Universidade de Taubaté, Taubaté, SP, Brasil. Submitted on March 06, 2019. Accepted for publication on December 13, 2019. Conflict of interests: none – Sponsoring sources: none. Correspondence to: Avenida Tiradentes, 500 – Centro 12030-180 Taubaté, SP, Brasil. E-mail: oscarpires50@gmail.com.br © Sociedade Brasileira para o Estudo da Dor Descritores: Duloxetina, Fentanil, Fluoxetina, Hiperalgesia, Pregabalina, Ratos. ORIGINAL ARTICLE ORIGINAL ARTICLE BrJP. São Paulo, 2020 jan-mar;3(1):14-8 ABSTRACT Keywords: Duloxetine, Fentanyl, Fluoxetine, Hyperalgesia, Pre- gabalin, Rats. BACKGROUND AND OBJECTIVES: Opioids are drugs used to relieve pain, but may cause increased pain sensitivity, known as opioid-induced hyperalgesia, which adversely affects pain ma- nagement. This study aimed to check if fentanyl, an opioid wi- dely used in the clinical practice, produces hyperalgesia that can be attenuated by duloxetine, fluoxetine and pregabalin. Efeitos da duloxetina, fluoxetina e pregabalina sobre a hiperalgesia induzida por fentanil em rattus novergicus Oscar César Pires1, Maria Luiza Dalcim1, Ana Luiza Montes Pigozzi1, Fabiana Mara Scarpelli de Lima Alvarenga Caldeira1, Marta Helena Rovani Pires1, Lafayette de Almeida Neto1, Irimar de Paula Posso1 RESUMO No grupo 1, os animais receberam 1mL de solução fi- siológica (SF) a 0,9% por via intraperitoneal (IP) e por gavagem; no grupo 2, fentanil na dose de 100µg.kg-1 IP e SF a 0,9% por gavagem; nos grupos 3, 4 e 5 os animais receberam fentanil na dose de 100µg.kg-1 IP e, por gavagem, receberam respectivamen- te duloxetina, 40mg.kg-1, fluoxetina, 40mg.kg-1 e pregabalina, 40mg.kg-1. A avaliação da hiperalgesia e sua atenuação foi feita pela aplicação de filamentos de Von Frey, na 2ª hora e nos dias 1, 3, 5 e 7, após o tratamento. l MÉTODOS: Trinta ratos Wistar machos, foram divididos em 6 grupos. No grupo 1, os animais receberam 1mL de solução fi- siológica (SF) a 0,9% por via intraperitoneal (IP) e por gavagem; no grupo 2, fentanil na dose de 100µg.kg-1 IP e SF a 0,9% por gavagem; nos grupos 3, 4 e 5 os animais receberam fentanil na dose de 100µg.kg-1 IP e, por gavagem, receberam respectivamen- te duloxetina, 40mg.kg-1, fluoxetina, 40mg.kg-1 e pregabalina, 40mg.kg-1. A avaliação da hiperalgesia e sua atenuação foi feita pela aplicação de filamentos de Von Frey, na 2ª hora e nos dias 1, 3, 5 e 7, após o tratamento. RESULTS: Two hours after the procedure, no differences were observed between G1 and G2, although G3, G4, and G5 sho- wed less hyperalgesia. On day one and day three, a greater hype- ralgesic effect was observed in G2 when compared to G1, G3, G4 and G5. On day five, there was a hyperalgesic effect on G2, and on day seven, there were no differences among the groups. CONCLUSION: The results suggest that fentanyl induces hype- ralgesia and the efficacy of duloxetine, fluoxetine, and pregabalin in reducing it. p RESULTADOS: Na 2ª hora pós-procedimento não foram ob- servadas diferenças entre G1 e G2, entretanto, G3, G4 e G5 se mostraram com menor hiperalgesia. No 1º e 3º dias foi obser- vado maior efeito hiperalgésico em G2 quando comparado com G1, G3, G4 e G5. No 5º dia foi observado efeito hiperalgésico no G2, e no 7º dia não houve diferenças entre os grupos. CONCLUSÃO: Os resultados sugerem que o fentanil induz hi- peralgesia e eficácia da duloxetina, fluoxetina e pregabalina na sua redução. p RESULTADOS: Na 2ª hora pós-procedimento não foram ob- servadas diferenças entre G1 e G2, entretanto, G3, G4 e G5 se mostraram com menor hiperalgesia. INTRODUCTION 1. Universidade de Taubaté, Taubaté, SP, Brasil. Pain is one of the most important and complex human expe- riences, associated with actual or potential tissue damage, and its treatment with opioids has increased substantially in recent years, making it’s prescription common in the United States1,2. However, the increase in prescriptions has been causing many problems, among which are the lack of knowledge regarding lon- g-term efficacy, abusive use and adverse events associated with prolonged use, including opioid-induced hyperalgesia (OIH), a Correspondence to: 14 Effects of duloxetine, fluoxetine and pregabalin on fentanyl-induced hyperalgesia in rattus novergicus BrJP. São Paulo, 2020 jan-mar;3(1):14-8 phenomenon for which paradoxically, opioids can induce or sen- sitize patients to acute pain3,4. In this sense, patients who receive high doses of opioids may experience severe acute pain after sur- gery, with an increased dose of analgesics, and anxiety for both the patient and the physician4. rotonergic projections from the brain stem descend through the spinal cord, where it is believed to be involved in the regulation of somatosensory perception. Alterations in the serotonergic and noradrenergic pathways modify both the cerebral perception of the sensory stimuli of the ascending pathways, as well as alter the mechanism of pain inhibition by the descending pathways. Depression and chronic pain share these neuronal serotonergic and noradrenergic pathways, which is why duloxetine has been shown to be effective in these two conditions15.f The mechanisms proposed to be responsible for OIH are mul- tiple, including changes in N-methyl-D-aspartate (NMDA) re- ceptors and second messengers, spinal cyclooxygenase (COX) activation, the release of excitatory amino acids, reduction of inhibitory neurotransmitters, descending facilitation and the an- ti-analgesic system2,3,5,6. h f Duloxetine has been shown to be effective in chronic pain condi- tions such as fibromyalgia, peripheral diabetic neuropathy, pain- ful symptoms of knee osteoarthritis, and chronic low back pain. There is also evidence of relief from painful symptoms associated with depression and generalized anxiety disorder16. The increased release of glutamate in the dorsal horn of the spinal cord and the consequent sustained increase in stimulus and res- ponse of NMDA receptors by removal of magnesium mediated by protein kinase-C seem to be important mechanisms involved in OIH7. These NMDA receptors can be activated by opioids, which act as excitatory neurotransmitters facilitating calcium in- take into the cell and central sensitization (CS). Calcium intake causes increased protein kinase-C activity, phosphorylation, and inactivation of opioid receptors, in addition to an increase in nitric oxide synthase8. The effect of fluoxetine on the serotonergic system (SRI) is well known, making evident the use of fluoxetine as a treatment op- tion for different chronic pain conditions such as fibromyalgia, chronic tension-type headache, migraine without aura, painful diabetic neuropathy, musculoskeletal pain, chronic pelvic pain, and coronary syndrome17. Due to the effects of SRI by elevating serotonin in the central nervous system (CNS), it is postulated that duloxetine and fluoxetine may be useful in attenuating OIH. Correspondence to: Studies in rodents have demonstrated that fentanyl cause OIH and suggested that the protein kinase Iiα (CaMKIIα) dependent of Ca2+/calmodulin in the lateral capsular division of the central nucleus of the amygdala (CeLC) and the spinal cord can play a key role in the modulation of the OIH12,13. Correspondence to: The involvement of glutamate neurotransmission in synaptic plasticity suggests that pregabalin may also be useful in atte- nuating OIH18, being a GABA analog drug, which selectively binds with high affinity to the calcium channels, widely dis- tributed in the CNS and peripheral, producing a modulating effect with a reduction of the excessive release of several excita- tory neurotransmitters.f OIH has been associated with an increase in cholecystokinin, a calcitonin gene-related peptide (CGRP), substance-P, and oci- ception in the rostral ventromedial medulla due to increased expression of excitatory opioid receptors, to the detriment of inhibitory opioid receptors5,9,10. The descending facilitatory pathways, mediated by opioids, loca- ted in the rostral ventromedial medulla, also seem to be involved in OIH due to neuroplastic changes, since exposure to morphine causes neuroplastic changes in the rostral ventromedial medulla, with increased release of dynorphin and primary afferents fiber neurotransmitters3,6,11. In this way, the administration of opioids would cause an increase in dynorphin, which may favor OIH5,6. There is evidence that spinal dynorphin is pro-nociceptive, cau- sing the release of excitatory neurotransmitters from primary afferent neurons, suggesting positive feedback, amplifying the sensory afferents6. In addition, prostaglandins, cytokines, and chemokines may also be relevant in the development of OIH, since opioids activate the release of cytokines, with increased C-fos protein in sensory neurons in the spinal cord. Other sys- tems that may be involved in OIH with reduced glycinergic inhi- bitory control are nitric oxide synthase and heme oxygenase2,3. Several clinical trials have documented its effect on pain relief and quality of life, including mood and sleep disorders, and are, therefore, indicated for the treatment of fibromyalgia, neuropa- thic pain, and generalized anxiety disorder18,19. fi Pregabalin has been shown to be effective in treating fibromyal- gia, with improvement in various sleep parameters. Pain reduc- tion was evidenced, regardless of anxiety or depression symp- toms, suggesting that the pain reduction caused by pregabalin results mainly from the direct effect of the treatment, and not the indirect effect from the improvement of anxiety and depression symptoms20. Behavioral tests such as the application of von Frey filaments and thermal hyperalgesia have been used to evaluate hyperalgesia in rats21. In this study, the test with von Frey filaments was used to assess whether fentanyl, an opioid widely used in clinical practi- ce, produces hyperalgesia that may suffer interference from the drugs duloxetine, fluoxetine, and pregabalin. RESULTS When comparing the average weight of the animals before the beginning of the experiment, there was no statistically significant difference between groups (p<0.05). In the 2nd hour after the surgical procedure, the pain intensity, assessed by von Frey fila- ments, is shown in figure 1, showing that there is no significant difference when comparing G1 with G2 (p=0.3759), but with statistical significance when they were compared to groups G3, G4, and G5 (p<0.05). On the first day after the surgical procedure, the pain intensity, assessed by von Frey filaments, is shown in figure 2, showing a significant difference between G1 and G2 (p <0.05) and between these and groups G3, G4 and G5 (p<0.01). The surgical procedure consisted of a 1.0cm long, longitudinal surgical incision in the right posterior paw, according to the pos- toperative pain model21. This incision was made with a scalpel with blade number 11, incising the skin and the plantar fascia region of the paw, starting 0.5cm from the edge of the calcaneus and extending towards the toes. Then, the plantar muscle was elevated and incised longitudinally, with its insertion intact. Af- ter hemostasis with slight pressure on the surgical area, all planes were approached and sutured with two separate stitches with 4-0 mono nylon needle thread.h On the third day after the surgical procedure, the pain intensity, assessed by von Frey filaments, is shown in figure 3, showing a significant difference between the G1 and G2 (p<0.05) and, between these and groups G3, G4 and G5 (p<0.01). On the 5th day after the surgical procedure, the pain intensity, assessed by von Frey filaments, is shown in figure 4, showing a significant difference between G2 and groups G1, G3, G4, and G5 (p<0.05). The animals were randomly divided into six groups to recei- ve similar volumes of drugs or 0.9% saline solution (SS). In group 1, the animals received 1mL of 0.9% SS by intrape- ritoneal (IP) and gavage; group 2 fentanyl (100μg.kg-1) (IP) in a single dose and 0.9% SS by gavage; Group 3 fentanyl (100μg.kg-1) (IP) in a single dose and duloxetine (40mg.kg-1) by gavage; group 4 fentanyl (100μg.kg-1) (IP) in a single dose and fluoxetine (40mg.kg-1) by gavage and group 5 fentanyl (100μg.kg-1) (IP) in a single dose and pregabalin (40mg.kg-1) by gavage. METHODS The duloxetine, an antidepressant from the class of serotonin- -norepinephrine reuptake inhibitors (SNRI), is indicated for the treatment of depressive disorder, generalized anxiety disorder, and chronic pain conditions as diabetic neuropathic pain, chro- nic fibromyalgia and chronic musculoskeletal pain14. The duloxetine, an antidepressant from the class of serotonin- -norepinephrine reuptake inhibitors (SNRI), is indicated for the treatment of depressive disorder, generalized anxiety disorder, and chronic pain conditions as diabetic neuropathic pain, chro- nic fibromyalgia and chronic musculoskeletal pain14. For the characteristics of the animal sample and convenience, it was used 30 male Wistar rats, weighing between 220 and 300g, allocated in number of 5 animals per compartment, where they remained for 15 days before the beginning of the experiment for adequate adaptation, treated with balanced commercial feed and water “ad libitum,” 12-hour light-dark cycle and room tempera- ture ranging between 19 and 25°C. i The role of serotonin and norepinephrine in the regulation of mood occurs through the ascending neuronal pathways, starting from the middle portion of the brain, extending to the limbic system and the prefrontal cortex. Besides, noradrenergic and se- i The role of serotonin and norepinephrine in the regulation of mood occurs through the ascending neuronal pathways, starting from the middle portion of the brain, extending to the limbic system and the prefrontal cortex. Besides, noradrenergic and se- 15 Pires OC, Dalcim ML, Pigozzi AL, Caldeira FM, Pires MH, Almeida Neto L and Posso IP ão Paulo, 2020 jan-mar;3(1):14-8 Pires OC, Dalcim ML, Pigozzi AL, Caldeira FM, Pires MH, Almeida Neto L and Posso IP BrJP. São Paulo, 2020 jan-mar;3(1):14-8 times: 2nd hour, 1, 3, 5 and 7 days after the surgical procedure and treatment administration. The IASP Ethical Standards, which regulates experiments carried out in animals (Committee for Research and Ethical Issues of the IASP, 1983), were followed to conduct the expe- rimental procedures. All experiments were carried out at the Laboratory of Pharmacology and Physiology at the University of Taubaté, SP. The project started after the approval by the Ethics Committee on the Use of Animals CEUA/UNITAU, under No. 03/2017. Statistical analysis ® The JMP® software from the SAS (Statistical Analysis System) Ins- titute was used, applying the Student’s t-test, comparing pair by pair, and adopting a significance level lower than 5% (p<0.05). To obtain mild anesthesia, the animals were placed in a 15x25x- 15cm transparent glass chamber with a transparent cover to allow the visualization of the animal, with a hole in the front and back to enable oxygen (O2), anesthetic gases and carbon dioxide, entering and exiting, respectively. The halogenated agent used in anesthetic induction was isoflurane (Isoforine®, Cristália, Itapira, Brazil), at a concentration of 4.0% in fraction of inspired oxygen (FiO2) of 1.0, administered by a calibrated vaporizer (Hospital HB) and maintained for three minutes, time necessary for the animal to present loss of postural re- flexes and inability to move in the chamber. Then, the animal was removed from the chamber and placed with the snout in a mask through which it received 4% isoflurane in O2 as in the anesthesia induction chamber.h RESULTS Student’s t-test showed no significant differences between groups (p>0.05) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * * Figure 2. Student’s t-test showed a significant difference when com- paring G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Figure 5. Student’s t-test showed no significant differences between groups (p>0.05) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * * Figure 2. Student’s t-test showed a significant difference when com- paring G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * * Figure 2. Student’s t-test showed a significant difference when com- paring G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) Figure 2. Student’s t-test showed a significant difference when com- paring G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) Figure 5. Student’s t-test showed no significant differences between groups (p>0.05) DISCUSSION G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * * Figure 3. Student’s t-test showed a significant difference when com- paring the G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) Opioids are important drugs for the treatment of pain. However, at the same time that they are initially analgesic and antihyperal- gesic, they can later cause hyperalgesia, making the patient more sensitive to pain2-5. OIH has been attributed to acute desensitization of receptors by derailing G protein from opioid receptors, activation of NMDA receptors, among other mechanisms2. OIH has been attributed to acute desensitization of receptors by derailing G protein from opioid receptors, activation of NMDA receptors, among other mechanisms2. A study has shown that the concomitant use of low doses of opioid antagonists and NMDA receptor antagonists can prevent or reduce the development of OIH, and that ketamine in low doses can modulate OIH7,22,23. A study has shown that the concomitant use of low doses of opioid antagonists and NMDA receptor antagonists can prevent or reduce the development of OIH, and that ketamine in low doses can modulate OIH7,22,23. A review proves that the mechanisms involved in the develop- ment of OIH include the glutamatergic system and NMDA re- ceptors, spinal cyclooxygenase activation, excitatory amino acids, dynorphins, cytokines, and chemokines, prostaglandins and do- wnward facilitation. In this sense, it is speculated that the modu- lation of hyperalgesia can be done with NMDA receptor anta- gonists, alpha-2 adrenergic agonists, selective serotonin reuptake inhibitors, cyclooxygenase inhibitors, and GABA analogs24. Figure 3. Student’s t-test showed a significant difference when com- paring the G1* with G2 (p<0.05) and with G3, G4 and G5* (p<0.01) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * Figure 4. Student’s t-test showed a significant difference when com- paring G2* with groups G1, G3, G4, and G5 (p<0.05). In accordance with the present results, a study using fentanyl in Sprague-Dawuley rats caused OIH and demonstrated a mitiga- ting effect by the drugs duloxetine and pregabalin12. f In the present study, in the 2nd hour after the surgical procedu- re, there were no differences between the control groups, which received SS by IP associated with SS by gavage compared to the group that received fentanyl by IP associated with SS by gavage. RESULTS On the 7th day after the surgical procedure, the pain intensity, assessed by von Frey filaments, is shown in figure 5, showing a significant difference between groups. Figure 1. Student’s t-test did not show a significant difference when comparing G1 with G2 (p=0.3759), but with statistical significance when compared with G3, G4 and G5 * (p<0, 05). G1 G2 G3 G4 G5 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * Figure 1. Student’s t-test did not show a significant difference when comparing G1 with G2 (p=0.3759), but with statistical significance G1 G2 G3 G4 G5 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 * * * y g g The evaluation of hyperalgesia was performed by applying the von Frey21 filament test. The animals were kept in a wooden chamber, with a 0.5cm checkered galvanized fabric floor. A mirror was attached under the floor to allow the researcher to observe the application of the filament and the reflex of limb removal. Before applying the filament, the animals were kept in the box for about 15 minutes for adaptation. Each of the fila- ments, in an ascending pressure order, was applied three times in a row with an interval of 3 to 5 seconds, moving on to the next filament, being considered a positive response when the animal removed the support of the injured limb from the floor by the application of the filament. It was considered zero pres- sure value when the animals presented the limb fully retracted; that is, there was no need for any stimulus for the animal to collect the limb support. The collected data were recorded on a specific data collection form for each animal at the following Figure 1. Student’s t-test did not show a significant difference when comparing G1 with G2 (p=0.3759), but with statistical significance when compared with G3, G4 and G5 * (p<0, 05). 16 BrJP. São Paulo, 2020 jan-mar;3(1):14-8 Effects of duloxetine, fluoxetine and pregabalin on fentanyl-induced hyperalgesia in rattus novergicus G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Figure 5. Student’s t-test showed no significant differences between groups (p>0.05) G1 G2 G3 G4 G5 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Figure 5. CONCLUSION 22. DuPen A, Shen D, Ersek M. Mechanisms of opioid-induced tolerance and hyperalge- sia. Pain Manag Nurs. 2007;8(3):113-21. 23. Joly V, Richebe P, Guignard B, Fletcher D, Maurette P, Sessler DI, et al. Remifenta- nil-induced postoperative hyperalgesia and its prevention with small-dose ketamine. Anesthesiology. 2005;103(1):147-55. The present study showed evidence that fentanyl produces OIH and is likely to have a mitigating effect mediated by serotonin and norepinephrine and by calcium channel blockage by the drugs duloxetine, fluoxetine, and pregabalin. gy 24. Leal P da C, Clivatti J, Garcia JB, Sakata RK. Opioid-induced hyperalgesia (HIO). Rev Bras Anestesiol. 2010;60(6):639-47. English, Portuguese, Spanish. 25. Mixcoatl-Zecuatl T, Jolivalt CG. A spinal mechanism of action for duloxetine in a model of painful diabetic neuropathy. Br J Pharmacol. 2011;164(1):159-69. model of painful diabetic neuropathy. Br J Pharmacol. 2011;164 DISCUSSION f On the 5th day after the surgical procedure, the group that received fentanyl maintained a higher pain response when compared to the other groups. However, although G1, which received SS, showed less hyperalgesic effect compared to the fentanyl group, it did not show any difference in comparison to the groups that received dulo- xetine, fluoxetine or pregabalin, still showing the presence of OIH. On the 7th day, there were no differences between the groups; that is, the possible residual effect of hyperalgesia induced by a single dose of fentanyl was not evident. 11. Gebhart GF. Descending modulation of pain. Neurosci Biobehav Rev. 2004;27(8):729-37. 12. Li Z, Li C, Yin P, Wang ZJ, Luo F. Inhibition of CaMKIIα in the central nucleus of amygdala attenuates fentanyl-induced hyperalgesia in rats. J Pharmacol Exp Ther. 2016;359(1):82-9. 13. Li Z, Yin P, Chen J, Jin S, Liu J, Luo F. CaMKIIα may modulate fentanyl-induced hyperalgesia via a CeLC-PAG-RVM-spinal cord descending facilitative pain pathway in rats. PLoS One. 2017;12(5):e0177412. l p g g p On the 7th day, there were no differences between the groups; that is, the possible residual effect of hyperalgesia induced by a single dose of fentanyl was not evident. 14. Pergolizzi JV Jr, Raffa RB, Taylor R Jr, Rodriguez G, Nalamachu S, Langley P. A review of duloxetine 60 mg once-daily dosing for the management of diabetic periphe- ral neuropathic pain, fibromyalgia, and chronic musculoskeletal pain due to chronic osteoarthritis pain and low back pain. Pain Pract. 2013;13(3):239-52. i osteoarthritis pain and low back pain. Pain Pract. 2013;13(3):239 Studies using duloxetine and fluoxetine to combat pain in ro- dents support the results of this study that 5-HT and NE play a critical role in attenuating persistent pain mechanisms, presu- mably through descending modulatory pathways from pain and consequently in OIH28- 30.h 15. Smith HS, Harris R, Clauw D. Fibromyalgia: an afferent processing disorder leading to a complex pain generalized syndrome. Pain Physician. 2011;14(2):E217-45. 16. Chappell AS, Ossanna MJ, Liu-Seifert H, Iyengar S, Skljarevski V, Li LC, et al. Dulo- xetine, a centrally acting analgesic, in the treatment of patients with osteoarthritis knee pain: a 13-week, randomized, placebo-controlled trial. Pain. 2009;146(3):253-60.fi 17. Dharmshaktu P, Tayal V, Kalra BS. Efficacy of antidepressants as analgesics: a review. J Clin Pharmacol. 2012;52(1):6-17. DISCUSSION The use of pregabalin on nociceptive behavior and SC in a model of trigeminal pain in rats, attenuating mechanical allodynia and SC on the model of trigeminal pain confirms its clinical use in the treatment of pain and, although there are few studies with OIH, there is evidence that it can be useful in controlling this type of pain31. 18. Micó JA, Prieto R. Elucidating the mechanism of action of pregabalin: α(2)δ as a therapeutic target in anxiety. CNS Drugs. 2012;26(8):637-48. 19. Bellato E, Marini E, Castoldi F, Barbasetti N, Mattei L, Bonasia DE, et al. Fibrom- yalgia syndrome: etiology, pathogenesis, diagnosis, and treatment. Pain Res Treat. 2012;2012:426130. 20. Crofford LJ, Rowbotham MC, Mease PJ, Russell IJ, Dworkin RH, Corbin AE, et al. Pregabalin for the treatment of fibromyalgia syndrome: results of a randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 2005;52(4):1264-73. 21. Brennan TJ, Vandermeulen EP, Gebhart GF. Characterization of a rat model of inci- sional pain. Pain. 1996;64(3):493-501. DISCUSSION f In the present study, in the 2nd hour after the surgical procedu- re, there were no differences between the control groups, which received SS by IP associated with SS by gavage compared to the group that received fentanyl by IP associated with SS by gavage. However, when comparing these with animals that received du- loxetine, fluoxetine, or pregabalin, it was found less hyperalgesia, demonstrating possible involvement of serotonin and norepine- phrine receptors and reduction of calcium-dependent pro-no- ciceptive neurotransmitters in spinal cord release, in agreement with other studies25-27. When the animals were evaluated on the 1st and 3rd day after the surgical procedure, the group that received fentanyl via IP showed a greater hyperalgesic effect and with a statistically significant diffe- Figure 4. Student’s t-test showed a significant difference when com- paring G2* with groups G1, G3, G4, and G5 (p<0.05). 17 BrJP. São Paulo, 2020 jan-mar;3(1):14-8 Pires OC, Dalcim ML, Pigozzi AL, Caldeira FM, Pires MH, Almeida Neto L and Posso IP Pires OC, Dalcim ML, Pigozzi AL, Caldeira FM, Pires MH, Almeida Neto L and Posso IP 7. Reznikov I, Pud D, Eisenberg E. Oral opioid administration and hyperalge- sia in patients with cancer or chronic nonmalignant pain. Br J Clin Pharmacol. 2005;60(3):311-8. rence in relation to the control group, evidencing OIH. However, the group that received fentanyl via IP, in addition to showing a significant difference in relation to the control with SS, showed a difference in relation to the duloxetine, fluoxetine and pregabalin group, which were different from the control group that received SS, suggesting effectiveness in reducing OIH. 8. Célèrier E, Rivat C, Jun Y, Laulin JP, Larcher A, Reynier P, et al. Long-lasting hype- ralgesia induced by fentanyl in rats: preventive effect of ketamine. Anesthesiology. 2000;92(2):465-72. 9. Simonnet G, Rivat C. Opioid-induced hyperalgesia: abnormal or normal pain? Neu- roreport. 2003;14(1):1-7. 10. Ossipov MH, Lai J, King T, Vanderah TW, Porreca F. Underlying mechanisms of pro- nociceptive consequences of prolonged morphine exposure. Biopolymers. 2005;80(2- 3):319-24. f On the 5th day after the surgical procedure, the group that received fentanyl maintained a higher pain response when compared to the other groups. However, although G1, which received SS, showed less hyperalgesic effect compared to the fentanyl group, it did not show any difference in comparison to the groups that received dulo- xetine, fluoxetine or pregabalin, still showing the presence of OIH. REFERENCES 26. Nakajima K, Obata H, Iriuchijima N, Saito S. 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Mechanisms of opioid-in- duced pain and antinociceptive tolerance: descending facilitation and spinal dynor- phin. Pain. 2001;92(1-2):5-9. 31. Cao Y, Wang H, Chiang CY, Dostrovsky JO, Sessle BJ. Pregabalin suppresses noci- ceptive behavior and central sensitization in a rat trigeminal neuropathic pain model. J Pain. 2013;14(2):193-204. 18
https://openalex.org/W4378905280	https://pubs.rsc.org/en/content/articlepdf/2023/cc/d3cc02164e	English	null	A practical synthesis of 1,3-disubstituted cubane derivatives	Chemical communications	2,023	cc-by	2,377	Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16:24 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16 This article is licensed under a Creative Commons Attribution 3.0 Unp DOI: 10.1039/d3cc02164e DOI: 10.1039/d3cc02164e rsc.li/chemcomm which is not commercial, is highly toxic and must be synthe- sised from expensive precursors.7 A robust multigram-scale synthesis of 1,3-disubstituted cubanes (previously only available on milligram-scale) is reported. The approach exploits a readily available enone intermediate previously used for the synthesis of 1,4-disubstituted cubanes, by introducing a novel Wharton transposition to access useful quantities of 1,3- disubstituted cubanes for diverse applications. The Pettit approach was very recently revisited by MacMillan and co-workers,4 exploiting bicyclic diazetidine 78 as a new cyclobutadiene precursor. However, cyclobutadiene is very reactive and cycloadduct 3 is air- and moisture-sensitive, thus the route is feasible only on small scales (the sequence was demonstrated on 1 mmol scale), limiting its practicality. On the other hand, Ueda and co-workers showed that enone 9b could be converted to cubane 5 (Scheme 1; B),9 but this route is not viable for scale-up, due to the diﬃculty in accessing the key enone 9b in appreciable quantities – it was obtained in only 4.5% yield (after exhaustive chromatography) as a side product of the synthesis of the regioisomeric enone 9a (a precursor to 1,4-cubane dicarboxylate 10, and available on kilogram scale). Ever since Eaton and Cole’s landmark synthesis of the cubane system,1 chemists have been fascinated by this highly strained, non-natural cage motif.2 More recently, attention has turned to developing applications of cubanes, particularly within medic- inal chemistry, where it has been suggested that cubanes may act as bioisosteres for the benzene ring.3–5 However, the vast majority of work on the applications of cubanes has been focused on 1,4-disubstituted cubanes (Fig. 1), as only these derivatives are available on multigram-scale. Other substitution patterns (such as 1,3-disubstituted cubanes), are accessible only in milligram quantities, and/or their synthesis requires long synthetic sequences often starting from the corresponding 1,4-disubstituted derivatives. The lack of access to multigram quantities of cubanes that bear different substitution patterns continues to preclude a full evaluation of the potential of cubanes in medicinal chemistry (as well as other areas), thus new synthetic approaches are required to bridge this gap. Herein, we demonstrate a robust approach to 5 that allows the straightforward multigram-scale synthesis of 1,3-cubane diester 11 as well as various novel 1,3-disubstituted cubane derivatives. This journal is © The Royal Society of Chemistry 2023 Chem. Commun., 2023, 59, 7971–7973 \| 7971 a Department of Chemistry, Lancaster University, Bailrigg, LA1 4YB, UK. E-mail: s.coote@lancaster.ac.uk b Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3cc02164e Cite this: Chem. Commun., 2023, 59, 7971 Nahin Kazi, ab Marine C. Aublette, ab Sarah L. Allinson b and Susannah C. Coote *a Received 3rd May 2023, Accepted 30th May 2023 Received 3rd May 2023, Accepted 30th May 2023 COMMUNICATION Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16:24 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16 This article is licensed under a Creative Commons Attribution 3.0 Unp A key component of our approach is the 1,3- transposition of enone 9a into enone 9b, thus enabling the preparation of both 1,4- and 1,3-disubstituted cubanes from a common, easily accessible intermediate. To start with, enone 9a was prepared from cyclopentanone according to Tsanaktsidis’s pilot scale synthesis10 using stan- dard laboratory glassware, followed by selective mono- deketalisation.11 The synthesis was routinely carried out on scales of several hundred grams (see the ESI† for details). 9a has previously been used in the synthesis of cubane 1,4- dicarboxylic acid,12 and to access the corresponding 1,3- disubstituted cubanes, we envisaged converting 9a into its isomeric enone 9b via a Wharton transposition sequence.13 Thus, nucleophilic epoxidation of enone 9a gave epoxide 12 as a single diastereoisomer in essentially quantitative yield, with no purification required (Scheme 1). The subsequent Wharton reaction required some optimisation, since in addition to 13, the corresponding debrominated allylic alcohol was produced in varying amounts depending on the reaction conditions. Limiting the number of equivalents of hydrazine employed Only two distinct direct routes to 1,3-disubstituted cubanes have been reported. The 3-step Pettit route (Scheme 1; A) features cyclobutadiene (2) as a key reagent, which undergoes Diels–Alder reaction with dibromoquinone 1.6 This is a parti- cularly elegant and extremely concise route, but is not scalable, as it relies on the cyclobutadieneiron(tricarbonyl) complex (6), Chem. Commun., 2023, 59, 7971–7973 \| 7971 This journal is © The Royal Society of Chemistry 2023 Scheme 2 Conversion of enone 9b into dimethyl 1,3-cubane dicarbox- ylate 11. en Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16:24 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Scheme 2 Conversion of enone 9b into dimethyl 1,3-cubane dicarbox- ylate 11. largely suppressed the debromination, but slowed the reaction. largely suppressed the debromination, but slowed the reaction. Thus, the optimized conditions involved gentle heating of a solution of 12 and hydrazine in ethanol in the presence of an basic resin for 72 hours, furnishing allylic alcohol 13 in 51% yield. Subsequent oxidation using Dess-Martin periodinane gave enone 9b in 92% yield, with the 3-step Wharton transposi- tion sequence being carried out successfully on decagram scale. This article is licensed under a Crea q g y g Next, enone 9b underwent facile [2+2] photocycloaddition upon irradiation at 300 nm, in acetone as both solvent and triplet sensitiser (Scheme 2). This journal is © The Royal Society of Chemistry 2023 References 1 P. E. Eaton and T. W. Cole, J. Am. Chem. Soc., 1964, 88, 962–964. 1 P. E. Eaton and T. W. Cole, J. Am. Chem. Soc., 1964, 88, 962–964. 2 K. F. Biegasiewicz, J. R. Griﬃths, G. P. Savage, J. Tsanaktsidis and R. Priefer, Chem. Rev., 2015, 115, 6719–6745. 3 B. A. Chalmers, H. Xing, S. Houston, C. Clark, S. Ghassabian, A. Kuo, B. Cao, A. Reitsma, C.-E. P. Murray, J. E. Stok, G. M. Boyle, C. J. Pierce, S. W. Littler, D. A. Winkler, P. V. Bernhardt, C. Pasay, J. J. De Voss, J. McCarthy, P. G. Parsons, G. H. Walter, M. T. Smith, H. M. Cooper, S. K. Nilsson, J. Tsanaktsidis, G. P. Savage and C. M. Williams, Angew. Chem., Int. Ed., 2016, 55, 3580–3585. Scheme 3 Selected synthetic manipulations of 17 to give five novel 1,3- disubstituted cubane derivatives. aNaOH (aq), MeOH bPhI(OAc)2, I2, C6H6, reflux cCDI, MeNH(OMe)HCl, CH2Cl2 dDPPA, Et3N, tBuOH, reflux eBH3 SMe2, THF, 0 1C to rt fAllylamine, reflux. CDI = carbonyldiimidazole, DPPA = diphenylphosphorylazide. , , y, J , y , C. J. Pierce, S. W. Littler, D. A. Winkler, P. V. Bernhardt, C. Pasay, 4 M. P. Wiesenfeldt, J. A. Rossi-Ashton, I. B. Perry, J. Diesel, O. L. Garry, F. Bartels, S. C. Coote, X. Ma, C. S. Yeung, D. J. Bennett and D. W. C. MacMillan, Nature, 2023, DOI: 10.1038/ s41586-023-06021-8. Finally, 1,3-cubane diester 11 underwent selective monosa- ponification to give carboxylic acid 17 (Scheme 3), which is a versatile intermediate en route to diverse 1,3-disubstituted cubanes. Thus, in addition to the cross-coupling methodology recently reported by the MacMillan group,4 17 also undergoes standard functional group interconversions, and five novel functionalised 1,3-disubstituted cubanes were prepared in short order, through iodination of the carboxylic acid moiety to give 18, formation of the corresponding Weinreb amide 19, Curtius rearrangement to give 20, and selective borane- mediated reduction to give alcohol 21. In addition, aminolysis of the ester moiety in 17 through heating in allylamine gave amide 22 in excellent yield (Scheme 3). 5 For reviews of cubanes and other hydrocarbon structures as bioi- sosteres: (a) M. A. M. Subbaiah and N. A. Meanwell, J. Med. Chem., 2021, 64, 14046–14128; (b) E. G. Tse, S. D. Houston, C. M. Williams, G. P. Savage, L. M. Rendina, I. Hallyburton, M. Anderson, R. Sharma, R. S. Obach and M. H. Todd, J. Med. Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16 This article is licensed under a Creative Commons Attribution 3.0 Unp The reaction was performed on decagram scale and cycloadduct 14 was obtained in essentially quantitative yield after simple evaporation of the solvent. As previously noted by Ueda,9 attempts to eﬀect a double Favorskii-type ring contraction (after deketalisation of 14) failed to yield diacid 5, hence two separate ring contraction reactions were carried out. Acid 15 was obtained in 78% yield upon heating in aqueous sodium hydroxide, and deprotection of the ketal in 15 was best performed by heating in trifluoroacetic acid. Attempts to perform this deprotection in sulfuric acid (as is common in the 1,4-cubane series10 and as reported by Ueda on small scales for the 1,3-cubane9) resulted in significant decomposition and an arduous aqueous extraction on multi- gram scales; pleasingly, both issues could be avoided simply by employing trifluoroacetic acid. The crude product was taken on directly into the second Favorskii-type ring contraction, which took place under more forcing conditions, and acid-mediated esterification of the resulting diacid in methanol gave 1,3-cubane diester 11 in 52% yield over the three steps – a significant improvement on the 25% yield obtained previously.9 Interestingly, Ueda and co-workers obtained a B1:1 mixture of 11 and open- cage derivative 16 (which presumably derives from initial Haller– Bauer reaction14 instead of closure of the cubane), thus decreas- ing the overall yield of cubane 11. This undesired reactivity was also observed under our conditions through monitoring the ring contraction reaction by 1H NMR spectroscopy (with solvent sup- pression), but to our surprise, the cubane core was regenerated under the acidic esterification conditions employed, and no trace of 16 was detected. Thus, the choice of esterification conditions is crucial in optimising the yield of 11, with the milder esterification conditions chosen by the Ueda group (diazomethane) leading to significant amounts of the undesired open-cage product 16. Fig. 1 Disubstituted cubanes; previous synthetic approaches to 1,3- disubstituted cubanes (A/B); this work (C). Fig. 1 Disubstituted cubanes; previous synthetic approaches to 1,3- disubstituted cubanes (A/B); this work (C). Scheme 1 1,3-Transposition sequence to convert enone 9a into enone 9b. Scheme 1 1,3-Transposition sequence to convert enone 9a into enone 9b. 9b. 7972 \| Chem. Commun., 2023, 59, 7971–7973 This journal is © The Royal Society of Chemistry 2023 Communication View Article Online View Article Online View Article Online Communication Communication Communication ChemComm ChemComm Scheme 3 Selected synthetic manipulations of 17 to give five novel 1,3- disubstituted cubane derivatives. References Chem., 2020, 63, 11585–11601; (c) T. A. Reekie, C. M. Williams, L. M. Rendina and M. Kassiou, J. Med. Chem., 2019, 62, 1078–1095; (d) S. D. Houston, T. Fahrenhorst-Jones, H. Xing, B. A. Chalmers, M. L. Sykes, J. E. Stok, C. F. Soton, J. M. Burns, P. V. Bernhardt, J. J. De Voss, G. M. Boyle, M. T. Smith, J. Tsanaktsidis, G. P. Savage, V. M. Avery and C. M. Williams, Org. Biomol. Chem., 2019, 17, 6790–6798; (e) K. J. Flanagan, S. S. R. Bernhard, S. Plunkett and M. O. Senge, Chem. – Eur. J., 2019, 25, 6941–6954; ( f ) P. K. Mykhailiuk, Org. Biomol. Chem., 2019, 17, 2839–2849; (g) J. Wlochal, R. D. M. Davies and J. Burton, Org. Lett., 2014, 16, 4094–4097. 6 J. C. Barborak, L. Watts and R. Pettit, J. Am. Chem. Soc., 1966, 88, 1328–1329. 7 (a) R. Pettit and J. Henery, Org. Synth., 1970, 50, 21; (b) M. Rosenblum and C. Gatsonis, J. Am. Chem. Soc., 1967, 89, 5074–5075. In summary, we have developed a multigram-scale synthesis of dimethyl 1,3-cubane dicarboxylate (11), which was previously available only on milligram scale, and demonstrated that 11 can be readily converted into a variety of diﬀerent 1,3- disubstituted cubane derivatives via acid-ester intermediate 17. Key to the success of the approach was the development of a Wharton 1,3-transposition sequence of readily available enone 9a into enone 9b, such that both 1,4- and 1,3- disubstituted cubanes can now be easily prepared from a single intermediate. This work will expedite investigations into the applications of 1,3-disubstituted cubanes, which have so far been frustrated by the paucity of accessible 1,3-disubstituted cubanes. Now that appreciable quantities are available, appli- cations are envisaged in particular within medicinal chemistry (e.g. as bioisosteres for meta-substituted benzene rings), but 8 T. K. Britten, P. D. Kemmitt, N. R. Halcovitch and S. C. Coote, Org. Lett., 2019, 22, 9232–9235. 9 T. Nigo, T. Hasegawa, Y. Kuwatani and I. Ueda, Bull. Chem. Soc. Jpn., 1993, 66, 2068–2072. 10 M. J. Falkiner, S. W. Littler, K. J. McRae, G. P. Savage and J. Tsanaktsidis, Org. Process Res. Dev., 2013, 17, 1503–1509. g 11 N. B. Chapman, J. M. Key and K. J. Toyne, J. Org. Chem., 1970, 35, 3860–3867. 12 S. Lal, A. Bhattacharjee, A. Chowdhury, N. Kumbhakarna and I. N. N. Namboothiri, Chem. – Asian J., 2022, 17, e202200489. 13 (a) J. J. Open Access Article. Published on 31 May 2023. Downloaded on 10/24/2024 6:16 This article is licensed under a Creative Commons Attribution 3.0 Unp aNaOH (aq), MeOH bPhI(OAc)2, I2, C6H6, reflux cCDI, MeNH(OMe)HCl, CH2Cl2 dDPPA, Et3N, tBuOH, reflux eBH3 SMe2, THF, 0 1C to rt fAllylamine, reflux. CDI = carbonyldiimidazole, DPPA = diphenylphosphorylazide. ChemComm diverse applications can easily be envisaged in a broad range of other areas (for example, in materials chemistry, supramole- cular chemistry and biochemistry). This work was supported by funding from North West Cancer Research (LFSL2018-23, Studentship to MCA). This journal is © The Royal Society of Chemistry 2023 References Li, in Name Reactions for Functional Group Transformations, ed. J. J. Li and E. J. Corey, John Wiley & Sons, Inc., 2007, ch. 2, pp 152–158; (b) P. S. Wharton and D. H. Bohlen, J. Org. Chem., 1961, 26, 3615–3616. 14 (a) M. Goverdhan and R. V. Venkateswaran, Tetrahedron, 2000, 56, 1399–1422; (b) J. P. Gilday and L. A. Paquette, Org. Prep. Proced. Int., 1990, 22, 167–201. Chem. Commun., 2023, 59, 7971–7973 \| 7973 This journal is © The Royal Society of Chemistry 2023
https://openalex.org/W2075168801	https://zenodo.org/records/2492919/files/article.pdf	English	null	Operative treatment of fractures of long bones	Transactions of the Royal Academy of Medicine in Ireland	1,902	public-domain	4,577	OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. At a time when aseptic surgery was in its infancy there may have been some justification for the unwillingness of sm'geons to mldertake what seemed g~at lisks, but now, when the pl~nciples of aseptic technique are thoroughly understood, and every modern hospital is equipped with the means of canting out this technlque thoroughly, we are no longer justified in ~amsting to what may without disparage- ment be spoken of as methods of haphazard, instead of boldly using those of scientific accuracy. I am not advocating that every case of simple fracture should be subjected to operation. Ordinary fractures, for instance of the tibia or fibula, Lm- accompanied by displacement, usually give excellent result~ when treated intelligently by the methods hitherto in vogue, but it does seem rather a reflection upon us that we should still regard with equs~nlmity a shortening of the limb mnount~ ing to one and a half inches as the result of a fracture of the shaft of the femm -, or be content with the permanent lame- ness and loss of function which so often accompany a Port's fractm~. Every surgeon is aware of the di~r of treating a fracture involving both bones of the forea~n o1" separation of the lower epiphysis of the humelms. In both these instances deformity and impairment of function are the too frequent results of ore" present methods, and I would plead, therefore, for a careful consideration of these methods and an inquiry as to the possibility of improving them. shrinks from the much smaller operation of exposing the broken ends of the shattered femur, and seeming perfeot coaptation of the broken surfaces by the application of simple mechanical appliances. This, I think, is hardly creditable to us, and shows that, notwithstanding all our talk about the entel~rise and boldness of modern surgery, our mh~ds are very apt to run in a groove, and with difficulty- do we extricate om~elves flvm the fetters of tradition. On no other hypothesis can our adherence to present methods be explained. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. By THOMAS MTLES, F.R.C.$. ; President of the Royal College oE Surgeons in Ireland. [Inaugural Address delivered at the Opening Meeting of the Section of Surgery, November 8, 1901.] [Inaugural Address delivered at the Opening Meeting of the Section of Surgery, November 8, 1901.] GENTLE~,--To-night I purpose bringing under your con- sideration a few points in connection with such a compara, tively commonplace subject as injury to the long bones. In recent years visceral surgery has perhaps too exclusively occupied the attention of operators, and with the brilliant result of such concentration of effort you are all familiar, but I think that in the interval perhaps an insufficient amount of attention has been devoted to the consideration of the subject that I now intend to place before you. While in the surgery of the thorax, abdomen, and head the enormous advantages of an aseptic technique have been fully availed of, we have, I feel, been rather remiss in applying the same principles to the treatment of fractured bones. It seems almost as if the boldness and enterprise which have achieved such remarkable results in abdominal surgery are considered entirely out of place when applied to the treatment of fractures. It still is considered compatible with good,surgery that a fracture of the shaft of the femur should be followed by a shortening of from one to two inches, and the man who, without a moment's hesitation, would open the abdomen to seek fox- a possible perforation of the stomach or intestine, H H Fractures of Long Bones. 114 shrinks from the much smaller operation of exposing the broken ends of the shattered femur, and seeming perfeot coaptation of the broken surfaces by the application of simple mechanical appliances. This, I think, is hardly creditable to us, and shows that, notwithstanding all our talk about the entel~rise and boldness of modern surgery, our mh~ds are very apt to run in a groove, and with difficulty- do we extricate om~elves flvm the fetters of tradition. On no other hypothesis can our adherence to present methods be explained. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. At a time when aseptic surgery was in its infancy there may have been some justification for the unwillingness of sm'geons to mldertake what seemed g~at lisks, but now, when the pl~nciples of aseptic technique are thoroughly understood, and every modern hospital is equipped with the means of canting out this technlque thoroughly, we are no longer justified in ~amsting to what may without disparage- ment be spoken of as methods of haphazard, instead of boldly using those of scientific accuracy. I am not advocating that every case of simple fracture should be subjected to operation. Ordinary fractures, for instance of the tibia or fibula, Lm- accompanied by displacement, usually give excellent result~ when treated intelligently by the methods hitherto in vogue, but it does seem rather a reflection upon us that we should still regard with equs~nlmity a shortening of the limb mnount~ ing to one and a half inches as the result of a fracture of the shaft of the femm -, or be content with the permanent lame- ness and loss of function which so often accompany a Port's fractm~. Every surgeon is aware of the di~r of treating a fracture involving both bones of the forea~n o1" separation of the lower epiphysis of the humelms. In both these instances deformity and impairment of function are the too frequent results of ore" present methods, and I would plead, therefore, for a careful consideration of these methods and an inquiry as to the possibility of improving them. 115 By ~.fR. T. MYLES. By ~.fR. T. MYLES. The surgical world owes a great debt of gratitude in this connection to the laboul~ of Mr. Arbuthnot Lane, who has for maaly years past laboured with but indiffel~nt success to im- press his own bold and sagacious views upon the general body of the profession. In /hnerica, where traditions are of less influence tha~l they al~ with us, his suggestions have met ~4th a ready acceptance, but it must be confessed that in 'Ilmland, as yet, little effort has been made to tread the path he has pointed out to us. It is true that in occasional cases, such as mltmited fractm~s, or those resulting in angular union, we adopt modern methods, but I think these isolated instances do. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. not relieve us of the reproach that in this pal~i- cular branch of surgery Dublin lags behind, instead of being "in the van, as it ought to be. ~Iy purpose to-night is not to dilate upon this subject at large, but rather to confine my attention to one or two cases ~illustrative of the subject under review, and from them to make any larger generalisations that may seem indicated. Quite recently two cases of rather rare injury were 1ruder my care at the Richmond Hospital, and were both submitted first to examination by the X-rays, and subsequently to opera- tion. They were both cases of fracture of the neck of the hmnelms, with dislocation of the head towards the axilla. As this particular injury is not discussed at any length in ~.he ordinary text-books, and as both the skiagl~ph and opera- tion taught me a good deal I did not know before, I feel that no apology on my part is necessal T for bringing the subject under your notice to-night. As you will see later on, the fundamental question that I have raised in the fnmt part of this paper was forced upon my attention in these two cases. My fnmt case was that of a woman, aged forty years, a patient in the North Dublin Union Infn~nary, sent to me by Dr. Caleb Powell, a Fellow of this College. The accident had occurred some months before, and a diagnoss of dislocation, Fractures of Loag Bones. 116 with some other indefinite injm'y to the upper end of the shaft, was made. The sholfldel:joint was ank~'losed, and the arm practically useless. An X-ray exanfination by Dr. R. Lane Joynt showed that, in addition to the dislocation, there was a fracture of the anatomical neck ~4thout impaetion. As there was both pain along the nerve tl"tu~s, and cedema of the hand and am1, and as the limb was practically useless, ,I determined to operate. This I did by an anterior incision, such as is usual in excision of the head of the hmnerus. The operation in this case, as in all other cases of old-standing uncomplicated dislocations, was very difficult, much more so than is usually fotmd to l)e the case in resections for disease of the bone. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. Tills difficulty was due to the altered relations of the parts, to the thickening of the capsule and ligamentous structures arolmd the joint, and to the genel~l shrinkage which takes place when a dislocation has existed for any length of time. To permit sufficient freedom of movement the long head of the biceps had to be divided and the capsule split very freely. When at length a good view of the parts was obtained, 'I found that the head of the humerus along its posterior narrow edge was attached by osseous mlion to the anterior edge of the glenoid fossa, and a nan-ow area of the renter of the scapula adjacent thereto. On the anterior edge of the glenoid fossa, eneroac.h~ng on the cartilage, was a distinct crescentic depression, into which the head was finbedded-- apparently the pressure of the latter had first set up a rarefying osteitis, followed later on by osseous union between the apposed parts. The bond of union had to be divided with a bone forceps before the head could be removed. Portion of the great tu~)erosity was removed also, and the case became one simply of resection. My second case was that of a man aged fort.y-two yearn, who fell about fourteen feet on to his head and shoulder. He was at once brought to the neighboming hospital, where By .'M_R. T. ]i[s 117 a diagnosis of dislocation was made and vigorous repeated effol~ were made under chloroform to reduce it. It is im- possible for me to say if the fractm~ and dislocation co-existed from the outset, or if, as he alleges, the fracture was produced by the vigorous efforts at reduction. I am inclined to think from the natm'e of the accident, a fall from such a height, that the two injmies were produced at the same moment, and that the repeated efforts made to effect reduction under anaesthesia were made after due recognition of the nature .of the lesion. This is lundered more probable by the fact that when he came under my care some months later even r crepitus was easily obtained, and must have been still more easily obtained a few hours after the accklent. On examination of the injm~d are.a a large bony mass ~ould be felt in the shoulder extending inwards beneath the r pi~)cess ; this was smooth, and felt like the head of the htune1~is. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. There was but little flattening of the deltoid or prominence of the acromion. The drctunfel~nce of the shoulde," was nearly one inch g1"eater thaa~ on the opposite side. He had practically no power whatever in the ann, and any movement caused pain. An X-ray photo taken by I)r. R. Lane Joyat showed that the head of the humeru,s was dislocated inwards, and that the main line of fractm'e was tln'ough the great tuberosity. In addition, a long splinter was detached from the outside of the shaft, extending for some three inches downwards. This case I also submitted to operation, but I fotmd it neces- sary, in addition to the long anterior incision, to divide part of the deltoid close to its insertion, so as to obtain sufficient room for the subsequent manipulations. I confess I was somewhat astonished on exposing the parts to find the head firmly al~l,-ylosed to the scapula, as in the forlner case. The seat of attachment, however, was lower down on the scapula, mid nmch further inwards. It was not a mere line of bony Fractures of Lon, g Bo~es. 118 1mien, as in the first case, but the posterior edge, and nearly half of the articular surface, was, as it were, imbedded in the- scapula and incorporated with it. All attempts to displace it were futile, and I had to remove it piecemeal with cutting forceps. On the other hand, the long splinter of bone was quite ununited, and showed no attempt at bony uuion. This is.certainly not what one would have expected from a priori reasoning. One of the dangers of this injury is said to be necrosis of the head, but in my two cases at least experience does not sup- port the t.heolies based on a p~-iori reasoning. It is un- doubtedly a strange fact that the detached head, instead of necrosing, should have become fn'mly united to the scapula, while the long splinter of the shaft was still unattached. ,In this case, also, I had to resect a part of the great tuberosi~- so as to obtain a smooth end to the humelxlS. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. These two cases are, to my mind, typical illustrations of my thesis, that the principles of modern surgel T are not applied to the treatment of fiuctures with an)r like the same bol&less that they are applied to other surgical aihnents, First comes the ever-present difficulty of diagnosis, li: think ,I am justified in making the assertion that ordhml"y methods of examination are not sttfficient to enable even the most experienced surgeon to make an accurate diagnosis of these complicated injuries about the shoulder. Evel T Ia~sh surgeon is bound to speak respectfully of the work done by the late Professor ttobert Smith, but no one, I think, now-a- days will be fotmd to assert that the admirable rules he has laid down for the diffel~ntial diagnosis of injuries about the shottlder cover the enth'e ground, o1" are in themselves suffi-- Cient to enable an ordilmi T surgeon to make an absolutely certain diagnosis. To meet the difficulty of teaching students, the teacher is By 'MR. T. '~YLES. 119 compelled to adopt a system of classification which divides these injuries into separate groups, and to endeavour to place any one injury in some one of these groups. Hence, as a logical consequence, has arisen the unnatural divi- sions of these fractures into intra-and extra-capsular, &c., with o1" without impaction. The learner, with his nmch smaller experience, is still more urgently impelled in the same direction, and when called to exan~ine one of these cases his first mental effort is to place it in group i or B. Now this dogmatic teaching is, I believe, responsible for very many errors in diagnosis, and I am com~nced that if men could approach the questions before them with a more open mb~d, unham- pel~i by the venerable dogmas of the past, such errors would be less frequent. Nature does not care much for our classifi- cations, and is not withheld by respect for sth'gical traditions from superimposing a dislocation upon any or every fracture. The first requirement in surgery is accurate diagnosis, audI do not believe that the precise rules laid dou~a by the great teachers in the past are sufficient in themselves to supply what we require. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. Every one of us has had experience of the great difficulties that attend the diagnosis of such injuries, and I hope I will not be thought to labour the point when I say that such injuries should not be treated at all until either an X-ray photograph has been made by a competent person, or a thorough examination has been made under an anaesthetic. The former course is far the more reliable and informing, but it is not always available. Even when an X-ray photograph has been taken it requh~s skill and ex- perience in such work to read aright the lesson it conveys. We must not fail to remember that such injuries may be very complicated, several fractures co-existing, each of which, adding its own PecUliar symptoms to the case, may more or less effect~_~"y mask its companions. Thus, for instance, 120 Fractures of Lang Bones. the head of the bone may be obviously dislocated, and yet the characteristic l~gidity of the dislocation be absent owing to fi~cture of the neck, or all attempts at accuracy of diagnosis may be frustrated by the enormous effusion of blood that so 5"equently accompanies any injury in this region. The difficulty of accurate diagnosis in such cases is well illustrated by the following case :--A pati'ent of mine in Jet,is-street Hospital, with the histo1% name of John Morley, was sent me fl~m Ballaghadereen, in the County Mayo. He had appaa~ntly an old-standing disloeation of the head of the humerus beneath the coracoid process, but on examlnlug the shoulder carefully one could find under the acromion a mass of bony consistence and about the size of a pigeon's egg, or a little larger. He said, moreover, that the dislocation had been fi-e- quently reduced, but always recurred. Now I l~ow I am quite sure of your sympathy when I tell you that I made a very bad error in this case, and one not due to ignorance alone, but rather to the influence of the dogmatic teaching I received in my youth. I was convinced that I had to deal with the very rare condition described by the late R. W. Smith, in which, super- )reposed on a disloeation, is a fi~cture of the great tuben)sity. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. JIt is some consolation to me now, in moments of reflection, to know that on that occasion, at least, I en-ed in the very best company, for my fi~end Professor Bennett, whose authority in all injm~es to bones no one here will question, kindly examined the case for me, and came to the same con- cluaion as I did. 'I operated on the man aal(l discovered that there was no fi-actm-e whatever; what I mistook for/.he detached tuber- osity was a calcm~ous mass in the subdeltoid bm~a. Now this is a very interesting case to me, and it is rendered still more so by the thought that even had the X-my method been available then it coukl not have helped us much, as the mass of lime salts woldd have thl~wn as deep a shadow as By M~. T. HYT.~,S. 121 bone. This is a condition that I have never seen descl~bed and, of com~e, is not included in any of the numerous classifications of injm T in this region. Having, perhaps, at too much length discussed the question of diagnosis, let me take up that of treatment. Having satis- fied o~u~elves that we are dealing with a case of fractm~ of the neck of the humerus complicated ~ith a dislocation, how should we treat it ? My answer to this question is--By immediate operation in the majolity of cases. I am con- vincod that no other method gives promise of an equally good result. What are the other methods, the traditional ones, if I may call them so ? First, to do nothing wlmtever t~ the fracture but begin to set up passive motion as soon as possible, so as to establish a false joint between the head, wifich is pretty certain to become fixed to the scapula, and tim upper end of the shaft. I need not waste your time by discussing-this method, which is in itself a confession of hnpotenco and does nothing to relieve the pain and cedema due to pressure of the displaced head on nelwes and veins. Secondly, to endea- vour to effect reduction of the displaced head, and then to treat the fractm~. EvelTone who has given any thought to the pathology of this condition will find it hard indeed to believe that such reduction is ever possible. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. The head cannot be grasped, it can only be pushed with the finger tips ; it cannot be rotated. ~-action cannot be made on the shaft, lest the few untorn fibres of pel~ostemn be severed and the vita~ty of the head endangered. Even should the head be forced back into place, one cannot be sure that it is properly applied to the broken end of the shaft. It may be tluu~ed topsy-tm'~', or it may be implanted in the shaft far below its nomnal position. However theolmtically attractive such a method may be, it is, I think, of little practical value. Thh~lly, comes resection 122 Fractures of Long Bo~es. of the head. As tl~ involves a cutting operation, it is not less sciious than the plan I have ,~commended of reduction after incision and exposure of the parts. In l~cent eases it would only be justified after the fom~er had been tried and failure resulted. In old-standing cases it is p,~etically the only method open to us. The pain and cedema of the arm and head cannot be relieved Lmtil the bony mass of the head is taken off the vein and brachial plexus. The l~sult of re- section all the same is, I believe, hmetionally better than that given by a false joint with the head ank-ylosed to the scapula. On s~mnning up these argmnents and weighing them carefully, I think one is folded to the" conclusion that when a dislocation of the head of the hmnerus complicated by fi~cture of the n~k occurs in a young, healthy man, especially one who has to live by manual labom', the sm-geon ought to. make up his mind to operate on it at the earliest possible date. Tl~s being granted, let us consider what method of operating is likely to prove the most useful for our propose. My own experience is too limited to make dognmtic assertions on the subject., but I am pl~tty well convinced by such ex- perience as I have had on the subject that the anterior incisiolt alone is not sufficient for us. It does not give a sufficiently fi~e exposure of the parts to enable the necessary manipula- tions to be caaTied out fi~ely and quickly. MY belief is that the cap formed by the deltoid muscle must be raised before one can get pl~per access to the parts. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. This can be done either, as suggested by Pl~fessor Kocher, by detaching the spine of the scapula along its entil~ length from the rest of the bone, disart:ieulating the aeromial end from the clavicle and throwing the ent/re mass down, or by detaching the deltoid insertion from the shaft of the humerus and raising the muscle. This latter seems to me the preferable method~ as it does not involve such a serious addition to an ah~ady grave injury as Professor Kocher's method, nor does it neces- By ,MR. T. MYLES. 123 sitate the subsequent wiring into position of the detached scapular spine. ~[y plan would be as follows :--An incision, starting from the clavicle above the eoracoid process would follow the line of separation between the deltoid and g,~at pectoral, as far as the insertion of the fomler. The peri- osteum here wonld have to be raised colwesponding to the line of attachment of the muscle, or even a small flap of the bone taken with it. The incision would then follow the hinder" edge of the deltoid as high as the point at which the circtml- flex nerve entel~ it. The muscle could then be tin'ned up, veKy fully, and the seat of injm-y thoroughly exposed. By means either of a screw inserted in it, o1" ~f'Gill's hook, the dislocated segment could, I believe, be brought into its normal position, and then wired on to the shaft. Such an operation can1 only be a success if performed very soon after the injm T occurs. If any time be allowed to elapse, the shrinkag e which takes place prevents either reduction of the dislocation or perfect coaptation of the segments. What are the objections to this comparatively simple and easily ~mderstood operation ? That it makes a grave injmT still gTaver, and puts in danger a life that was not previously imperilled. Surely, to say that such an operation impelils a patient's life is really a confession of inability to carry out an operation in accordance with modem principles. The operation should not be dangerous, and never will be in the hands of a careful, competent, and cleanly surgeon. An operator who feels he cannot guarantee his work against infection in such a case should not operate at all ; he should seek some other outlet for his energies more suitable to his capacity. OPERATIVE TREATMENT OF FRACTURES OF LONG BONES. Again, say opponents of reform, this injury does not threaten a man's life, thel~fore we are not justified in putting him ta~ any risk whatever. Is danger to a man's life the greatest danger he can face, compared to which all others sink into. 124 Fractures of Loug Bones. ns gnifieance ? Is the poverty, due to inability to labour with his hands, to which a man the victim of such an injm'y as that under review is inevitably doomed, of no importance ? Are the persistent shooting pains, the paralysis and (edema of hand and aaTn of no consequence ? q My object to-night will have been achieved if I succeed in impressing upon my hearers how open to reproach at present is the attitude of most Izish surgeons in hesitating to apply to the treatment of injured bones and joints the same boldness and scientific sldll which they unhesitatingly apply to the head, thorax, and abdomen. I feel assmBd it will not be many yeaa~ before we will see as great a l~volution in the treatment of such cases as we have witnessed during the last twenty years in the treatment of penetrating wounds and other in- juries of the abdomen, and those who come after us will look back with contempt.uo~s pity on the unreasoning fatuity of the men of our day, who, with the light of modern know- ledge shining brightly on their path, wel~ so fettered by routine and the teaching of tradition that they shrank thnidly from s that path, and left to another generation the glory of reaping the harvest which it should have been their pride and privilege to garner. Surely no one who has ever seen a wor-ldng man with an old-standing dislocation and fracture of the upper end of the humerus will hesitate for a moment in urging such a one to face any and every risk to be relieved .of his incapacity. Such cases are not commonly seen in the general hospitals, but they can be-seen in abmldanco in the workhouse hospitals, where these poor creatures are com- pelled, though often yet in the very plane of life, to spend the remainder of their days through inability to eal~ their own ]i~ing by the labour of their hands. That such a state of affairs should still exist is a reproach we should labour diligently :~o remove.
https://openalex.org/W2005421646	https://europepmc.org/articles/pmc6271709?pdf=render	English	null	Synthesis and Antibacterial Activity of Analogs of 5-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiazoli-din-4-one	Molecules/Molecules online/Molecules annual	2,014	cc-by	4,715	77 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules OPEN ACCESS 77 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules OPEN ACCESS Molecules 2014, 19, 13577-13586; doi:10.3390/molecules190913577 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Synthesis and Antibacterial Activity of Analogs of 5-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo- 1,3-thiazoli-din-4-one Nguyen Tien Cong 1, Huynh Thi Nhan 1, Luong Van Hung 1, Tran Dinh Thang 2,* and Ping-Chung Kuo 3,* 1 Department of Chemistry, Ho Chi Minh City University of Education, Ho Chi Minh City 70000, Vietnam; E-Mails: congchemist@gmail.com (N.T.C.); nhanhuynhsp@gmail.com (H.T.N.); luonghung365@gmail.com (L.V.H.) 2 Department of Chemistry, Vinh University, Vinh 42000, Vietnam 3 Department of Biotechnology, National Formosa University, Yunlin 63201, Taiwan * Authors to whom correspondence should be addressed; E-Mails: thangtd@vinhuni.edu.vn (T.D.T.); pcckuoo@sunws.nfu.edu.tw (P.-C.K.); Tel.: +886-5-6315491 (P.-C.K.); Fax: +886-5-6315502 (P.-C.K.). Received: 30 June 2014; in revised form: 26 August 2014 / Accepted: 27 August 2014 / Published: 1 September 2014 Abstract: In an effort to develop new antimicrobial agents, 3-(4-methylcoumarin-7- yloxyacetylamino) 2 thioxo 1 3 thiazolidin 4 one (4) was synthesized by reaction of OPEN ACCESS Molecules 2014, 19, 13577-13586; doi:10.3390/molecules190913577 Keywords: coumarin; 2-thioxo-1,3-thiazolidin-4-one; spectral elucidation; antibacterial activity Keywords: coumarin; 2-thioxo-1,3-thiazolidin-4-one; spectral elucidation; antibacterial activity Synthesis and Antibacterial Activity of Analogs of 5-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo- 1,3-thiazoli-din-4-one Nguyen Tien Cong 1, Huynh Thi Nhan 1, Luong Van Hung 1, Tran Dinh Thang 2,* and Ping-Chung Kuo 3,* 1 Department of Chemistry, Ho Chi Minh City University of Education, Ho Chi Minh City 70000, Vietnam; E-Mails: congchemist@gmail.com (N.T.C.); nhanhuynhsp@gmail.com (H.T.N.); luonghung365@gmail.com (L.V.H.) 2 Department of Chemistry, Vinh University, Vinh 42000, Vietnam 2 Department of Chemistry, Vinh University, Vinh 42000, Vietnam 3 Department of Biotechnology, National Formosa University, Yunlin 63201, Taiwan 3 Department of Biotechnology, National Formosa University, Yunlin 63201, Taiwan * Authors to whom correspondence should be addressed; E-Mails: thangtd@vinhuni.edu.vn (T.D.T.); pcckuoo@sunws.nfu.edu.tw (P.-C.K.); Tel.: +886-5-6315491 (P.-C.K.); Fax: +886-5-6315502 (P.-C.K.). Received: 30 June 2014; in revised form: 26 August 2014 / Accepted: 27 August 2014 / Published: 1 September 2014 Received: 30 June 2014; in revised form: 26 August 2014 / Accepted: 27 August 2014 / Published: 1 September 2014 Abstract: In an effort to develop new antimicrobial agents, 3-(4-methylcoumarin-7- yloxyacetylamino)-2-thioxo-1,3-thiazolidin-4-one (4) was synthesized by reaction of thiocarbonylbisthioglycolic acid with ethyl (4-methyl-2-oxo-2H-chromen-7-yloxy)aceto- hydrazide (3), which was prepared in turn from 7-hydroxy-4-methylcoumarin (1). The condensation of compound 4 with different aromatic aldehydes afforded a series of 5-(arylidene)-3-(4-methylcoumarin-7-yloxyacetyl-amino)-2-thioxo-1,3-thiozolidin-4-one analogs 5a–h. The structures of these synthetic compounds were elucidated on the basis of IR, 1H-NMR and 13C-NMR spectral data and ESI-MS spectrometric analysis. Compounds 5a–h were examined for their antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. ywords: coumarin; 2-thioxo-1,3-thiazolidin-4-one; spectral elucidation; antibacterial it Keywords: coumarin; 2-thioxo-1,3-thiazolidin-4-one; spectral elucidation; antibacterial activity Molecules 2014, 19 Molecules 2014, 19 13578 1. Introduction Bacterial disease control, including the food safety issue, has continuously attracted researchers’ attention from various fields. The use of preservatives and pathogen antagonists had been reported as a means of protecting the microbiological safety of fresh and processed food products [1–4]. Although some antagonists exhibited significant inhibition of bacterial growth, they were too toxic to be utilized long term. In the present study, we hoped to explore new lead compounds with natural skeletons which could be modified for further investigation as antimicrobial agents applied to food preservation. 7-Hydroxy-4-methylcoumarin derivatives with heterocyclic moieties possess diverse biological properties such as antibacterial [5–7], antifungal [8,9], anticancer [10], enzyme-inhibitory [11], and antioxidant activities [7,9]. On the other hand, thiazolidin-4-ones are important compounds due to their broad range of biological activities including anticancer [12–14], virus-inhibitory [15], HIV-inhibitory [16], and enzyme-inhibitory activities [14]. These observations prompted our interest in synthesizing some new 7-hydroxy-4-methylcoumarin derivatives bearing 2-thioxo-1,3-thiozolidin-4-one substituents and evaluate their antibacterial potential. 2. Results and Discussion 2.1. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h 2.1. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h The synthetic route for the preparation of the target compounds was presented in Scheme 1. Compounds 1–3 were synthesized according to the corresponding published procedures [6,7,9–11] and characterization of these synthetic compounds was achieved by comparison of their physical and spectral data with those reported in the previous literature [17]. Then compound 3 was reacted with thiocarbonylbisthioglycolic acid in ethanol to obtain new compound 4, which was converted into the series of N-(5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)- acetamides 5a–h by Knoevenagel condensation. In the IR spectrum of compound 4, in addition to the lactone and amide carbonyl group absorption at 1709 cm−1, the stretching band in a high frequency region (1771 cm−1) indicated the presence of C=O bonds in a thiazolidine ring. A new signal with intensity of 2H appearing at 4.47 ppm in the 1H-NMR spectrum was attributed to the methylene group of the thiazolidine ring. Another signal also with intensity of 2H appearing in the downfield region at 4.96 ppm was attributed to the oxymethylene protons (OCH2). In terms of unexpected results, the signals of the methylene protons in 4 were split instead of being a singlet. The splitting of these signals could be explained by a non-first order splitting effect. Comparing the 13C-NMR spectra of 3 [7] and 4, three more signals appeared in 4, two of which were at around 160 ppm corresponding to the signals of the carbon atoms in the thioxo group and carbonyl group, whereas the last one was at 33.4 ppm corresponding to the signal of the saturated carbon atom of the thiazolidine ring. The spectral data of 4 as well as the agreement of the predicted mass with molecular mass determined by HR-MS confirmed that a 4-oxo-2-thioxothiazolidine ring was formed. 13579 Molecules 2014, 19 Scheme 1. Synthetic route for the preparation of compounds 5a–h. 2.1. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h HO OH H3C C CH2 C OC2H5 O O O O OH CH3 (1) ClCH2COOC2H5 O O OCH2COOC2H5 CH3 H2N NH2 O O OCH2CONHNH2 CH3 S C SCH2COOH SCH2COOH O O O CH3 NH N O S O S CHO X O O O CH3 NH N O S O S (2) (3) (4) (5a-h) (a) (b) (c) X =4-Cl X = 4-OCH3 X = 4-NO2 (d) X = 4-N(CH3)2 (e) X = 4-OH (f) X = 2-NO2 (g) X = 4-Br (h) X = 4-H X I h IR f d 5 h h hif f h b i f h b l CH3 HO ClCH2COOC2H5 CH3 OCH2CONHNH2 (2) CHO X S O O O CH3 NH N O S O S (5a-h) (b) (c) X =4-Cl X = 4-OCH3 X = 4-NO2 (d) X = 4-OH (f) X = 2-NO2 (g) (h) X = 4-H X (4) In the IR spectrum of compounds 5a–h, there were shifts of the absorption of the carbonyl group at 1771 cm−1 to lower frequencies (1721–1755 cm−1), in agreement with the formation of a conjugated system between the carbonyl group and the benzylidene moiety. Comparison of the 1H-NMR spectra of 5a–h with the 1H-NMR of 4 showed not only the disappearance of the methylene proton’s signal at 4.47 ppm, but also appearance of additional aromatic proton signals at 6.84–8.36 ppm and methylidene proton signals at 7.82–8.86 ppm. The signal of the oxymethylene protons (OCH2) in compounds 5a–h appeared at 5.00–5.04 ppm. The signals of these protons, like the signals of the methylene protons OCH2 in compound 4, were also split by a non–first order splitting effect. Therefore, they did not appear as singlets. Molecules 2014, 19 13580 Molecules 2014, 19 diameters were 15 mm to 20 mm) against certain bacteria including Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria). In addition, the synthetic compounds 3 and 4 did not show any significant inhibition of bacterial growth in our preliminary screening and therefore the data were not included. Table 1. Antimicrobial inhibition zone diameters (d) of compounds 5a–h. Bacteria Conc. X 4-N(CH3)2 (5a) 4-OH (5b) 4-OCH3 (5c) 4-H (5d) 4-Br (5e) 4-Cl (5f) 2-NO2 (5g) 4-NO2 (5h) d (mm) a Escherichia coli 0.1% 14 15 13 14 15 12 12 15 0.2% 16 17 16 16 16 15 15 18 Pseudomonas aeruginosa 0.1% 13 19 12 17 15 15 14 18 0.2% 15 23 15 19 18 17 16 20 Bacillus subtilis 0.1% 15 15 14 15 18 14 13 14 0.2% 17 19 16 18 21 18 16 17 Staphylococcus aureus 0.1% 13 14 14 16 17 14 15 16 0.2% 15 16 18 17 20 16 17 18 a D − d ≥ 25 mm: Very high activity; D − d ≥ 20 mm: High activity; D − d ≥ 15 mm: Average activity; D − d ≤ 15 mm: Low activity. Each experiment was performed in triplicate. Table 1. Antimicrobial inhibition zone diameters (d) of compounds 5a–h. a D − d ≥ 25 mm: Very high activity; D − d ≥ 20 mm: High activity; D − d ≥ 15 mm: Average activity; D − d ≤ 15 mm: Low activity. Each experiment was performed in triplicate. The minimum inhibitory concentration (MIC) value is a measure to define the antibacterial activity of a compound and is defined as the lowest concentration of drug that inhibits visible growth. Compounds 5a–h were subjected to examination of their MIC values according to the reported method [19] and the data are shown in Table 2. The two-fold microdilution broth method was used and all of the tested samples demonstrated inhibitory effects in a concentration-dependent manner. However, only 5c, 5g and 5h exhibited any significant inhibition against S. aureus with MIC values of 50 μg/mL. Table 2. The minimum inhibitory concentrations (MICs) of 5a–h against bacteria. Bacteria MIC (μg/mL) 4-N(CH3)2 (5a) 4-OH (5b) 4-OCH3 (5c) 4-H (5d) 4-Br (5e) 4-Cl (5f) 2-NO2 (5g) 4-NO2 (5h) Escherichia coli - a - - - - - - - P. Molecules 2014, 19 aeruginosa - - - - - - - - Bacillus subtilis - - - - - - - - Staphylococcus aureus - - 50 - - - 50 50 a MIC > 50 μg/mL and not determined. Table 2. The minimum inhibitory concentrations (MICs) of 5a–h against bacteria. Table 2. The minimum inhibitory concentrations (MICs) of 5a–h against bacteria. Bacteria MIC (μg/mL) 4-N(CH3)2 (5a) 4-OH (5b) 4-OCH3 (5c) 4-H (5d) 4-Br (5e) 4-Cl (5f) 2-NO2 (5g) 4-NO2 (5h) Escherichia coli - a - - - - - - - P. aeruginosa - - - - - - - - Bacillus subtilis - - - - - - - - Staphylococcus aureus - - 50 - - - 50 50 a MIC > 50 μg/mL and not determined. a MIC > 50 μg/mL and not determined. 2.2. Determination of the in Vitro Antimicrobial Activity Compounds 5a–h were examined for antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria) at concentrations of 0.1% and 0.2% according to the reported method with minor modifications [18]. As shown in Table 1, most of the compounds 5a–g at 0.1% exhibited low antimicrobial activity, with antimicrobial inhibition zone diameters of less than 15 mm. However, at the concentration of 0.2%, most of these compounds showed average activity (the antimicrobial 3.2. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h 3.2.1. Synthesis of 4-Oxo-2-thioxothiazolidine Derivatives 3.2.1. Synthesis of 4-Oxo-2-thioxothiazolidine Derivatives Compounds 1–3 were prepared using the corresponding reported methods [6,7,9–11] as shown in Scheme 1. 3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta- mide (4) 3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta- mide (4) 3.1. General Procedures All starting materials were purchased from Merck (Darmstadt, Germany) and used without purification. Melting points were measured in open capillary tubes on a Gallenkamp melting point 13581 Molecules 2014, 19 apparatus. The structures of all compounds were confirmed by IR, NMR and HR-MS spectra. IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer using KBr pellets. The 1H-NMR spectra were recorded on a Bruker Avance spectrometer at 500 MHz using DMSO-d6 as solvent, while the 13C-NMR, HSQC, HMBC spectra were recorded at 125 MHz. The data are given in parts per million (ppm) and are referenced to an internal standard of tetramethylsilane (TMS, δ 0.00 ppm). The spin-spin coupling constants (J) are given in Hz. Peak multiplicities are reported as s (singlet), d (doublet), dd (double-doublet), t (triplet), q (quartet), and m (multiplet). The MS spectra were recorded on a Bruker microTOF-Q 10187 spectrometer or on a Varian FT-ICR-MS 910 spectrometer. 3.2. Synthesis of 7-Hydroxy-4-methylcoumarin Derivatives 5a–h 3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta- mide (4) N-(5-(4-Hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetamide (5b): Brown yellow powder; yield: 64%; mp: 284–285 °C; IR (ν, cm−1): 3340, 2920, 2851, 1732, 1709, 1678, 1572, 1510, 1483, 1397, 1364, 1242, 1229; 1H-NMR (δ, ppm): 10.70 (1H, s, OH), 2.41 (3H, s, CH3), 5.01 (2H, OCH2), 6.25 (1H, s, 3-H), 7.05 (1H, d, 4J = 2.5, 8-H), 7.06 (1H, dd, 3J = 9.0, 4J = 2.5, 6-H), 7.73 (1H, d, 3J = 8.5, 5-H), 7.86 (1H, s, =CH-), 11.7 (1H, s, NH), 7.58 (2H, d, 3J = 9.0) and 6.95 (2H, d, 3J = 9.0) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.4 (4-C), 18.1 (CH3), 126.5 (5-C), 112.7 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH2), 166.1 (–NH–CO–CH2), 163.2 (>N–CO), 114.3 (>C= thiazolidine ring), 190.1 (C=S), 135.7 (=CH–), 123.8, 133.7 and 116.7 (Cbenzene ring), 161.2 (Carom–OH); HR-ESI-MS: 469.0497 (M+H), calcd. for (C22H16N2O6S2): 468.0450. N-(5-(4-Methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetamide (5c): Yellow powder; yield: 71.0%; mp: 239–240 °C; IR (ν, cm−1): 3205, 3080, 2936, 1736, 1699, 1618, 1510, 1479, 1389, 1314, 1258, 1229, 1180, 1134; 1H-NMR (δ, ppm): 6.26 (1H, d, 4J = 1.0, 3-H), 2.41 (3H, d, 4J = 1.0, CH3), 7.72 (1H, 3J = 8.5, 5-H), 7.08 (1H, dd, 3J = 8.5; 4J = 2.5, 6-H), 7.05 (1H, d, 4J = 2.5, 8-H), 5.02 (2H, OCH2), 11.70 (1H, s, NH), 7.92 (1H, s, =CH-), 7.68 (2H, d, 3J = 8.5) and 7.15 (2H, d, 3J = 8.5) (Hbenzene ring), 3.85 (3H, s, OCH3); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH2), 166.0 (–NH–CO–CH2), 163.2 (>N–CO), 115.6 (>C= thiazolidine ring), 190.0 (C=S), 135.2 (=CH–), 125.2, 133.3 and 115.2 (Cbenzene ring), 161.9 (Carom–OCH3), 55.6 (OCH3); HR-ESI-MS: 505.0494 (M+Na), calcd. for (C23H18N2O6S2): 482.0606. 3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta- mide (4) A mixture of (4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (3, 0.01 mol) and thio-carbonylbisthioglycolic acid (0.01 mol) in ethanol (5 mL) was refluxed for 8 h. After cooling the resulting solid was filtered off, dried and recrystallized from HOAc/DMF to give compound 4 as a yellowish powder in 74.0% yield; mp: 248–249 °C; IR (ν, cm−1): 3258, 3100, 2901, 1771, 1709, 1622, 1491, 1425, 1391, 1358, 1298, 1245; 1H-NMR (ppm) δ 11.41 (1H, s, NH), 7.71 (1H, d, 3J = 9.0 Hz, H-5), 7.04 (1H, dd, 3J = 9.0 Hz, 4J = 2.5 Hz, H-6), 7.01 (1H, d, 4J = 2.5 Hz, H-8), 6.23 (1H, s, H-3), 4.96 (2H, OCH2), 4.47 (2H, CH2 thiazolidine ring), 2.39 (3H, s, CH3); 13C-NMR (ppm) δ 199.7 (C=S), 170.1 (NH–CO–CH2), 165.8 (>N–CO), 160.3 (C-7), 160.0 (O–C=O), 154.4 (C-9), 153.3 (C-4), 126.5 (C-5), 112.5 (C-6), 113.8 (C-10), 111.6 (C-3), 101.8 (C-8), 66.0 (OCH2), 33.4 (SCH2), 18.1 (CH3); HR-ESI-MS m/z 365.0266 [M+H]+ (calcd. for C15H13N2O5S2, 365.0266). 3.2.3. General Procedure for Synthesis of N-(5-Arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl- 2-oxo-2H-chromen-7-yloxy)acetamides 5a–h Equimolar amounts of 4 (5.0 mmol), anhydrous sodium acetate (5.0 mmol) and an appropriate aromatic aldehyde (5.0 mmol) in glacial acetic acid (5 mL) were refluxed for 5 h. The reaction mixture was cooled and the solid separated was filtered and recrystallized to give compounds 5a–h. N-{5-[4-(Dimethylamino)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}-2-(4-methyl-2-oxo-2H-chromen- 7-yloxy)acetamide (5a): Red powder; Yield: 69.0; mp. 265–266 °C; IR (ν, cm−1): 3314, 3088, 2915, 1721, 1616, 1574, 1530, 1441, 1385, 1302, 1263; 1H-NMR (δ, ppm): 3.06 (6H, s, –N(CH3)2), 2.41 (3H, s, CH3), 5.00 (2H, OCH2); 6.25 (1H, s, 3-H), 7,05 (1H, d, 4J = 2.5, 8-H), 7.07 (1H, dd, 3J = 9.0, 4J = 2.5, 6-H), 7.72 (1H, d, 3J = 8.5, 5-H), δ 7.79 (1H, s, =CH-), 11.6 (1H, s, NH), 7.50 (2H, d, 3J = 9.0) and 6.84 (2H, d, 3J =9.0) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 13582 Molecules 2014, 19 Molecules 2014, 19 (OCH2), 166.0 (–NH–CO–CH2), 163.2 (>N–CO), 113.9 (>C= thiazolidine ring), 189.6 (C=S), 136.3 (=CH–), 119.6, 133.6, 112.6 and 112.3 (Cbenzene ring), 152.2 (Carom–N(CH3)2), 39.6 (–N(CH3)2); HR-ESI-MS: 496.1001 (M+H), calcd. for (C24H21N3O5S2): 495.0923. Molecules 2014, 19 Molecules 2014, 19 119.9 (>C= thiazolidine ring), 189.8 (C=S), 133.8 (=CH–),131.8 and 132.6 (Cbenzene ring), 125.1 (Carom–Br); HR-ESI-MS: 530.9671 (M+H) and 532.9651 [M+H+2], calcd. for (C22H15BrN2O5S2): 529.9606 and 531.9606. 119.9 (>C= thiazolidine ring), 189.8 (C=S), 133.8 (=CH–),131.8 and 132.6 (Cbenzene ring), 125.1 (Carom–Br); HR-ESI-MS: 530.9671 (M+H) and 532.9651 [M+H+2], calcd. for (C22H15BrN2O5S2): 529.9606 and 531.9606. N-(5-(4-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetamide (5f): Yellow powder; yield: 70.0%; mp: 275–276 °C; IR (ν, cm−1): 3215, 3084, 2,934, 1736, 1697, 1605, 1584, 1489, 1476, 1441, 1387, 1370, 1269, 1258, 1242, 1132; 1H-NMR (δ, ppm): 6.30 (1H, s, 3-H), 2.41 (3H, s, CH3), 7.74 (1H, d, 3J = 8.5; 5-H), 7.07 (1H, dd, 3J = 8.5, 4J = 2.5, 6-H), 7.05 (1H, d, 4J = 2.5, 8-H), 5.02 (2H, OCH2), 11.7 (1H, s, NH), 7.97 (1H, s, =CH–), 7.72 (2H, d, 3J = 8.5) and 7.64 (2H, d, 3J = 8.5) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0 (OCH2), 166.1 (–NH–CO–CH2), 163.0 (>N–CO), 119.8 (>C= thiazolidine ring), 189.8 (C=S), 136.1 (=CH–), 131.5, 132.5 and 129.6 (Cbenzene ring), 133.7 (Carom–Cl); HR-ESI-MS: 509.0004 (M+Na), calcd. for (C22H15ClN2O5S2): 486.0111. 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(5-(2-nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetamide (5g): Pale pink powder; yield: 57.0%; mp: 260–261 °C; IR (ν, cm−1): 3316, 3080, 2940, 1755, 1717, 1616, 1522, 1483, 1387, 1343, 1298, 1271; 1H-NMR (δ, ppm): 6.30 (1H, s, 3-H), 2.41 (3H, s, CH3), 7.74 (1H, d, 3J = 8.5, 5-H), 7.07 (1H, dd, 3J = 8.5, 4J = 2.5, 6-H), 7.05 (1H, d, 4J = 2.5, 8-H), 5.03 (2H, OCH2), 11.6 (1H, s, NH), 8.22 (1H, s, =CH–), 7.81 (1H, d, 3J = 7.5), 7.79 (1H, dd, 3J1 = 3J2 = 7.5), 7.91 (1H, dd, 3J1 = 3J2 = 7.5) and 8.26 (1H, d, 3J = 7.5) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.6 (5-C), δ 112.6 (6-C), 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0 (OCH2), 166.1 (–NH–CO–CH2), 162.3 (>N–CO), 123.4 (>C= thiazolidine ring), 190.3 (C=S), 134.8 (=CH–),147.8 (Carom–NO2), 131.7, 125.7, 129.6, 132.3 and 128.3 (Cbenzene ring); HR-ESI-MS: 498.04295 (M+H), calcd. for (C22H15N3O7S2): 497.0351. Molecules 2014, 19 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(5-(4-nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetamide (5h): Yellow powder; yield: 60%; mp: 273–274 °C; IR (ν, cm−1): 3235, 3084, 2940, 1736, 1697, 1609, 1526, 1474, 1442, 1387, 1346, 1269, 1238, 1200; 1H-NMR (δ, ppm): 6.30 (1H, s, 3-H), 2.41 (3H, s, CH3), 7.73 (1H, d, 3J = 9.0, 5-H), 7.07 (1H, dd, 3J = 9.0, 4J= 2.5, 6-H), 7.04 (1H, d, 4J = 2.5, 8-H), 5.04 (2H, OCH2), 11.7 (1H, s, NH), 8.08 (1H, s, =CH–), 8.36 (2H, d, 3J = 8.5) and 7.95 (2H, d, 3J = 8.5) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.0 (OCH2), 166.1 (–NH–CO–CH2), 162.9 (>N–CO), 123.4 (>C= thiazolidine ring), 189.6 (C=S), 132.2 (=CH–), 147.9 (Carom–NO2), 138.7, 124.4 and 131.7 (Cbenzene ring); HR-ESI-MS: 520.0239 (M+Na), calcd. for (C22H15N3O7S2): 497.0351. 3.2.2. Synthesis of 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-thioxothiazo-lidin-3-yl)aceta- mide (4) N-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide (5d): Yellow powder; yield: 54.0%; mp: 249–250 °C; IR (ν, cm−1): 3376, 3080, 1740, 1719, 1622, 1564, 1479, 1427, 1370, 1296, 1256, 1220; 1H-NMR (δ, ppm): 2.41 (3H, s, CH3), 5.02 (2H, OCH2); 6.25 (1H, s, 3-H), 7.05 (1H, d, 4J = 2.5, 8-H), 7.08 (1H, dd, 3J = 8.5, 4J = 2.0, 6-H), 7.74 (1H, d, 3J = 8.5, 5-H), 7.96 (1H, s, =CH-), 7.69 (2H, d, 3J =7.0) and 7.57 (3H, m) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), δ 160.3 (7-C), 101.9 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH2), 166.1 (–NH–CO–CH2), 163.1 (>N–CO), 119.8 (>C= thiazolidine ring), 190.1 (C=S), 135.1 (–CH=), 132.7, 130.9, 131.4 and 129.6 (Cbenzene ring); HR-ESI-MS: 453.0478 (M+H), calcd. for (C22H16N2O5S2): 452.0501. N-(5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetamide (5e): Yellow powder; yield: 73.0%; mp: 279–280 °C; IR (ν, cm−1): 3214, 3100, 1736, 1697, 1618, 1605, 1578, 1487, 1440, 1387, 1269, 1258, 1240; 1H-NMR (δ, ppm): 2.41 (3H, s, CH3), 5.02 (2H, OCH2); 6.30 (1H, s, 3-H), 7.04 (1H, d, 4J = 2.5, 8-H), 7.06 (1H, dd, 3J = 8.5, 4J = 2.5, 6-H), 7.74 (1H, d, 3J = 8.5, 5-H), 7.97 (1H, s, =CH–), 7.76 (2H, d, 3J = 9.0) and 7.62 (2H, d, 3J = 9.0) (Hbenzene ring); 13C-NMR (δ, ppm): 160.0 (O–C=O), 111.6 (3-C), 153.3 (4-C), 18.1 (CH3), 126.5 (5-C), 112.6 (6-C), 160.3 (7-C), 101.8 (8-C), 154.5 (9-C), 113.9 (10-C), 66.1 (OCH2), 166.1 (–NH–CO–CH2), 163.0 (>N–CO), 13583 3.4. Minimum Inhibitory Concentration (MIC) Determination The amount of growth in the wells containing test samples was compared with the amount of growth in the control wells when determining the growth end points. When a single skipped well occurred, the highest MIC was read. Each experiment was performed in triplicate. Streptomycin and tetracyclin were used as positive controls for Gram-positive bacteria and Gram-negative bacteria, respectively. 3.3. Determination of the in Vitro Antimicrobial Activity The compounds 5a–h at concentrations of 0.1% and 0.2% were examined for antimicrobial activity against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25923) (Gram-negative bacteria), Bacillus subtilis (ATCC 11774) and Staphylococcus aureus (ATCC 11632) (Gram-positive bacteria) according to the reported method with minor modifications [18]. A mixture of meat extract 13584 Molecules 2014, 19 (5.0 g), peptone (5.0 g), NaCl (5.0 g), agar (20.0 g) and distilled water (1000 mL) was stirred to dissolve the ingredients and then sterilized in an autoclave to give the MPA environment for growing the bacteria. The mixture was poured to Petri dishes. The Petri dishes then were put in a sterile cabinet for 24 h. After infusion of the particular bacteria into the MPA environment in the Petri dishes, a hole was drilled in the center of the dish. DMSO solution (0.1 mL) of the particular chemical at a concentration of 0.1% or 0.2% was dripped into the hole. The samples were placed in a refrigerator for 4–8 h, and then incubated at room temperature for 24 h. The inhibiting zone was measured by the (D − d) value expressed in millimeters (mm), where D was the diameter of inhibited zone and d was the diameter of the hole. The evaluation was based on the following criteria: D − d ≥ 25 mm: very strong antibacterial activity; D − d ≥ 20 mm: strong antibacterial activity; D − d ≥ 15 mm: medium antibacterial activity; D − d ≤ 15 mm: weak antibacterial activity. Each experiment was performed in triplicate. Author Contributions Nguyen Tien Cong, Huynh Thi Nhan, and Luong Van Hung performed the research and recorded the spectra. Tran Dinh Thang and Ping-Chung Kuo designed research and wrote the paper. 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Microbiology: Essentials and Applications, 2nd ed.; McGraw-Hill: New York NY USA 1996; pp 375 406 18. Egorov, N.S. Antibiotics A Scientific Approach; Mir Publishers: Moscow, Russia, 1985; pp. 76 340. 19. McKane, L.; Kandel, J. Microbiology: Essentials and Applications, 2nd ed.; McGraw-Hill: New York, NY, USA, 1996; pp. 375–406. 19. McKane, L.; Kandel, J. Microbiology: Essentials and Applications, 2nd ed.; McGraw-Hill: New York, NY, USA, 1996; pp. 375–406. e Availability: Samples of the compounds 1–4 and 5a–h are available from the authors. Sample Availability: Samples of the compounds 1–4 and 5a–h are available from the authors. Sample Availability: Samples of the compounds 1–4 and 5a–h are available from the authors. © 2014 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 license (http://creativecommons.org/licenses/by/3.0/). © 2014 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 license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W3026180772	https://cardiothoracicsurgery.biomedcentral.com/track/pdf/10.1186/s13019-020-01148-5	English	null	Normalization of the right heart and the preoperative factors that influence the emergence PAH after surgical closure of atrial septal defect	Journal of cardiothoracic surgery	2,020	cc-by	5,481	(2020) 15:105 (2020) 15:105 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 https://doi.org/10.1186/s13019-020-01148-5 Supomo et al. Journal of Cardiothoracic Surgery https://doi.org/10.1186/s13019-020-01148-5 Normalization of the right heart and the preoperative factors that influence the emergence PAH after surgical closure of atrial septal defect Normalization of the right heart and the preoperative factors that influence the emergence PAH after surgical closure of atrial septal defect Supomo Supomo1* , Agung Widhinugroho2 and Aditya Agam Nugraha3 Supomo Supomo1* , Agung Widhinugroho2 and Aditya Agam Nugraha3 © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Surgical closure of atrial septal defect (ASD) is contraindicated in the condition with severe pulmonary arterial hypertension (PAH), whereas ASD closure in an effective intervention to normalize the structure and function of the right heart after previously experiencing volume overload due to shunting from the defect. This study aimed to evaluate normalization of the right heart and emergence of PAH after surgical closure of ASD. Methods: This retrospective study was carried out in 45 patients over 18 years who had undergone surgical closure of ASD. The study has the aim to evaluate the morphological and functional parameters before and after the surgical approach and the preoperative factors that influenced the development of pulmonary arterial hypertension (PAP) after the ASD closure. Results: The majority of subjects were female (73.3%) although there were no significant differences between males and females from the various parameters. The average of mPAP in the group that experienced PAH was higher than non-PAH group after ASD closure (p = 0.019, 31.23 ± 12.70 mmHg vs 24.07 ± 13.08 mmHg). Significant differences were found in the Right Atrium (RA) dimension, Right Ventricle (RV) dimension, Tricuspid Regurgitation Velocity (TRV) and Tricuspid Annular Plane Systolic Excursion (TAPSE) between before and at 6 months after ASD closure (p = 0.000, p = 0.000, p = 0.000, p = 000, respectively). The sensitivity of the predictive model to estimate PAH at 6 months after surgical closure of ASD was 58%, with a specificity of 62.5%. Conclusion: Structural and functional normalization of the right heart occurs at 6 months after surgical closure of ASD with the decrease of RA and RV dimensions and improvement from tricuspid regurgitation. Emergence of PAH after ASD closure was influenced by higher mPAP before surgical approach. Keywords: Atrial Septal defect, ASD, Surgical closure, ASD closure, Normalization * Correspondence: supomo.tkv@mail.ugm.ac.id * Correspondence: supomo.tkv@mail.ugm.ac.id 1Department of Surgery, Thoracic, Cardiac and Vascular Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Kesehatan Street No. 1, Yogyakarta 55281, Indonesia Full list of author information is available at the end of the article Background prediction was mPAP obtained using predictive models to estimate mPAP after ASD closure by Supomo [7], and mPAP prediction = (0.24 x age) + (0.06 x mPAP before sur- gery) + (0.17 x RA dimension) + (0.47 x RV dimension) – 13.79. Pulmonary arterial hypertension (PAH) was defined as mean arterial pressure (mPAP) above 25 mmHg [2]. Atrial Septal Defect (ASD) can go undiagnosed until adulthood [1, 2], and is one of the most common con- genital heart diseases in adults [3]. Defect of the atrium often leads to volume overload in the right heart coupled with the potential risk of right heart failure, pulmonary arterial hypertension (PAH) and atrial arrhythmia [1, 4].. ASD closure is the current best treatment for ASD pa- tients with stable hemodynamics [1]. ASD closure is usually done at infancy while still asymptomatic to pre- vent complications from shunt flow [3–5]. Early surgical closure of ASD has a good long-term outcome [2]. The Medical and Health Research Ethics Committee (MHREC) of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada / Dr. Sardjito General Hospital, Yogyakarta, Indonesia (KE/1240/10/2019) ap- proved this study. Informed consent was obtained from each patient before surgical closure of ASD. p g Statistical analysis on all data was performed using SPSS version 23 (IBM Corp., Chicago). Continuous vari- ables were in the form of mean ± standard deviation, while continuous variables with non-parametric data were presented in the median. Characteristic data between males and females were compared using student’s t-test to determine any significant difference between the variables as shown in Table 1, whereas non-parametric variables used the Mann-Whitney test. Evaluation of the independ- ent variables that affect the development of PAH was done by comparing the preoperative condition of the PAH and non-PAH group before and at 6 months after ASD closure used the Mann Whitney test as shown in Table 2. PAH at 6 months after ASD closure was classified as mPAP > 25 mmHg based on the TTE examination at 6 months after surgical approach. The analysis results were considered to be significantly different if p < 0.05. Comparison of pre- operative and 6 months postoperative data was performed using paired student’s t-test and for non-parametric data using the Wilcoxon test (Table 3). Background Predicted mPAP using Supomo [7] predictive model was compared with postop- erative mPAP from TTE using unpaired student’s t-test, Pulmonary arterial (PA) pressure improvement can occur after ASD closure [6]. However, severe PAH can worsen the condition of the patients undergoing ASD closure because it requires partial shunting for blood flow to reduce right heart pressure [3]. Accordingly, severe PAH is contraindicated for surgical closure of ASD be- cause of the risk of persistent PAH after ASD closure [7]. ASD is often followed by right heart enlargement due to volume overload [8]. Evidence of right heart enlarge- ment from Transthoracic Echocardiography (TTE) can be used as an indication of ASD closure [4, 9]. Volume overload of the right atrium (RA) and right ventricle (RV) is known to be a consequence of the condition of untreated ASD, and the presence of persistent shunting can have an effect on the appearance of cardiac arryth- mias [10]. This study aimed to evaluate the normalization of the right heart and preoperative factors that influence the development of PAH after ASD closure. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Page 2 of 6 Page 2 of 6 Page 2 of 6 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Methods This retrospective study was carried out in 45 patients over 18 years who had undergone surgical closure of ASD. The study has the aim to evaluate the morpho- logical and functional parameters before and after the surgical approach and the preoperative factors that influ- enced the development of pulmonary arterial hyperten- sion (PAP) after the ASD closure. Preoperative factors seen from the morphology and functional parameters before ASD closure by TTE examination were compared between PAH and non-PAH group at 6 months after ASD closure. Table 1 Characteristics of data between male and female before surgery (N = 45 subjects) Variables Male (n = 12) Female (n = 33) p Age (years) 33.45 ± 9.89 34.68 ± 9.90 0.753 BMI (kg/m2) 18.09 ± 2.98 19.60 ± 4.21 0.239 LA Dimension (mm) 35.81 ± 7.93 33.03 ± 7.58 0.425 RA Dimension (mm)* 47 (43–64) 47 (41–65) 0.198 RV Dimension (mm) 48.81 ± 9.43 43.50 ± 7.24 0.062 EF (%) 68.36 ± 8.27 69.24 ± 7.64 0.326 mPAP (mmHg) 28.76 ± 16.98 28.62 ± 11.66 0.985 RA Pressure (mmHg)* 8 (3–15) 8 (3–15) 0.809 TRV (m/s) 2.91 ± 0.89 3.07 ± 0.94 0.598 TAPSE (mm) 25.72 ± 6.46 27.71 ± 5.14 0.144 PAH 7 (58.3) 23 (69.7) 0.496 =non parametric; BMI = Body Mass Index; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = Right Ventricle; TRV = Tricuspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium; PAH = Pulmonary Arterial Hypertension Table 1 Characteristics of data between male and female before surgery (N = 45 subjects) Data retrospectively taken from the Medical Record Installation of Dr. Sardjito General Hospital included: age, sex, body mass index (BMI), mean pulmonary arter- ial pressure (mPAP), left atrium (LA) dimension, diam- eter of ASD, right atrium (RA) dimension, right ventricle (RV) dimension, right atrium (RA) pressure, tricuspid re- gurgitation velocity (TRV), tricuspid annular plain sys- tolic excursion (TAPSE), and ejection fraction (EF) from the results of TTE by cardiologists before surgery and 6 months after surgical closure of ASD. The mPAP Supomo et al. Methods Journal of Cardiothoracic Surgery (2020) 15:105 Page 3 of 6 Table 2 Comparison of preoperative factors in pulmonary arterial hypertension group and non-pulmonary arterial hypertension group before and at 6 months after ASD closure Preoperative factors Before Surgery p 6 Months After Surgical Closure p Pulmonary Hypertension (N = 30) Non-PAH (N = 15) Pulmonary Hypertension (N = 29) Non-PAH (N = 16) Age (years) 36.12 ± 11.11 31.14 ± 5.91 0.049 35.32 ± 10.37 32.57 ± 8.74 0.144 BMI (kg/m2) 19.67 ± 4.25 18.28 ± 3.20 0.194 19.27 ± 3.83 19.00 ± 4.23 0.621 EF (%) 68.95 ± 8.74 69.07 ± 5.78 0.804 69.19 ± 8.54 68.64 ± 6.29 0.946 TRV (m/s) 3.28 (0.53–4.30) 2.74 (1.47–3.89) 0.057 3.27 (0.53–4.30) 3.00 (0.59–4.30) 0.551 TAPSE (mm) 27.36 ± 6.05 26.78 ± 4.64 0.682 27.40 ± 5.82 26.71 ± 5.15 0.994 RA Dimension (mm) 50.04 ± 6.49 47.35 ± 6.03 0.180 49.32 ± 6.73 48.64 ± 5.91 0.640 RV Dimension (mm) 45.80 ± 7.38 43.57 ± 9.52 0.109 45.44 ± 7.42 44.21 ± 9.58 0.379 mPAP (mmHg) 35.84 ± 10.40 15.84 ± 5.32 0.000 31.23 ± 12.70 24.07 ± 13.08 0.019 RA Pressure (mmHg)* 8 (3–15) 8 (3–8) 0.549 8 (3–15) 8 (3–8) 0.916 LA dimension (mm) 34.12 ± 9.19 33.28 ± 4.03 0.440 34.92 ± 8.37 31.85 ± 6.06 0.171 Size of ASD (mm) 26.52 ± 8.47 27.14 ± 10.48 0.941 28.88 ± 7.73 22.93 ± 10.38 0.050 * = non parametric; ** = Significant difference; BMI = Body Mass Index; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = Right Ventricle; TRV = Tricuspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium; ASD = Atrial Septal Defect Table 2 Comparison of preoperative factors in pulmonary arterial hypertension group and non-pulmonary arterial hypertension group before and at 6 months after ASD closure * = non parametric; ** = Significant difference; BMI = Body Mass Index; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = Right Ventricle; TRV = Tricuspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium; ASD = Atrial Septal Defect cant difference; BMI = Body Mass Index; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = d Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium; ASD = Atrial Septal Defect while calculating the sensitivity and specificity of formula compared with the gold standard of mPAP from TTE. Methods (15.84 ± 5.32 mmHg) before ASD closure (p = 0.000), while at 6 months after surgery, the preoperative mPAP in PAH and non-PAH group were 31.23 ± 12.70 mmHg and 24.07 ± 13.08 mmHg (p = 0.019), respectively. Higher mPAP before ASD closure was found in PAH group at 6 months after ASD closure. Whereas BMI, EF, TRV, RA dimension, LA dimension, RV dimension, RA pres- sure and TAPSE did not show any significant differences between PAH and non-PAH group before and at 6 months after ASD closure. * = Significant Difference; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = Right Ventricle; TRV on Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium * = non parametric; = Significant Difference; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; Tricuspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium non parametric; = Significant Difference; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = R uspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium Discussion The current gold standard to assess PA pressure to diag- nose PAH is using the right heart catheterization (RHC), but TTE is more often used to diagnose PAH because of its availability [11]. This study used TTE as a tool to ac- cess the condition of the heart in ASD patients before surgery and at 6 months after surgical closure of ASD. PAH in congenital heart disease can occur due to pro- gressive vascular remodelling [11]. Defect closure in ASD is indicated in the left to right shunt with the evi- dence of the right heart enlargement because of volume overload [9, 12, 13]. The closure of ASD causes protec- tion of the right heart from volume overload and can also reduce the pulmonary arterial pressure [3]. In our study, most of the patients had an enlarged right heart as indicated by RA and RV dilatation from TTE examin- ation. However, ASD closure is contraindicated in high pulmonary vascular resistant (PVR) conditions or in pa- tients with pulmonary arterial pressure more than two- thirds of systemic arterial pressure [9, 12]. ASD closure is contraindicated in severe PAH, but there is currently no PA pressure and pulmonary vascu- lar resistance index (PVRI) level set as a limit for ASD closure [7, 19]. Besides PAP, another parameter used is evidence of shunt left to right, with pulmonary vascular resistance (PVR) less than two-thirds of systemic vascu- lar resistance (SVR) [19]. This study showed a slight de- crease in pulmonary arterial pressure after ASD closure but the decrease was not statistically significant. Persistent elevated PAP can occur after ASD closure [16]. In ASD patients with an increase in PAP and Qp but who have normal PVR or only slightly increased, there is usually only minimal change in pulmonary vascular, so it is good to do surgical closure of ASD [20]. Conversely, complete change in pulmonary vascular can happen in the condi- tions with high increases in PAP and high PVR, so that the surgical closure of ASD is contraindicated [20]. Surgical closure of ASD is an effective and safe method to correct the defect, with RV volume overload character- ized by RA and RV dilatation as the most common criteria used as indication for ASD closure [13]. In our study, there were significant differences in RA and RV diameters before and at 6 months after surgical closure of ASD. Results The average preoperative mPAP was higher in PAH (35.84 ± 10.40 mmHg) than non-PAH Table 3 Comparison of the variables before and after ASD closure Variables Before After p 95%CI Mean Differences RA Dimension (mm) 48.84 ± 6.23 36.96 ± 6.03 0.000 9.77–13.99 11.87 ± 7.01 RV Dimension (mm) 44.89 ± 8.11 34.80 ± 5.50 0.000 7.71–12.46 10.09 ± 7.91 LA Dimension (mm) 34.13 ± 7.26 32.75 ± 6.75 0.184 −0.677-3.43 1.37 ± 6.84 TRV (m/s) 2.95 ± 0.98 2.23 ± 0.60 0.000 0.40–1.04 0.72 ± 0.89 TAPSE (mm) 27.13 ± 5.42 14.46 ± 4.39 0.000 10.69–14.64 12.66 ± 6.57 mPAP (mmHg)* 31.52 (5.65–52.45) 27.20 (2.50–51.00) 0.101 RA Pressure (mmHg)* 8.00 (3.00–15.00) 3.00 (3.00–15.00) 0.403 EF (%) * 69.50 (49.00–85.00) 69.00 (56.00–79.00) 0.404 * = non parametric; ** = Significant Difference; mPAP = Mean Pulmonary Arterial Pressure; EF = Ejection Fraction; RA = Right Atrium; RV = Right Ventricle; TRV = Tricuspid Regurgitation Velocity; TAPSE = Tricuspid Annular Plane Systolic Excursion; LA = Left Atrium Table 3 Comparison of the variables before and after ASD closure Page 4 of 6 Page 4 of 6 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Comparison between estimated mPAP using the Supomo [7] predictive models and mPAP from TTE examination did not show any significant difference (p = 0.105) and used a cut off of 25 mmHg in PA pressure to categorize the patient’s condition as PAH. The diagnos- tic test to assess PAH at 6 months after ASD closure showed that the sensitivity of the Supomo [7] prediction model was 56% with a specificity of 62.5%. ASD with an average reduction of 12.66 ± 6.57 mm. This result is similar to the findings of Akula et al. [15] which showed a decrease in TAPSE after ASD closure. This de- crease in TAPSE indicates an improvement in the func- tion of the right ventricle [15]. RV will accommodate smaller volumes after ASD closure due to the decreased dimension of RV, and this has an effect on improving stroke volume from the RV to pulmonary artery [14, 15]. Results Tricuspid regurgitation (TR) often occurs in adult ASD patients because of the shunt caused by ASD that is a consequence of right ventricle volume overload [16, 17] Our study showed similar results that all adult ASD patients experienced tricuspid regurgitation as seen from the TTE examination. Improvement from regurgitation state of tricuspid valve in long-term follow-up occurs after the closure of ASD, because of reverse remodelling that occurs after ASD closure [16–18]. The condition of regurgitation of the heart valve that includes the tricus- pid valve will increase cardiovascular morbidity, reduce functional capacity, and increases the risk of heart failure and even death [16]. Results The baseline characteristics of a total of 45 patients be- fore ASD closure are shown in Table 1 by comparing between males and females. No significant difference in the patients’ condition before ASD closure was found between males and females, while the majority of pa- tients were female with as many as 73.3% of the total subjects. Bivariate analyses of RA dimension, RV dimension, LA dimension, RA pressure, TRV, TAPSE, EF and mPAP before and at 6 months after ASD closure are shown in Table 3. There were significant differences in RA dimen- sion, RV dimension, TRV and TAPSE before and at 6 months after ASD closure (p = 0.000, p = 0.000, p = 0.000, p = 000, respectively). Meanwhile, other variables such as LA dimension, RA pressure, mPAP and EF did not show any significant difference between before and at 6 months after ASD closure. The analyses of the preoperative parameters in PAH and non-PAH group before and at 6 months after sur- gery are shown in Table 2. There were 30 patients with PAH before surgery, and 29 patients at 6 months after surgery who experienced PAH. The average age of the patients who experienced PAH before and at 6 months after surgery were 36.12 ± 11.11 years and 35.32 ± 10.37 years, respectively. Authors’ contributions S (Supomo), AW (Agung Widhinugroho) and AAN (Aditya Agam Nugraha) conceived the study. S and AW collected the data, and AW and AAN analyzed the data. S, AW and AAN drafted the manuscript. All authors read and approved the final manuscript. Availability of data and materials All data are included in the submission. The raw data are available from the corresponding author on reasonable request. References K M 1. Komar M, Przewlocki T, Olszowska M, Sobien B, Podolec P. The benefit of atrial septal defect closure in elderly patients. Clin Interv Aging. 2014;9: 1101–7. Abbreviations l ASD: Atrial Septal Defect; PAH: Pulmonary Arterial Hypertension; mPAP: Mean Pulmonary Arterial Pressure; TTE: Transthoracic Echocardiography; RA: Right Atrium; RV: Right Ventricle; TRV: Tricuspid Regurgitation Velocity; TAPSE: Tricuspid Annular Plane Systolic Excursion; EF: Ejection Fraction; BMI: Body Mass Index; RHC: Right Heart Catheterization; PVR: Pulmonary Vascular Resistance; PVRI: Pulmonary Vascular Resistance Index; SVR: Systemic Vascular Resistance 8. Kumar P, Sarkar A, Kar SK. Assessment of ventricular function in patients of atrial septal defect by strain imaging before and after correction. Ann Card Anaesth. 2019;22(1):41–6. 10. Kucinska B, Werner B, Wróblewska-Kałużewska M. Assessment of right atrial and right ventricular size in children after percutaneous closure of secundum atrial septal defect with Amplatzer septal occluder. Arch Med Sci. 2010;6(4):567–72. Competing interests The authors have no conflict of interest. Conclusions 2. Humenberger M, Rosenhek R, Gabriel H, et al. Benefit of atrial septal defect closure in adults: impact of age. Eur Heart J. 2010;32(5):553–60. 2. Humenberger M, Rosenhek R, Gabriel H, et al. Benefit of atrial septal defect closure in adults: impact of age. Eur Heart J. 2010;32(5):553–60. 2. Humenberger M, Rosenhek R, Gabriel H, et al. Benefit of atrial septal defect closure in adults: impact of age. Eur Heart J. 2010;32(5):553–60. 3. Arafa OS, Aboelazm TH, Mostafa SA, Elemam AM, Abdelhakim A. Transcatheter closure of atrial septal defect preserves right ventricular function. EC Cardiology. 2015;2(1):71–85. Surgical closure of ASD is an intervention that can normalize the anatomy and function of the right heart. Emergence of PAH after ASD closure was influenced by higher mPAP before surgical approach. Improvements to the atrial and ventricular dimensions occur at 6 months after ASD closure. Tricuspid regurgitation (TR), which often occurs in ASD, has significantly improved TR velocity at 6 months after ASD closure. Further study with more accurate parameters is needed to confirm the findings of our study. g 3. Arafa OS, Aboelazm TH, Mostafa SA, Elemam AM, Abdelhakim A. Transcatheter closure of atrial septal defect preserves right ventricular function. EC Cardiology. 2015;2(1):71–85. 4. Kort HW, Balzer DT, Johnson MC. Resolution of right heart enlargement after closure of secundum atrial septal defect with transcatheter technique. J Am Coll Cardiol. 2001;38(5):1528–32. 5. Castaldi B, Vida VL, Argiolas A, et al. Late electrical and mechanical remodeling after atrial septal defect closure in children: surgical versus percutaneous approach. Ann Thorac Surg. 2015;100(1):181–6. 5. Castaldi B, Vida VL, Argiolas A, et al. Late electrical and mechanical remodeling after atrial septal defect closure in children: surgical versus percutaneous approach. Ann Thorac Surg. 2015;100(1):181–6. 6. Balint OH, Samman A, Haberer K, et al. Outcomes in patients with pulmonary hypertension undergoing percutaneous atrial septal defect closure. Heart. 2008;94(9):1189–93. 5. Castaldi B, Vida VL, Argiolas A, et al. Late electrical and mechanical remodeling after atrial septal defect closure in children: surgical versus percutaneous approach. Ann Thorac Surg. 2015;100(1):181–6. 6. Balint OH, Samman A, Haberer K, et al. Outcomes in patients with pulmonary hypertension undergoing percutaneous atrial septal defect closure. Heart. 2008;94(9):1189–93. 7. Supomo S, Arjana AZ, Darmawan H. Predictive model for secundum atrial septal defect closure with pulmonary artery hypertension in adult: when to close. Heart Surg Forum. 2018;21(2):108–11. Discussion The right ventricle and atrial di- mensions are related to the presence of PAH because in the PAH, the right ventricular and atrial dilatation often occur [2, 21]. Low sensitivity and specificity of the pre- dictive model to predict PAH can be caused by PAH is a complex process that is not only influenced by the pa- rameters examined in the previous study. Besides being influenced by the right heart dimensions, the emergence of PAH in ASD also influenced by the size of defect, re- spiratory disease, NYHA class III/IV, tricuspid regurgita- tion and the presence of arrhythmias [2, 6, 21], where these parameters have not been analyzed in previous studies. Persistent PAH can occur because of persistent elevated levels of PVR after ASD closure [2]. Although there is a decrease in PA pressure postoperatively, it rarely reaches normal values, especially in patients with advanced PAH before ASD closure [2]. effect on vascular hypertrophy and vasoconstriction in pulmonary arteries [21]. The right ventricle and atrial di- mensions are related to the presence of PAH because in the PAH, the right ventricular and atrial dilatation often occur [2, 21]. Low sensitivity and specificity of the pre- dictive model to predict PAH can be caused by PAH is a complex process that is not only influenced by the pa- rameters examined in the previous study. Besides being influenced by the right heart dimensions, the emergence of PAH in ASD also influenced by the size of defect, re- spiratory disease, NYHA class III/IV, tricuspid regurgita- tion and the presence of arrhythmias [2, 6, 21], where these parameters have not been analyzed in previous studies. Persistent PAH can occur because of persistent elevated levels of PVR after ASD closure [2]. Although there is a decrease in PA pressure postoperatively, it rarely reaches normal values, especially in patients with advanced PAH before ASD closure [2]. Consent for publication Not applicable. One limitation of this study is that there was no randomization of subjects, because all ASD patients from 2017 to 2019 who underwent surgical closure of ASD were included in this study and the parameters used were only from TTE examination. Besides that, we had not in- cluded stratification of age, type and size of ASD, presence or absence of congenital heart disorder concomitant and the treatment that had been obtained in the analysis. Fur- ther study can use parameters that are the current gold standards such as using the right heart catheterization (RHC) to evaluate pulmonary arterial pressure and also need of complementary studies to evaluate the long-term evolution of PAH after the ASD closure. Funding Thi This research received no specific funding from any agencies in the public, commercial, or not-for-profit sectors. Ethics approval and consent to participate The present study was approved by The Medical and Health Research Ethics Committee (MHREC) of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito General Hospital (KE/1240/10/2019). Additionally, written informed consent was obtained from all the participants. Discussion There was a decrease in diameter of about 10 mm in both RA and RV diameters. Interventions in the form of closure of the ASD, including both surgical and transcatheter, have good efficacy in the normalization of the right heart chamber [2–5, 14]. Previous studies showed that there was a significant decrease in both horizontal and vertical dimensions of the RA before and after ASD closure [2, 3, 15]. In addition to the normalization of the right heart chamber, there is also a functional improvement of the right heart that can be seen from the improvement of TAPSE parameters in TTE after ASD closure, and this im- provement can reduce pulmonary arterial pressure that was elevated before the closure [3, 15]. In our study, the value of pulmonary artery pressure at 6 months after ASD closure was still above 25 mmHg. A predictive model of mean pulmonary arterial pressure by Supomo et al. [7] used several parameters from TTE examination before and at 6 months after surgical clos- ure was retested in this study and showed no significant difference between mPAP from TTE and mPAP that was estimated with the predictive model (p = 0.105). The sensitivity of the diagnostic test for the development of pulmonary arterial hypertension at 6 months after ASD closure was 58% and the specificity was 62.5% by using a cut-off of 25 mmHg to define the patient’s condition as PAH [2]. Aging is a risk of PAH in ASD patients because it is a reflection of the duration of the shunt flow due to ASD [21]. The presence of shunt flow in ASD can cause an increase in pulmonary flow and has a prolonged In our study, right heart function described by TAPSE and TR velocity had significant differences between be- fore and at 6 months after ASD closure. TAPSE is the standard parameter for evaluating right ventricular func- tion quantitatively [15]. In our study, we found a de- crease in TAPSE at 6 months after surgical closure of Page 5 of 6 Page 5 of 6 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 effect on vascular hypertrophy and vasoconstriction in pulmonary arteries [21]. 9. Fraisse A, Latchman M, Sharma SR, et al. Atrial septal defect closure: indications and contra-indications. J Thorac Dis. 2018;10(24):2874–81. 11. Nashat H, Montanaro C, Li W, et al. Atrial septal defects and pulmonary arterial hypertension. J Thorac Dis. 2018;10(24):2953–65. Author details 1 f 1Department of Surgery, Thoracic, Cardiac and Vascular Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Kesehatan Street No. 1, Yogyakarta 55281, Indonesia. 2Department of Surgery, General Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia. 3Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia. Received: 15 January 2020 Accepted: 10 May 2020 Received: 15 January 2020 Accepted: 10 May 2020 Acknowledgements l f l We are also grateful to Mr. Erik from English Service Center, Universitas Gadjah Mada and highly acknowledge his contribution for proofreading of our manuscript. 11. Nashat H, Montanaro C, Li W, et al. Atrial septal defects and pulmonary arterial hypertension. J Thorac Dis. 2018;10(24):2953–65. Page 6 of 6 Supomo et al. Journal of Cardiothoracic Surgery (2020) 15:105 12. Liava’a M, Kalfa D. Surgical closure of atrial septal defects. J Thorac Dis. 2018; 10(24):2931–9. 13. Rao PS, Harris AD. Recent advances in managing septal defects: atrial septal defects. F1000Research. 2017; 6: 2042–50. 14. Balcı KG, Balcı MM. Aksoy MM, et al remodeling process in right and left ventricle after percutaneous atrial septal defect closure in adult patients. Turk Kardiyol Dern Ars Arch Turk Society of Cardiology. 2015;43(3):250–8. 14. Balcı KG, Balcı MM. Aksoy MM, et al remodeling process in right and left ventricle after percutaneous atrial septal defect closure in adult patients. Turk Kardiyol Dern Ars Arch Turk Society of Cardiology. 2015;43(3):250–8. 15. Akula VS, Durgaprasad R, Velam V, Kasala L, Rodda M, Erathi HV. Right ventricle before and after atrial septal defect device closure. Echocardiography. 2016;33(9):1381–8. 15. Akula VS, Durgaprasad R, Velam V, Kasala L, Rodda M, Erathi HV. Right ventricle before and after atrial septal defect device closure. Echocardiography. 2016;33(9):1381–8. 16. Takaya Y, Akagi T, Kijima Y, Nakagawa K, Ito H. Functional tricuspid regurgitation after transcatheter closure of atrial septal defect in adult patients: long-term follow-up. JACC Cardiovasc Interv. 2017;10(21):2211–8. 16. Takaya Y, Akagi T, Kijima Y, Nakagawa K, Ito H. Functional tricuspid regurgitation after transcatheter closure of atrial septal defect in adult patients: long-term follow-up. JACC Cardiovasc Interv. 2017;10(21):2211–8. 17. Giamberti A, Chessa M, Ballotta A, et al. Functional tricuspid valve regurgitation in adults with congenital heart disease: an emerging problem. J Heart Valve Dis. 2011;20(5):565–70. 17. Giamberti A, Chessa M, Ballotta A, et al. Functional tricuspid valve regurgitation in adults with congenital heart disease: an emerging problem. J Heart Valve Dis. 2011;20(5):565–70. 18. Chen L, Shen J, Shan X, Wang F, Kan T, et al. Improvement of tricuspid regurgitation after transcatheter ASD closure in older patients. Herz. 2018; 43(6):529–34. 19. Jain S, Dalvi B. Atrial septal defect with pulmonary hypertension: when/how can we consider closure? J Thorac Dis. 2018;10(24):2890–8. 19. Jain S, Dalvi B. Atrial septal defect with pulmonary hypertension: when/how can we consider closure? J Thorac Dis. 2018;10(24):2890–8. 20. Acknowledgements l f l D'Alto M, Romeo E, Argiento P, et al. Hemodynamics of patients developing pulmonary arterial hypertension after shunt closure. Int J Cardiol. 2013; 168(4):3797–801. 21. Yong G, Khairy P, De Guise P, et al. Pulmonary arterial hypertension in patients with transcatheter closure of secundum atrial septal defects: a longitudinal study. Circ Cardiovasc Interv. 2009;2(5):455–62. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4206030424	https://www.pnnl.gov/main/publications/external/technical_reports/PNNL-21986.pdf	English	null	Performance Assessment of Bi-Directional Knotless Tissue-Closure Devices in Juvenile Chinook Salmon Surgically Implanted with Acoustic Transmitters, 2009 - Final Report	null	2,012	public-domain	21,222	PNNL-21986 PNNL-21986 Performance Assessment of Bi-Directional Knotless Tissue- Closure Devices in Juvenile Chinook Salmon Surgically Implanted with Acoustic Transmitters, 2009 Performance Assessment of Bi-Directional Knotless Tissue- Closure Devices in Juvenile Chinook Salmon Surgically Implanted with Acoustic Transmitters, 2009 Final Report November 2012 November 2012 November 2012 PNNL-21986 Summary The purpose of this report is to assess the performance of bi-directional knotless tissue-closure devices1 for use in tagging juvenile salmon. This study is part of an ongoing effort at Pacific Northwest National Laboratory (PNNL) to reduce unwanted effects of tags and tagging procedures on the survival and behavior of juvenile salmonids, by assessing and refining suturing techniques, suture materials, and tag burdens. The objective of this study was to compare the performance of the knotless (barbed) suture, using three different suture patterns (treatments: 6-point, Wide “N”, Wide “N” Knot), to the current method of suturing (MonocrylTM monofilament, discontinuous sutures with a 2×2×2×2 knot) used in monitoring and research programs with a novel antiseptic barrier on the wound (“Second Skin”). This experiment was conducted at 12 and 17°C, which is similar to temperatures experienced in river. Seven questions were addressed in this experiment: Seven questions were addressed in this experiment: 1. Does one suture pattern and its associated needle type have a greater mortality rate as measured by the number of fish deaths per treatment group? 2. Does one suture pattern and its associated needle type yield better (i.e. greater) acoustic transmitter (AT) or passive integrated transponder (PIT) retention as measured by the number of dropped ATs or PITs? 3. Does one suture pattern and its associated needle type have a greater potential for tag loss and physiological stress as measured by incision gaping? 4. Is one suture pattern and associated needle type more functional than the others based on the number of sutures that can be identified as functioning at Site 1, Site 2, and where it applies at Site 3? 5. Does one suture pattern and its associated needle type have a greater amount of tissue trauma as measured by wound ulceration and redness? 6. Is there transmitter bulging vary with fish size or suture treatment? 7. Is fish size a confounding variable to any of the above measures? On October 14 and 15, 2009, juvenile subyearling Chinook salmon were implanted with Juvenile Salmon Acoustic Telemetry System (JSATS) micro-ATs (each 12 mm long × 5 mm wide × 4 mm high, 0.43 g in air) and PITs. Incisions were closed with either a bi-directional knotless tissue-closure device or MonocrylTM monofilament. The study was conducted at the PNNL Aquatics Research Laboratory. 1 MonodermTM, QuillTM, Angiotech Pharmaceuticals, Vancouver, BC. Performance Assessment of Bi-Directional Knotless Tissue-Closure Devices in Juvenile Chinook Salmon Surgically Implanted with Acoustic Transmitters, 2009 Final Report CM Woodley KA Wagner AJ Bryson Pacific Northwest National Laboratory Marine Sciences Laboratory Sequim, Washington 98382 Summary Test fish were examined on post-surgical days 7, 14, 21, and 28 for survival, tag loss, functional suture, incision openness, and redness and ulceration in the area of the incision. After external examination on day 28, fish were euthanized and necropsied for internal assessment of suture and tag effects. The elevated ambient water temperature, 17°C, greatly affected all factors analyzed, thus likely indicating multiplicative stress from the surgeries and the long-term holding. The categorical factors examined significantly varied among suture pattern and treatment types and were expressed differently iii between the two temperature treatments. Overall, in 12°C the performance index indicated that the Second Skin overall performed better than the other treatments, although not consistently superior across the examined factors. For the 17°C group, performance index indicated that the Wide “N” overall performed better than the other treatments, although not consistently superior across the examined factors. Both the Second Skin and Wide “N” treatments had more tags dropped than with the 6-Point treatment. For purposes of biotelemetry, in 17°C, the Second Skin treatment had the greatest gaping (days 21 and 28) and tag loss (overall), which may be of a concern when tracking juvenile Chinook in warmer water. We attribute this to the ability of the knotless (barbed) suture to maintain closure in the middle of the incision even when overall the ends of the suture were not functional. The simple interrupted suture pattern overlaid with the antiseptic does not provide structure or reliance to the middle of the incision, but rather to 1/3 and 2/3 across the incision. Conversely, the knotless (barbed) suture provided more structure initially across the whole incision as expected. However, as the suture became less functional, specifically the ends began to slide out of place; the middle section of the incision still remained closed with the device in place. Ultimately, the question remains whether bi-directional knotless tissue-closure devices are as effective as or more effective than traditional sutures for incision closure in juvenile Chinook salmon. Based on the suture retention and suture rigidity, bi-directional knotless sutures would likely be more suitable for use with larger adult fish and/or fish with large scales. Several surgery factors should be considered prior to use in field conditions. Tissue type or tissue consistency when exposed to thermal stress and suture geometry can influence retention/loss of the bi-directional knotless tissue-closure device (Ingle and King 2010; Jefferies et al. 2012). Summary When the sutures are embedded in tissue there are two primary modes of failure—peeling or bending of the barb. Peeling occurs when the barb pulls away from the suture; bending occurs when the barb pulls back without breaking off. Bent barbs remain intact attached to the suture, but will eventually release from the surrounding tissue (Ingle and King 2010). A more flexible suture, barb geometry, or even number of barbs per suture may be required for better anchoring in juvenile Chinook salmon tissue. iv Acknowledgments This project was funded by the U.S. Army Corps of Engineers (USACE), Portland, Oregon. We thank M. Brad Eppard, the USACE contracting officer’s representative, for his oversight and guidance; and Richard S. Brown as the Project Investigator for the 2009 Tag Effects Study. We also thank the following Pacific Northwest National Laboratory staff and interns for their assistance with this research: Carmina Arimescu, Jim Boyd, Scott Carpenter, Thomas Carlson, Jessica Carter, Kathleen Carter, Kate Deters, Marybeth Gay, Kasey Knox, Tyrell Monter, Eric Oldenburg, Jennifer Panther, Andy Solcz, and Mark Weiland. Lastly, we would like to thank our thorough reviewer Amoret Bunn, editor Susan Ennor, and document production specialist Kathy Neiderhiser for their excellent comments and detailed editing. Animal facilities were certified by the Association for Assessment and Accreditation of Laboratory Animal Care; animals were handled in accordance with federal guidelines for the care and use of laboratory animals, and protocols were approved by the Institutional Animal Care and Use Committee (#2009-07), Battelle–Pacific Northwest Division. Trade names referenced do not imply endorsement by the U.S. Government. v Acronyms and Abbreviations °C degree(s) Celsius (or Centigrade) AT acoustic transmitter ANOVA analysis of variance F F-test statistic FCRPS Federal Columbia River Power System FL fork length g gram(s) gal gallon(s) h hour(s) JDA John Day Dam JSATS Juvenile Salmon Acoustic Telemetry System L liter(s) g gram(s) m3 cubic meter(s) mg milligram(s) mg/L milligram(s) per liter mm millimeter(s) mm2 square millimeter(s) MS-222 tricaine methanesulfonate N replicates NaHCO3 sodium bicarbonate P p-value; probability of test statistic PIT passive integrated transponder rkm river kilometer(s) SD standard deviation SYC subyearling Chinook salmon WW wet weight vii Contents Summary ............................................................................................................................................... iii Acknowledgments ................................................................................................................................. v Acronyms and Abbreviations ............................................................................................................... vii 1.0 Introduction .................................................................................................................................. 1.1 1.1 Background .......................................................................................................................... 1.1 1.2 Purpose and Scope ............................................................................................................... 1.3 1.3 Report Contents and Organization ....................................................................................... 1.4 2.0 Methods and Materials ................................................................................................................. 2.1 2.1 Fish Acquisition and Fish Maintenance ............................................................................... 2.1 2.2 Suture Pattern Mechanics ..................................................................................................... 2.1 2.3 Surgical Procedures .............................................................................................................. 2.4 2.4 Response Examinations ........................................................................................................ 2.5 2.5 Statistical Analysis ............................................................................................................... 2.7 3.0 Results .......................................................................................................................................... 3.1 3.1 Fish Size ............................................................................................................................... 3.1 3.2 Mortalities ............................................................................................................................ 3.3 3.3 Tag Loss ............................................................................................................................... 3.3 3.4 Incision Openness ................................................................................................................ 3.4 3.5 Functional Suture ................................................................................................................. 3.7 3.6 Ulceration and Redness ........................................................................................................ 3.10 3.7 Transmitter Bulge ................................................................................................................. 3.16 3.8 Performance Index ............................................................................................................... 3.16 4.0 Discussion ..................................................................................................................................... 4.1 5.0 References .................................................................................................................................... 5.1 ix Figures 3.6 3.7 Total Incision Openness Plotted Against the Wet Weight of Individual Fish, Day 7 at 17°C ............................................................................................................................................ 3.7 3.8 Example of Ulceration and Redness Caused by Suture Tearing ................................................. 3.10 3.9 Total Ulceration by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C....................... 3.11 3.10 Total Ulceration by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C....................... 3.12 3.11 Total Redness by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C .......................... 3.14 3.12 Total Redness by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C .......................... 3.15 4.1 Photos Taken from Day 7 Response Examinations that Show Ulceration and Redness Often Associated with the Barbed Suture Using the Wide “N” and 6-Point Suture Patterns ........................................................................................................................................ 4.4 Tables 2.1 Suture Patterns and Replicates of SYC in Each Treatment ........................................................ 2.2 2.2 Sample Sizes for Each Treatment at 12°C by Sample Day......................................................... 2.6 2.3 Sample Sizes for Each Treatment at 17°C by Sample Day......................................................... 2.7 3.1 Mortality Frequency for Fish in Each Treatment Group ............................................................ 3.3 3.2 AT Retention for Each Treatment ............................................................................................... 3.3 3.3 Incision Openness on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature ................................................................................................................................ 3.5 3.4 Number of Functional Sutures by Entry/Exit Point for Each Treatment at 12°C ....................... 3.8 3.5 Number of Functional Sutures by Entry/Exit Point for Each Treatment at 17°C ....................... 3.9 3.6 Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12°C ................. 3.11 Figures 1.1 Knotless Suture Geometry .......................................................................................................... 1.3 2.1 Schematic of the Four Suture Patterns ........................................................................................ 2.3 2.2 Day 0 Suture Patterns Demonstrating the Final Product of the 6-Point, Wide “N”, and the Second Skin ........................................................................................................................... 2.4 2.3 Wound Redness and Ulceration Differentiation ......................................................................... 2.6 3.1 Wet Weights by Fork Lengths of all Study Fish ......................................................................... 3.1 3.2 Wet Weights of Study Fish for Each Treatment at 12°C ............................................................ 3.2 3.3 Wet Weights of Study Fish for Each Treatment at 17°C ............................................................ 3.2 3.4 Total Incision Openness by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C ................................................................................................................................ 3.5 3.5 Total Incision Openness Plotted Against the Wet Weight of Individual Fish, Day 7 at 12°C ............................................................................................................................................ 3.6 3.6 Total Incision Openness by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 17°C ................................................................................................................................ 3.6 3.7 Total Incision Openness Plotted Against the Wet Weight of Individual Fish, Day 7 at 17°C ............................................................................................................................................ 3.7 3.8 Example of Ulceration and Redness Caused by Suture Tearing ................................................. 3.10 3.9 Total Ulceration by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C....................... 3.11 3.10 Total Ulceration by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C....................... 3.12 3.11 Total Redness by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C .......................... 3.14 3.12 Total Redness by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C .......................... 3.15 4.1 Photos Taken from Day 7 Response Examinations that Show Ulceration and Redness Often Associated with the Barbed Suture Using the Wide “N” and 6-Point Suture Patterns ........................................................................................................................................ 4.4 2.1 Schematic of the Four Suture Patterns ........................................................................................ 2.3 2.2 Day 0 Suture Patterns Demonstrating the Final Product of the 6-Point, Wide “N”, and the Second Skin ........................................................................................................................... 2.4 2.3 Wound Redness and Ulceration Differentiation ......................................................................... 2.6 3.1 Wet Weights by Fork Lengths of all Study Fish ......................................................................... 3.1 3.2 Wet Weights of Study Fish for Each Treatment at 12°C ............................................................ 3.2 3.3 Wet Weights of Study Fish for Each Treatment at 17°C ............................................................ 3.2 3.4 Total Incision Openness by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C ................................................................................................................................ 3.5 3.5 Total Incision Openness Plotted Against the Wet Weight of Individual Fish, Day 7 at 12°C ............................................................................................................................................ 3.6 3.6 Total Incision Openness by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 17°C ................................................................................................................................ 1.0 Introduction Current acoustic telemetry studies require invasive surgical techniques for transmitter implantation. Ongoing efforts have focused on reducing this invasiveness to address telemetry and survival model assumptions. Prior research has indicated that suture material and technique can be destructive to fish tissue, externally and internally (Wagner et al. 2000; Deters et al. 2010). Recovery from surgery, including an upregulated immune system response to tissue damage, may result in the “tagged” fish (“tagged” herein referring to a fish that underwent surgical intracoelomic implantation) not being equivalent to or representative of the population of interest due to an altered physical and physiological state of the tagged fish. Researchers at the Pacific Northwest National Laboratory (PNNL) have been conducting research on suturing techniques, suture materials, and tag burdens in an effort to reduce the unwanted effects of tags and tagging procedures (Deters et al. 2010; Panther et al. 2010; Carter et al. 2011; Cooke et al. 2011). In 2009, we began investigating the knotless (barbed) suture and suture patterns to determine if this technique is a viable alternative to the current method (MonocrylTM monofilament, discontinuous sutures with a 2×2×2×2 knot) used in river monitoring and research programs. On October 14 and 15, 2009, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha; SYC) were implanted with Juvenile Salmon Acoustic Telemetry System (JSATS) micro-acoustic transmitters (ATs; each 12 mm long × 5 mm wide × 4 mm high, 0.43 g in air), and passive integrated transponders (PITs). A bi-directional knotless tissue-closure device (MonodermTM, QuillTM, Angiotech Pharmaceuticals, Vancouver, BC) was used to close the incision. In this study, the effects of three suture patterns using the barbed suture material were examined over 28 days and compared to the currently accepted method of wound closure with a “Second Skin” (CavilonTM, No-Sting BarrierTM, 3M, St. Paul, MN) applied over the wound. The study was conducted at PNNL’s Aquatics Research Laboratory in Richland, Washington. Test fish were examined for suture and tag effects on post-surgical days 7, 14, 21, and 28. Fish were also examined for internal assessment of suture and tag effects on Day 28. Tables 2.1 Suture Patterns and Replicates of SYC in Each Treatment ........................................................ 2.2 2.2 Sample Sizes for Each Treatment at 12°C by Sample Day......................................................... 2.6 2.3 Sample Sizes for Each Treatment at 17°C by Sample Day......................................................... 2.7 3.1 Mortality Frequency for Fish in Each Treatment Group ............................................................ 3.3 3.2 AT Retention for Each Treatment ............................................................................................... 3.3 3.3 Incision Openness on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature ................................................................................................................................ 3.5 3.4 Number of Functional Sutures by Entry/Exit Point for Each Treatment at 12°C ....................... 3.8 3.5 Number of Functional Sutures by Entry/Exit Point for Each Treatment at 17°C ....................... 3.9 3.6 Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12°C ................. 3.11 x 3.7 Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 17°C ................. 3.12 3.8 Frequency and Mean Area of Redness for Each Treatment at 12°C .......................................... 3.13 3.9 Frequency and Mean Area of Redness for Each Treatment at 17°C .......................................... 3.14 3.10 Performance Index Based on Rank of Each Measured Treatment Observation at 12°C ............ 3.17 3.11 Performance Index Based on Rank of Each Measured Treatment Observation at 17°C ............ 3.18 xi 1.1 Background Telemetry applications for fish range from monitoring fine spatial movements and habitat preferences to monitoring large-scale migratory patterns and passage survival (Skalski 1998; Scruton et al. 2007). In the Columbia and Snake rivers, scientists have identified acoustic telemetry as an essential technology for observing behavior and estimating survival of juvenile salmonids passing through the main-stem Federal Columbia River Power System (FCRPS) and associated side channels (Faber et al. 2001; McComas et al. 2005; Ploskey et al. 2008; Clemens et al. 2009). Hydroelectric dams provide various routes of passage where mortality becomes pathway-specific depending on the physical properties of the technical installation; i.e., route through turbines, spillways, bypass structures, etc. (Coutant and Whitney 2000; Muir et al. 2001; Skalski et al. 2002; Weiland et al. 2009). In addition, impoundments and passage facilities may delay juvenile salmonid outmigration, conceivably increasing exposure to predators and contributing to disease. Because of the direct and indirect threats to salmonids caused by impoundments, telemetry and survival models are used to monitor passage. Both telemetry and survival models, though, assume tagged animals (whether external or internally implanted devices are used) to be representative of the population under evaluation; and not to exhibit behavioral, physiological, or survival differences when compared to the untagged populations. 1.1 Acoustic transmitters (ATs), when used in fish survival studies, are often surgically implanted into the coelomic cavity of the fish. Surgical implantation is a well-established method for studying fish movements and survival through structures, but this technique has disadvantages (Bridger and Booth 2003; Bauer and Loupal 2007; Chittenden et al. 2009; Frost et al. 2010; Gheorghiu et al. 2010). The tag or the surgical procedure may potentially alter the behavior, growth or survival of the fish (LaCroix et al. 2004; Chittenden et al. 2009; Stephenson et al. 2010). In addition, transmitter loss (or shedding) can occur due to foreign body rejection response (often referred to as “tag expulsion”), poor tissue apposition causing the transmitter to exit the incision (Panther et al. 2010), or application of external mechanical forces, such as pressure (Stephenson et al. 2010). If transmitters are expelled, a false mortality rate occurs; or if the tagging process decreases fish fitness or contributes to mortality, fish are no longer representative of the population under investigation. 1.1 Background Poor surgical procedures, including prolonged exposure to anesthetic (Congleton 2006; Rombough 2007), “unsanitary” conditions1 (Harms 2005; Leaper 2010), poor surgical techniques resulting in tissue trauma or incision gaping (Fontenot and Neiffer 2004; Harms 2005), or inefficient post-implantation recovery time (Harms 2005) can result in altered behavior, growth, and/or survival. After insertion of a telemetry device (e.g., an AT) into the coelomic cavity of a fish, the incision must be closed to prevent transmitter expulsion and pathogen entry, minimize changes in physiological state caused by osmotic stress, and support tissue healing (Jepsen et al. 2002; Mulcahy 2003). Based on prior research, synthetic monofilaments may elicit less tissue inflammation and promote more rapid incision healing than silk sutures (Cooke et al. 2003; Jepson 2008; Deters et al. 2009). For example, rainbow trout (O. mykiss) experienced less tissue inflammation from synthetic monofilament than from braided silk sutures (Wagner et al. 2000). Similarly, Deters et al. (2010) found that wound inflammation and ulceration were generally lower with synthetic monofilament compared to braided sutures in yearling juvenile Chinook (held at water temperatures of 12 and 17°C). As a result of studies like these, the Columbia Basin Surgical Protocol Steering Committee has recommended the use of absorbable synthetic monofilament suture material tied in a simple interrupted suture pattern for closing surgical incisions in fish (CBSPSC 2011). Wound closure in fish is a process involving several actions to produce a functional suture. A functional suture is defined as a suture in the fish that is knotted, has appropriate tension across the wound, and does not tear through the body wall of the fish (modified from Deters et al. 2009). Non- functional sutures result in slow tissue healing, osmotic stress, tissue damage, or possible premature mortality (Fontenot and Neiffer 2004; Harms 2005; Greenburg and Clark 2009). Ideally, the suture material should be placed in the tissue so that the incision margins are and remain approximated, thereby minimizing open spaces and aiding in healing (Lin et al. 1996; Wagner et al. 2000; Bridger and Booth 2003; Fontenot and Neiffer 2004). Excessive suture tension on tissue can cause ischemic areas that reduce or slow revascularization; increase stretching, tearing, and necrosis; and ultimately slow healing. Improperly tied knots can become untied, thereby releasing wound margins, slowing healing, and allowing transmitter loss. 1 Aseptic or sterile surgeries are not feasible because a fish’s mucous coat (barrier) is its first line of defense and should not be compromised. Surgical scrubs and disinfectants used on terrestrial animals could harm or degrade the mucous barrier and/or damage the skin and gills of fish. However, PNNL surgeries are conducted in a manner to be as “aseptic as possible.” 1.1 Background A) Knotless suture region where barbs transition from one direction to the other (accessed Angiotech March 15, 2011; http://www.angioedupro.com/Quill/index.php?ID=Photos). B) Individual barbs compared to the main suture shaft (photo credit and description Leung 2003). Figure 1.1. Knotless Suture Geometry. A) Knotless suture region where barbs transition from one direction to the other (accessed Angiotech March 15, 2011; http://www.angioedupro.com/Quill/index.php?ID=Photos). B) Individual barbs compared to the main suture shaft (photo credit and description Leung 2003). 1.1 Background Large knots can be a point source for tissue irritation due to the concentrated amount of foreign material making up the knot (van Rijssel et al. 1989). Functional sutures and practices 1 Aseptic or sterile surgeries are not feasible because a fish’s mucous coat (barrier) is its first line of defense and should not be compromised. Surgical scrubs and disinfectants used on terrestrial animals could harm or degrade the mucous barrier and/or damage the skin and gills of fish. However, PNNL surgeries are conducted in a manner to be as “aseptic as possible.” 1.2 to reduce tissue damage are needed to ensure the retention of intracoelomic transmitters, and reduce any behavioral or physiological differences between tagged fish and run-of-the-river populations. Currently, a novel bi-directional knotless tissue-closure device (MonodermTM, QuillTM, Angiotech Pharmaceuticals, Vancouver, BC) has been shown to streamline wound closure and decrease healing time. Knotless tissue-closure devices are easy to handle, reduce instrument handling and surgical time, enable the use of continuous stitching rather than interrupted sutures and knots, and most importantly provide uniformly distributed tension across the wound rather than at specific entry and exits points of the suture coming through the tissue (Sadick et al. 1994; Shermak et al. 2009). Similar to synthetic absorbable monofilament, MonodermTM is an absorbable monofilament (i.e., the copolymer material degrades in vivo over time). Degradation occurs by hydrolysis of the ester links in the polymer backbone, until dissolution and full absorption occurs (Angiotech 2011). QuillTM tissue-closure devices are based on the reconstruction of a traditional suture material where the suture has tissue retainers (barbs) arranged around the shaft that protrude at ~45° from the main suture shaft (Figure 1.1). Tissue retainers allow the suture to be pulled through the tissue, and then anchor itself, much like a porcupine quill or stingray barb, eliminating the need for a knot. Once anchored, the barbs distribute the suture tension across a larger area minimizing ischemic pressure points. The knotless design eliminates the potential for unraveling, and reduces the amount of foreign material against the tissue, which can cause irritation and allow fungal and bacterial growth. Figure 1.1. Knotless Suture Geometry. A) Knotless suture region where barbs transition from one direction to the other (accessed Angiotech March 15, 2011; http://www.angioedupro.com/Quill/index.php?ID=Photos). B) Individual barbs compared to the main suture shaft (photo credit and description Leung 2003). Figure 1.1. Knotless Suture Geometry. 1.2 Purpose and Scope The objective of the study reported herein was to assess the performance of the bi-directional knotless tissue-closure device in relation to a currently accepted technique for wound closure in juvenile salmon. SYC were implanted with a JSATS AT and PIT, and the incisions were closed with separate treatments consisting of four suture patterns; three patterns using the knotless suture material and one suture pattern using MonocrylTM monofilament and covered with “Second Skin” (further described below). This study 1.3 was conducted at water temperatures of 12 and 17°C, which are similar to those experienced in river. The wounds and suture performance were examined on 7, 14, 21, and 28 days post-surgery for suture loss, incision openness, redness, and ulceration in the area of the incision. The fish were continuously monitored for moribund behavior or mortalities and/or tag loss. On the 28th day post-implantation, fish were euthanized and necropsied to confirm the presence of the AT, PIT, and sutures, and to quantify the internal effects of tagging. Seven questions were addressed in this experiment: Seven questions were addressed in this experiment: 1. Does one suture pattern and its associated needle type have a greater mortality rate as measured by the number of fish deaths per treatment group? 2. Does one suture pattern and its associated needle type yield higher AT or PIT retention as measured by the number of dropped ATs or dropped PITs? 3. Does one suture pattern and its associated needle type have a greater potential for tag loss and physiological stress as measured by incision gaping? 4. Is one suture pattern and associated needle type more functional than the others based on the number of sutures that can be identified as functioning at Site 1, Site 2, and where it applies at Site 3? 5. Does one suture pattern and its associated needle type have a greater amount of tissue trauma as measured by wound ulceration and redness? 6. Is there transmitter bulging vary with fish size or suture type? 7. Is fish size a confounding variable? 2.1 Fish Acquisition and Fish Maintenance Subyearling Chinook salmon (N = 583) raised at the Priest Rapids hatchery and transferred to PNNL (Richland, WA) were used for this study. Fish were randomly sorted into one of two water temperature treatments; 12 or 17°C and held on a 12-h light to 12-h dark photoperiod during the acclimation (a 3°C change over 3 days) and experimental periods. Fish were housed in two 890-L circular fiberglass tanks supplied with aerated well water during acclimation for temperature treatments. Fish were fed Biodiet pellets (Bio-Oregon, Inc., Longview, WA) daily at a rate of 1.1% of their body weight. Food was restricted 24 h prior to and 6 h after surgery or weekly exams. SYC were observed several times daily to determine if there were injuries, abnormal behavior, or mortalities. Tanks were siphoned daily to remove fecal matter and debris and to recover any ATs or PITs that may have been shed. Each tank outflow was fitted with a net bag to prevent shed tags from being lost. All methods were approved by the Institutional Animal Care and Use Committee (IACUC Protocol 2009-07). 2.0 Methods and Materials This study, conducted over 29 days in fall 2009, involved fish acquisition, surgical implantation of ATs and PITs, examination of responses to implantation, and statistical analyses, as described below. 1.3 Report Contents and Organization The ensuing sections of this report describe the study methods and materials (Section 2.0), results (Section 3.0), and discussion (Section 4.0). References for sources cited in the text are listed in Section 5.0. 1.4 2.2 Suture Pattern Mechanics • Control. These fish underwent the same handling procedure as treatment fish but were not surgically implanted. These fish were used to gauge mortality rates between treatment groups. • Control. These fish underwent the same handling procedure as treatment fish but were not surgically implanted. These fish were used to gauge mortality rates between treatment groups. . Suture Patterns and Replicates of SYC in Each Treatment. All needles were 3/8 circle diamond point with an 18-mm circumference. Table 2.1. Suture Patterns and Replicates of SYC in Each Treatment. All needles were 3/8 circle diamond point with an 18-mm circumference. Table 2.1. Suture Patterns and Replicates of SYC in Each Treatment. All needles were 3/8 circle diamond point with an 18-mm circumference. Temperature Treatment Knots Used Number of Entry and Exit Points Sample Size 12°C 6-Point 0 3 65 Wide “N” 0 2 67 Wide “N” Knot 1 2 66 Second Skin 2 2 66 Control NA NA 22 17°C 6-Point 0 3 69 Wide “N” 0 2 66 Wide “N” Knot 1 2 71 Second Skin 2 2 69 Control NA NA 22 Depending on the suture pattern, there are several entry and exit points. The 6-Point suture pattern shown in Figure 2.1A has two points where the needle entered the skin (point E1, E2) and four points where it exited the skin (points X1, X2, X3, X4). The first exit came from the needle that was passed into the cavity via the incision, exiting at point X1 until the middle point of the barbed suture was halfway through the exit point. Next, the surgeon used the internal portion of the suture to exit at point X2, cutting the suture 3 mm from the exit point (i.e., leaving a 3-mm tail). The suture remaining outside of exit point X1 extended across the wound and entered the tissue at entry point E1. The needle passed into the body cavity at point E1 and extended across the wound exiting at point X3, before extending across the wound and entering at point E2 and exiting at point X4. The excess suture at point X4 was cut leaving a 3-mm tail (Figure 2.1A). The Wide “N” pattern (Figure 2.1B) has four entry and exit points with wider suture angles across the wound than those of the 6-Point treatment. 2.2 Suture Pattern Mechanics On the day of surgery, fish were assigned randomly to one of five treatments. All surgical treatments were performed using a 3/8 circle diamond point needle with 18-mm circumference. Treatment groups were as follows (Table 2.1): • 6-Point Continuous Suture treatment (herein referred to as “6-Point”). This pattern had smaller angles across the incision and more insertion points than other treatments. The first point of insertion was in the middle of the incision, and involved pulling the suture through opposing sides and ensuring the barbs were anchored in both directions. • 6-Point Continuous Suture treatment (herein referred to as “6-Point”). This pattern had smaller angles across the incision and more insertion points than other treatments. The first point of insertion was in the middle of the incision, and involved pulling the suture through opposing sides and ensuring the barbs were anchored in both directions. • Wide “N” Continuous Suture treatment (herein referred to as Wide “N”). This pattern had wider angles across the incision and fewer insertion points than the 6-Point treatment. The first point of insertion was in the middle of the incision and involved pulling the suture through the opposing sides and ensuring barbs were anchored in both directions. • Wide “N” Knot Suture treatment (herein referred to as Wide “N” Knot). This pattern had the same angles across the wound as the Wide “N” treatment. This technique used a small knot at the end of the suture rather than no knots as with Wide “N”. Barbs gripped in one direction, opposite the knot. Single square knots were used and placed on the suture prior to use. This technique is faster than placing a knot using a traditional suture and eliminates tissue tearing caused by knot and suture tension. • Two simple interrupted sutures were secured using a 2×2×2×2 knot pattern with MonocrylTM monofilament and Second Skin applied over the wound (herein referred to as Second Skin). This is the currently accepted technique for wound closure. Second Skin is a product used to create a 2.1 fast-drying, non-sticky barrier film that forms a breathable, transparent coating on the skin. The film is hypoallergenic, non-cytotoxic, and will not sting even when applied to damaged or denuded human skin. fast-drying, non-sticky barrier film that forms a breathable, transparent coating on the skin. The film is hypoallergenic, non-cytotoxic, and will not sting even when applied to damaged or denuded human skin. 2.2 Suture Pattern Mechanics The needle entered through the incision, exiting the skin at point X1 until the middle point of the barbed suture was halfway through the skin. Next, the surgeon used the internal piece of suture to exit at point X2, and the remaining suture was cut 3 mm from the entry point, leaving a 3-mm tail. The remaining suture outside of exit point X1 extended across the wound and entered the tissue at entry point E1. The needle was passed back into the body cavity at point E1 and extended across the wound at point X3. The excess suture at point X3 was cut leaving a 3-mm tail (Figure 2.2B). 2.2 . Schematic of the Four Suture Patterns. A) 6-Point, B) Wide “N”, C) Wide “N” Knot, an D) Second Skin. Brown dashed lines represent internal suture areas. Brown solid lines represent external suture areas. Brown curved lines represent the needle. The brown so dot is the knot tied for the Wide “N” Knot and Second Skin treatments. Black dotted lin section the incision into points for description purposes. The 6-Point pattern has three points, and the Wide “N”, Wide “N” Knot, and Second Skin have two points. The arrow indicate the point of first insertion. Letters indicate entry (E1-E2) and exit (X1-X4) sites. Figure 2.1. Schematic of the Four Suture Patterns. A) 6-Point, B) Wide “N”, C) Wide “N” Knot, and D) Second Skin. Brown dashed lines represent internal suture areas. Brown solid lines represent external suture areas. Brown curved lines represent the needle. The brown solid dot is the knot tied for the Wide “N” Knot and Second Skin treatments. Black dotted lines section the incision into points for description purposes. The 6-Point pattern has three points, and the Wide “N”, Wide “N” Knot, and Second Skin have two points. The arrows indicate the point of first insertion. Letters indicate entry (E1-E2) and exit (X1-X4) sites. Figure 2.1. Schematic of the Four Suture Patterns. A) 6-Point, B) Wide “N”, C) Wide “N” Knot, and D) Second Skin. Brown dashed lines represent internal suture areas. Brown solid lines represent external suture areas. Brown curved lines represent the needle. The brown solid dot is the knot tied for the Wide “N” Knot and Second Skin treatments. Black dotted lines section the incision into points for description purposes. 2.2 Suture Pattern Mechanics The 6-Point pattern has three points, and the Wide “N”, Wide “N” Knot, and Second Skin have two points. The arrows indicate the point of first insertion. Letters indicate entry (E1-E2) and exit (X1-X4) sites. 2.3 The Wide “N” Knot (Figure 2.1C) pattern used one segment of the suture with a knot tied at the end, denoted by the circle at point E1. The needle passed through the body wall into the cavity at point E1, exiting at point X1, and the suture was pulled until the knot met the fish scales at point E1. The needle was passed back into the body cavity at point E2 and extended across the wound, exiting at point X1. Excess suture was cut at point X2. The Second Skin pattern (Figure 2.1D) used two simple interrupted sutures with a knot for each tied in a 2×2×2×2 knot pattern. The 2×2×2×2 knot pattern consisted of four double throws in alternating directions. The needle made two separate entry (E1 and E2) and two separate exits (X1 and X2) and put pressure on two areas of the wound rather than evenly across the wound. Figure 2.2. Day 0 Suture Patterns Demonstrating the Final Product of the 6-Point (A, left photo), Wide “N” (B, center photo), and the Second Skin (C, right photo). The Wide “N” and Wide “N” Knot suture patterns have a similar pattern with larger angles between sutures and fewer entry/exit points (photo B) than the 6-Point pattern (see Section 2.2, Figure 2.1 for pattern mechanics).1 A B C A C B A B Figure 2.2. Day 0 Suture Patterns Demonstrating the Final Product of the 6-Point (A, left photo), Wide “N” (B, center photo), and the Second Skin (C, right photo). The Wide “N” and Wide “N” Knot suture patterns have a similar pattern with larger angles between sutures and fewer entry/exit points (photo B) than the 6-Point pattern (see Section 2.2, Figure 2.1 for pattern mechanics).1 1 The suture ends are longer in the photos to be visible to the reader; the ends should be no longer than 3 mm. 2.4 Response Examinations All fish were examined 7, 14, 21, and 28 days post-surgery (herein referred to as Day 7, Day 14, Day 21, and Day 28). Each fish was anesthetized with 80 mg/L of MS-222 for examination. Fish were removed from the bath, fork length (FL; mm) and wet weight (WW; g) were measured, and the fish were placed on a foam pad, ventral side up. Maintenance anesthetic of up to 40 mg/L of MS-222 was supplied to the fish in the same manner as for surgery. The incision, suture, and surrounding area were examined through a stereomicroscope (0.65× magnification; Stemi 2000-CS; Zeiss AG, Jena, Germany) connected to a computer for photographing wounds. The incision area was partitioned into paired suture points, i.e., having an entry and exit point pair (Figure 2.1). The 6-Point configuration had three points, while the Wide “N”, Wide “N” Knot, and Second Skin had two points each. On days 7, 14, 21, and 28, the presence of suture material was noted for each point and marked as a binary response (present “1” or absent “0”), and for suture tension consistency (yes or no). The area of redness, ulceration, and incision openness (mm2) were outlined and quantified using the “ImageJ” image processing program (public domain software, National Institute of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/; Figure 2.3). If there was more than one area on the fish with either redness or ulceration, individual measurements were summed for the analyses. Sutures were deemed non-functional if they were absent or lacked tension to properly close the incision. Redness was differentiated from ulceration by the consistency of the wound and area affected. Redness scores would include erythema (pink area in Figure 2.3), not ulcerations (maroon-hashed area, Figure 2.3). Ulceration scores would include the maroon-hashed area (inner circles in A and B, Figure 2.3) but exclude redness (pink area, Figure 2.3). Redness and/or ulcerations, if more than one occurrence per fish, would be summed (i.e., redness in Figure 2.3, two area would be summed into a single value). This approach allowed for the distinction between red inflamed areas and areas with exposed underlying tissue. Throughout the study, fish were randomly sacrificed for future histological examination. These fish were removed from any further analyses. All other fish were necropsied after the Day 28 examination. On Day 28, suture presence was noted (present or absent), as well as any tag bulging. 2.3 Surgical Procedures Surgeries on SYC were split into 2 days: October 14 and 15, 2009. Three surgeons performed all surgeries. Fish were anesthetized and handled similarly regardless of treatment. A buffered anesthetic (with 80 mg/L NaHCO3) was prepared using aerated well water and tricaine methanesulfonate (MS-222; 80 mg/L). Prior to surgery, fish were anesthetized in buckets until loss of equilibrium was observed (Stage 4; Summerfelt and Smith 1990). Anesthetized fish were immediately weighed (WW; g), measured (FL, mm), and both flanks were photographed. Water temperature was monitored and new water was acquired if the temperature varied more than 2°C from the respective experimental temperature—12 or 17°C. Fish were randomly assigned to one of five treatment groups: 6-Point, Wide “N”, Wide “N” Knot, Second Skin, or Control. All suture treatment groups underwent surgical implantation, while the Control fish bypassed the surgery stations, and then were placed into 5-gal perforated recovery buckets (5 fish per bucket), aerated with well water, and monitored during recovery from anesthesia. 2.4 Fish receiving surgical implants (PITs and ATs) were placed on the surgery table and given a maintenance anesthetic dose (well water containing 40 mg/L of MS-222) through silicone rubber tubing from a gravity-fed bucket. Each surgeon controlled the dose during the procedure by mixing well water with the maintenance anesthetic water. With the fish ventral side up, a 5- to 7-mm incision was made along the linea alba, between the pectoral fin and pelvic girdle. Incisions were closed using an absorbable bi-directional knotless monofilament tissue-closure device (MonodermTM, QuillTM, Angiotech Pharmaceuticals, Vancouver, BC) or MonocrylTM monofilament. The suture patterns and approach for insertion are described in Section 2.2, Suture Patterns. After surgery, a photo was taken of the closed incision and fish were placed in fresh aerated water to recover. Once the fish regained equilibrium they were placed in one of two circular tanks and provided with flow-through well water. Over the experimental period, water temperatures fluctuated within one degree of the desired temperatures, 12 or 17°C. 2.4 Response Examinations At the end of the study, all characteristics were ranked to give an overall performance index for each treatment (1 = best 2.5 and 4 = worst). See Table 2.2 and Table 2.3 for a full list of sample sizes for incision openness, ulceration, and redness analyses with fish removed for mortalities or histological examination. Figure 2.3. Wound Redness and Ulceration Differentiation. A) Only the pink area outer ring would be included in the redness score. Any redness in the ulcerated area was included in the ulceration score, not the redness score. B) The redness score would include the pink areas for each noted wound or affected area by adding the total pink areas together. Ulcerations, if more than one, would be summed similarly. Figure 2.3. Wound Redness and Ulceration Differentiation. A) Only the pink area outer ring would be included in the redness score. Any redness in the ulcerated area was included in the ulceration score, not the redness score. B) The redness score would include the pink areas for each noted wound or affected area by adding the total pink areas together. Ulcerations, if more than one, would be summed similarly. Table 2.2. Sample Sizes for Each Treatment at 12°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Table 2.2. Sample Sizes for Each Treatment at 12°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Table 2.2. Sample Sizes for Each Treatment at 12°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. 2.4 Response Examinations The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Table 2.3. Sample Sizes for Each Treatment at 17°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Table 2.3. Sample Sizes for Each Treatment at 17°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Day 6-Point Wide “N” Wide “N” Knot Second Skin Total Day 7 Mortality 8 5 7 6 26 Histology 0 0 0 0 0 N 61 61 64 62 248 Day 14 Mortality 10 9 4 9 32 Histology 1 2 2 1 6 N 49 49 58 53 209 Day 21 Mortality 14 9 14 11 48 Histology 1 3 2 2 8 N 34 37 42 39 152 Day 28 Mortality 6 5 6 6 23 Histology 2 1 2 2 7 N 26 31 34 31 122 2.4 Response Examinations Day 6-Point Wide “N” Wide “N” Knot Second Skin Total Day 7 Mortality 2 1 2 1 6 Histology 0 0 0 0 0 N 63 66 64 65 258 Day 14 Mortality 0 1 0 0 1 Histology 2 2 2 2 8 N 61 63 62 63 249 Day 21 Mortality 0 0 0 0 0 Histology 2 2 2 2 8 N 59 61 60 61 241 Day 28 Mortality 0 0 0 0 0 Histology 1 1 1 1 4 N 58 60 59 60 237 2.6 2.6 Table 2.3. Sample Sizes for Each Treatment at 17°C by Sample Day. The table includes the number of fish removed from statistical analyses for mortalities or later histological analyses. Sample sizes were used for statistical analysis of incision openness, ulceration, and redness. Day 6-Point Wide “N” Wide “N” Knot Second Skin Total Day 7 Mortality 8 5 7 6 26 Histology 0 0 0 0 0 N 61 61 64 62 248 Day 14 Mortality 10 9 4 9 32 Histology 1 2 2 1 6 N 49 49 58 53 209 Day 21 Mortality 14 9 14 11 48 Histology 1 3 2 2 8 N 34 37 42 39 152 Day 28 Mortality 6 5 6 6 23 Histology 2 1 2 2 7 N 26 31 34 31 122 2.5 Statistical Analysis Categorical covariates included four suture treatments (6-Point, Wide “N”, Wide “N” Knot, and Second Skin), four exam days (Day 7, Day 14, Day 21, and Day 28), and two holding temperatures (12 and 17°C). The response variables—mortality, tag retention, and functional suture (suture presence and tension)—at exam day and at necropsy were treated as binomial data because the variable could either be present or absent in each fish. The variables redness, ulceration, and openness were continuous data. For questions 1, 2, 4, and 6 (Section 1.1), the response variable was categorical. For these questions, a Chi Squared Test (χ2) was used to test for an association between the four suture treatments and the categorical response variable. For question 7, the response variable was continuous, so analysis of variance (ANOVA) was used to test for differences between treatments. For question 3 and 5, the response variable was measured as a categorical and continuous response, so both χ2 and ANOVA were used. Table 2.3. Sample Sizes for Each Treatment at 17°C by Sample Day. 2.5 Statistical Analysis Categorical covariates included four suture treatments (6-Point, Wide “N”, Wide “N” Knot, and Second Skin), four exam days (Day 7, Day 14, Day 21, and Day 28), and two holding temperatures (12 and 17°C). The response variables—mortality, tag retention, and functional suture (suture presence and tension)—at exam day and at necropsy were treated as binomial data because the variable could either be present or absent in each fish. The variables redness, ulceration, and openness were continuous data. For questions 1, 2, 4, and 6 (Section 1.1), the response variable was categorical. For these questions, a Chi Squared Test (χ2) was used to test for an association between the four suture treatments and the categorical response variable. For question 7, the response variable was continuous, so analysis of variance (ANOVA) was used to test for differences between treatments. For question 3 and 5, the response variable was measured as a categorical and continuous response, so both χ2 and ANOVA were used. 2.7 3.0 Results Fish size and temperature effects related to suture pattern or type outcomes are described in the following sections. Mortality rate, AT or PIT retention, incision openness (gaping), suture functionality, occurrence of redness and/or ulceration, and tag bulging are considered and ranked according to a performance index. 3.1 Fish Size For all SYC, FL was a significant predictor of WW (N = 580, F(1, 578) = 2708.48, P < 0.0001). The linear relationship between FL and WW can be described as WW = -33.44679 + 0.4476421*FL (R2 = 0.83; Figure 3.1). The SYC FL ranged from 96 to 117 mm (x̄ =107.8 ± 4.8) and the WW ranged from 9.3 to 21.3 (x̄ = 14.8 ± 2.4 g). The WW was significantly higher for fish in the 12°C treatment compared to the 17°C treatment (N = 580, F(1, 578) = 115.83, P < 0.0001). WW did not significantly vary with suture treatment in the 12°C treatment (N = 284, F(4, 279) = 1.82, P = 0.1247; Figure 3.2) or 17°C treatment (N = 296, F(4, 291) = 0.30, P = 0.8758; Figure 3.3), so fish could be pooled for the following analyses. Fork Length (mm) 90 95 100 105 110 115 120 Wet Weight (g) 8 10 12 14 16 18 20 22 FL (mm) vs WW (g) Regression Line Figure 3.1. Wet Weights (g) by Fork Lengths (mm) of all Study Fish. Each filled circle (●) represents an individual fish at the beginning of the study. Fork Length (mm) 90 95 100 105 110 115 120 Wet Weight (g) 8 10 12 14 16 18 20 22 FL (mm) vs WW (g) Regression Line Figure 3.1. Wet Weights (g) by Fork Lengths (mm) of all Study Fish. Each filled circle (●) represents an individual fish at the beginning of the study. 3.1 6 Point Wide "N" Wide "N" Knot Second Skin Control Wet Weight (g) 8 10 12 14 16 18 20 22 Figure 3.2. Wet Weights (g) of Study Fish for Each Treatment at 12°C. Each box represents the median and upper and lower quartiles for study fish WW at the beginning of the experiment. The whiskers represent the 5th and 95th percentiles and the “•” indicate points that are outside of the reaminder of the data. 6 Point Wide "N" Wide "N" Knot Second Skin Control Wet Weight (g) 8 10 12 14 16 18 20 22 Control Figure 3.2. Wet Weights (g) of Study Fish for Each Treatment at 12°C. Each box represents the median and upper and lower quartiles for study fish WW at the beginning of the experiment. 3.1 Fish Size The whiskers represent the 5th and 95th percentiles and the “•” indicate points that are outside of the reaminder of the data. 6 Point Wide "N" Wide "N" Knot Second Skin Control Wet Weight (g) 8 10 12 14 16 18 20 22 Figure 3.3. Wet Weights (g) of Study Fish for Each Treatment at 17°C. Each box represents the median and upper and lower quartiles for study fish WW at the beginning of the experiment. The whiskers represent the 5th and 95th percentiles and the “•” indicate points that are outside of the reaminder of the data. 6 Point Wide "N" Wide "N" Knot Second Skin Control Wet Weight (g) 8 10 12 14 16 18 20 22 Figure 3.3. Wet Weights (g) of Study Fish for Each Treatment at 17°C. Each box represents the median and upper and lower quartiles for study fish WW at the beginning of the experiment. The whiskers represent the 5th and 95th percentiles and the “•” indicate points that are outside of the reaminder of the data. 3.2 3.2 Mortalities To address whether suture pattern and type influenced mortality rates, we examined the mortality frequency among treatment groups (Table 3.1). Overall experimental mortality was low (3.0%) for fish held at 12°C and did not significantly vary between treatment groups (N = 266, χ2 = 0.68, P = 0.9552; Table 3.1). Mortalities for fish held at 12°C occurred from 1 to 14 days post-surgery (median = 1 day post-surgery). At 17°C, overall experimental mortality was relatively high (48.5%) and significantly varied between treatment groups (N = 274, χ2 = 11.96, P = 0.0177; Table 3.1); however, this relationship was mainly driven by the Control group and when the Control group was removed from analysis, mortality did not vary between treatment groups (N = 252, χ2 = 2.411621; P = 0.4915). At 17°C, the 6-Point treatment group suffered the highest mortality rate over the range of the study, followed by the Second Skin, Wide “N” Knot, Wide “N”, and Control treatment groups. Mortalities occurred from 0 to 27 days post-surgery (Median = 15 days post-surgery). Table 3.1. Mortality Frequency for Fish in Each Treatment Group. Frequency of occurrence as a percentage is shown in parentheses for each treatment. Temperature Mortality 6-Point Wide “N” Wide “N” Knot Second Skin Control 12°C No 58 60 59 60 21 Yes 2 (3.3%) 2 (3.2%) 2 (3.3)% 1 (1.6%) 1 (4.5%) 17°C No 26 31 34 32 18 Yes 38 (59.4%) 28 (47.5%) 31 (47.7%) 32 (50.0%) 4 (18.2%) ble 3.1. Mortality Frequency for Fish in Each Treatment Group. Frequency of occurrence percentage is shown in parentheses for each treatment. 3.4 Incision Openness To determine whether one suture pattern or type had a greater influence on tag loss or physiological stress, we examined incision openness (surface area; mm2) on days 7, 14, 21, and 28 (Table 3.3). On Day 7 at 12°C, the 6-Point, Wide “N”, and Wide “N” Knot treatment groups had fish with incision openness; the greatest average openness occurred in the Wide “N” treatment group (range = 0 to 3.82 mm2; Table 3.3). Incision openness significantly varied with treatments (N = 258, χ2 = 13.8356, P = 0.0031; Figure 3.4, Figure 3.5). Post hoc analyses revealed that the Wide “N” treatment group had significantly more incision openness than the Second Skin treatment group. On Day 7 at 17°C, all treatment groups had fish with incision openness; the greatest average openness occurred in the Wide “N” treatment group (range = 0 to 11.29 mm2). Incision openness significantly varied by treatment (N = 246, χ2 = 32.5104, P < 0.0001; Figure 3.6, Figure 3.7). Post hoc analyses revealed that fish in the Wide “N” treatment group had significantly greater incision openness than those in the 6-Point, Wide “N” Knot, and Second Skin treatment groups (all P < 0.05). On Day 14 at 12°C, the 6-Point, Wide “N”, and Wide “N” Knot treatment groups had fish with incision openness; the greatest average openness occurred in the Wide “N” treatment group (range = 0 to 5.13 mm2; Table 3.2). At 12°C, incision openness significantly varied between treatment groups (N = 249, χ2 = 8.5494; P = 0.0359; Figure 3.4), but post hoc analyses revealed no differences between them. On Day 14 at 17°C, all treatment groups had fish with incision openness; the greatest average openness occurred in the Wide “N” treatment group (range = 0 to 10.25 mm2). At 17°C, incision openness did not vary by treatment group (N = 209, χ2 = 0.4815, P = 0.9229; Figure 3.6). On Day 21 at 12°C, the Second Skin treatment group was the only group to have fish with incision openness (range = 0 to 1.42 mm2; Table 3.2). On Day 21 at 17°C, all treatments had fish with incision openness; the greatest average incision openness occurred in the Wide “N” treatment group (range = 0 to 11.22 mm2). 3.3 Tag Loss To address whether one suture pattern and type had a greater rate of tag loss, we analyzed the number of dropped AT and PIT tags. At 12°C, no ATs or PITs were dropped; therefore, statistical analyses were not performed. At 17°C, AT loss was relatively high (8.1%) and a total of 10 ATs were dropped by Day 28. Five fish in the Second Skin treatment group, three fish in the Wide “N” treatment group, one fish in the Wide “N” Knot treatment group, and one fish in the 6-Point treatment group dropped ATs between days 14 and 28 (Median = Day 28). The frequency of dropped ATs was not significantly different between treatment groups at 17°C (N = 123, χ2 = 4.41, P = 0.2208; Table 3.2). At 17°C, there were no dropped PITs in live fish; however dropped PITs occurred in dead fish, but these were not factored into the frequency of tag loss. Table 3.2. AT Retention for Each Treatment. Frequency of occurrence as a percentage is shown in parentheses for each treatment group. Temperature Tag Retention 6-Point Wide “N” Wide “N” Knot Second Skin 12°C Not Dropped 58 60 59 60 Dropped 0 0 0 0 17°C Not Dropped 25 28 33 27 Dropped 1 (3.8%) 3 (9.7%) 1 (2.9%) 5 (15.6%) AT Retention for Each Treatment. Frequency of occurrence as a percentage is shown in parentheses for each treatment group. Table 3.2. AT Retention for Each Treatment. Frequency of occurrence as a percentag parentheses for each treatment group. 3.3 3.4 Incision Openness Incision openness did not vary by treatment group at 12°C (N = 241, χ2 = 2.9508, P = 0.3993; Figure 3.4) or 17°C (N = 152, χ2 = 3.1135, P = 0.3745; Figure 3.6). On Day 28 at 12°C, the Second Skin treatment group was the only one that had fish with incision openness (range 0 to 1.95 mm2; Table 3.2). Incision openness did not vary by treatment group for fish at 12°C (N = 237, χ2 = 2.9500, P = 0.3994; Figure 3.4). On Day 28 at 17°C, all treatment groups had fish with incision openness; the greatest average openness occurred in the Second Skin treatment group (range = 0 to 7.59 mm2), but incision openness did not significantly vary by treatment group (N = 122, χ2 = 5.7495, P = 0.1245; Figure 3.6). 3.4 Table 3.3. Incision Openness (mm2) on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature. Average incision openness ± SD and frequency of occurrence as a percentage is shown in parentheses for each treatment. Temperature Observation Day 6-Point Wide “N” Wide “N” Knot Second Skin 12°C 7 0.05 ± 0.37 (1.6%) 0.21 ± 0.68 (12.1%) 0.09 ± 0.53 (3.1%) 0.0 (0%) 14 0.01 ± 0.10 (1.6%) 0.21 ± 0.82 (9.4%) 0.03 ± 0.26 (1.6%) 0.0 (0%) 21 0.0 (0%) 0.0 (0%) 0.0 (0%) 1.42 ± 0.0 (1.6%) 28 0.0 (0%) 0.0 (0%) 0.0 (0%) 0.03 ± 0.25 (1.6%) 17°C 7 0.02 ± 0.18 (1.6%) 1.25 ± 2.43 (29.5%) 0.31 ± 1.16 (7.8%) 0.56 ± 4.40 (3.2%) 14 0.08 ± 0.32 (6.1%) 0.26 ± 1.50 (4.1%) 0.09 ± 0.46 (3.4%) 0.15 ± 0.76 (5.7%) 21 0.05 ± 0.29 (2.9%) 0.37 ± 1.88 (5.4%) 0.01 ± 0.09 (2.4%) 0.24 ± 0.75 (10.3%) 28 0.09 ± 0.44 (7.7%) 0.63 ± 1.91 (16.1%) 0.08 ± 0.32 (5.9%) 1.14 ± 2.31 (22.9%) Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Figure 3.4. 3.4 Incision Openness Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Table 3.3. Incision Openness (mm2) on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature. Average incision openness ± SD and frequency of occurrence as a percentage is shown in parentheses for each treatment. Temperature Observation Day 6-Point Wide “N” Wide “N” Knot Second Skin 12°C 7 0.05 ± 0.37 (1.6%) 0.21 ± 0.68 (12.1%) 0.09 ± 0.53 (3.1%) 0.0 (0%) 14 0.01 ± 0.10 (1.6%) 0.21 ± 0.82 (9.4%) 0.03 ± 0.26 (1.6%) 0.0 (0%) 21 0.0 (0%) 0.0 (0%) 0.0 (0%) 1.42 ± 0.0 (1.6%) 28 0.0 (0%) 0.0 (0%) 0.0 (0%) 0.03 ± 0.25 (1.6%) 17°C 7 0.02 ± 0.18 (1.6%) 1.25 ± 2.43 (29.5%) 0.31 ± 1.16 (7.8%) 0.56 ± 4.40 (3.2%) 14 0.08 ± 0.32 (6.1%) 0.26 ± 1.50 (4.1%) 0.09 ± 0.46 (3.4%) 0.15 ± 0.76 (5.7%) 21 0.05 ± 0.29 (2.9%) 0.37 ± 1.88 (5.4%) 0.01 ± 0.09 (2.4%) 0.24 ± 0.75 (10.3%) 28 0.09 ± 0.44 (7.7%) 0.63 ± 1.91 (16.1%) 0.08 ± 0.32 (5.9%) 1.14 ± 2.31 (22.9%) Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Figure 3.4. Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C. Data points overlap at 0.00 mm2. Table 3.3. Incision Openness (mm2) on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature. Average incision openness ± SD and frequency of occurrence as a percentage is shown in parentheses for each treatment. 3.4 Incision Openness Temperature Observation Day 6-Point Wide “N” Wide “N” Knot Second Skin 12°C 7 0.05 ± 0.37 (1.6%) 0.21 ± 0.68 (12.1%) 0.09 ± 0.53 (3.1%) 0.0 (0%) 14 0.01 ± 0.10 (1.6%) 0.21 ± 0.82 (9.4%) 0.03 ± 0.26 (1.6%) 0.0 (0%) 21 0.0 (0%) 0.0 (0%) 0.0 (0%) 1.42 ± 0.0 (1.6%) 28 0.0 (0%) 0.0 (0%) 0.0 (0%) 0.03 ± 0.25 (1.6%) 17°C 7 0.02 ± 0.18 (1.6%) 1.25 ± 2.43 (29.5%) 0.31 ± 1.16 (7.8%) 0.56 ± 4.40 (3.2%) 14 0.08 ± 0.32 (6.1%) 0.26 ± 1.50 (4.1%) 0.09 ± 0.46 (3.4%) 0.15 ± 0.76 (5.7%) 21 0.05 ± 0.29 (2.9%) 0.37 ± 1.88 (5.4%) 0.01 ± 0.09 (2.4%) 0.24 ± 0.75 (10.3%) 28 0.09 ± 0.44 (7.7%) 0.63 ± 1.91 (16.1%) 0.08 ± 0.32 (5.9%) 1.14 ± 2.31 (22.9%) Table 3.3. Incision Openness (mm2) on Days 7, 14, 21, and 28 by Suture Treatment Group and Holding Temperature. Average incision openness ± SD and frequency of occurrence as a percentage is shown in parentheses for each treatment. Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Figure 3.4. Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C. Data points overlap at 0.00 mm2. Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Figure 3.4. Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C. 3.4 Incision Openness Data points overlap at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 1 2 3 4 5 6 Figure 3.4. Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 12°C. Data points overlap at 0.00 mm2. 3.5 Wet Weight (g) 10 12 14 16 18 20 22 Incision Openness (mm2) 0 1 2 3 4 5 Figure 3.5. Total Incision Openness (mm2) Plotted Against the Wet Weight of Individual Fish, Day 7 at 12°C. WW was likely not a determinative factor in the incision openness. Data points only overlapped at 0.00 mm2. Wet Weight (g) 10 12 14 16 18 20 22 Incision Openness (mm2) 0 1 2 3 4 5 Figure 3.5. Total Incision Openness (mm2) Plotted Against the Wet Weight of Individual Fish, Day 7 at 12°C. WW was likely not a determinative factor in the incision openness. Data points only overlapped at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Figure 3.6. Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 17°C. Data points only overlapped at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Incision Openness (mm2) 0 10 20 30 40 Figure 3.6. 3.4 Incision Openness Total Incision Openness (mm2) by Treatment at 7, 14, 21, and 28 Days Post-Surgery for Fish Held at 17°C. Data points only overlapped at 0.00 mm2. 3.6 Wet Weight (g) 8 10 12 14 16 18 20 22 Incision Openness (mm2) 0 10 20 30 40 Wet Weight (g) 8 10 12 14 16 18 20 22 Incision Openness (mm2) 0 10 20 30 40 Figure 3.7. Total Incision Openness (mm2) Plotted Against the Wet Weight of Individual Fish, Day 7 at 17°C. WW was likely not a determinative factor in the incision openness. Data points only overlapped at 0.00 mm2. Figure 3.7. Total Incision Openness (mm2) Plotted Against the Wet Weight of Individual Fish, Day 7 at 17°C. WW was likely not a determinative factor in the incision openness. Data points only overlapped at 0.00 mm2. 3.5 Functional Suture We examined sutures to determine if one suture pattern and its associated needle type resulted in more functional sutures (i.e., present and maintained proper tension) than others at Site 1, Site 2, and if applicable, Site 3 (Figure 2.1 and Table 3.4 and Table 3.5). At Site 1 on Day 7 at 12°C, the Wide “N” treatment group had the fewest functional sutures (77.8%), followed by the 6-Point (81.3%), Wide “N” Knot (100%), and Second Skin (100%) treatment groups (N = 209, χ2 = 33.42, P < 0.001; Table 3.4). At 17°C, the Wide “N” treatment group had the fewest functional sutures (86.7%), followed by the 6-Point (91.6%), Wide “N” Knot (96.7%), and Second Skin (100%) treatment groups (N = 192, χ2 = 9.87, P = 0.0197; Table 3.5). At Site 1 on Day 14 at 12°C, the Wide “N” treatment group had the fewest functional sutures (66.6%), followed by the 6-Point (81.1%), Wide “N” Knot (98.1%), and Second Skin (100%) treatment groups (N = 189, χ2 = 30.89, P < 0.0001; Table 3.4). At 17°C, the 6-Point treatment group had the fewest functional sutures (71.9%), followed by the Wide “N” (82.4%), Wide “N” Knot (96.0%), and Second Skin (97.4%) treatment groups (N = 138, χ2 = 14.92, P = 0.0019; Table 3.5). At Site 1 on Day 21 at 12°C, the Wide “N” treatment group had the fewest functional sutures (50.0%), followed by the 6-Point (67.4%), Wide “N” Knot (100%), and Second Skin (100%) treatment groups (N = 173, χ2 = 57.32, P < 0.0001; Table 3.4). At 17°C, the 6-Point treatment group had the fewest functional sutures (56.3%), followed by the Wide “N” (87.5%), Second Skin (88.9%), and Wide “N” Knot (93.8%) treatment groups (N = 83, χ2 = 10.29, P = 0.0162; Table 3.5). At Site 1 on Day 28 at 12°C, the 6-Point treatment group had the fewest functional sutures (73.5%), followed by the Wide “N” (83.3%), Wide “N” Knot (93.2%), and Second Skin (98.3%) treatment groups 3.7 (N = 143, χ2 = 14.90, P = 0.0019; Table 3.4). At 17°C, the 6-Point treatment group had the fewest functional sutures (28.6%), followed by the Wide “N” (33.3%), Wide “N” Knot (57.9%), and Second Skin (88.9%) treatment groups (N = 47, χ2 = 10.90, P = 0.0123; Table 3.5). Table 3.4. Number of Functional Sutures by Entry/Exit Point for Each Treatment at 12°C. 3.5 Functional Suture The last two rows indicate the presence of the suture internally, either in the body cavity or embedded in tissue. Observation Day Site Functional at Entry/Exit Point 6-Point Wide “N” Wide “N” Knot Second Skin 7 1 Yes 39 28 60 65 1 No 9 8 0 0 2 Yes 56 23 32 65 2 No 0 13 12 0 3 Yes 34 NA NA NA 3 No 12 NA NA NA 14 1 Yes 43 14 52 62 1 No 10 7 1 0 2 Yes 50 15 29 63 2 No 4 6 11 0 3 Yes 37 NA NA NA 3 No 16 NA NA NA 21 1 Yes 29 10 50 60 1 No 14 10 0 0 2 Yes 40 7 22 59 2 No 3 9 11 1 3 Yes 22 NA NA NA 3 No 10 NA NA NA 28 1 Yes 25 5 41 58 1 No 9 1 3 1 2 Yes 33 5 20 58 2 No 4 1 6 2 3 Yes 15 NA NA NA 3 No 12 NA NA NA Necropsy Suture present 1 0 1 0 Suture not present 24 48 16 1 NA = Not Applicable. Table 3.4. Number of Functional Sutures by Entry/Exit Point for Each Treatment at 12°C. The last two rows indicate the presence of the suture internally, either in the body cavity or embedded in tissue. At Site 2 on Day 7 at 12°C, the Wide “N” treatment group had the fewest functional sutures (63.9%), followed by the Wide “N” Knot (72.7%), 6-Point (100%) and Second Skin (100%) treatment groups (N = 201, χ2 = 52.32, P < 0.0001; Table 3.4). At 17°C, the Wide “N” treatment group had the fewest functional sutures (77.8%), followed by the Wide “N” Knot (88.1%), 6-Point (100%), and Second Skin (100%) treatment groups (N = 178, χ2 = 23.29, P < 0.0001; Table 3.5). At Site 2 on Day 7 at 12°C, the Wide “N” treatment group had the fewest functional sutures (63.9%), followed by the Wide “N” Knot (72.7%), 6-Point (100%) and Second Skin (100%) treatment groups (N = 201, χ2 = 52.32, P < 0.0001; Table 3.4). At Site 2 on Day 7 at 12°C, the Wide “N” treatment group had the fewest functional sutures (63.9%), followed by the Wide “N” Knot (72.7%), 6-Point (100%) and Second Skin (100%) treatment groups (N = 201, χ2 = 52.32, P < 0.0001; Table 3.4). At 17°C, the Wide “N” treatment group had the fewest functional sutures (77.8%), followed by the Wide “N” Knot (88.1%), 6-Point (100%), and Second Skin (100%) treatment groups (N = 178, χ2 = 23.29, P < 0.0001; Table 3.5). 3.5 Functional Suture At 17°C, the Wide “N” treatment group had the fewest functional sutures (77.8%), followed by the Wide “N” Knot (88.1%), 6-Point (100%), and Second Skin (100%) treatment groups (N = 178, χ2 = 23.29, P < 0.0001; Table 3.5). 3.8 Table 3.5. Number of Functional Sutures by Entry/Exit Point for Each Treatment at 17°C. The last two rows indicate whether the suture was seen internally either in the body cavity or embedded in tissue. Table 3.5. Number of Functional Sutures by Entry/Exit Point for Each Treatment at 17°C. The last two rows indicate whether the suture was seen internally either in the body cavity or embedded in tissue. Observation Day Site Functional at Entry/Exit Point 6-Point Wide “N” Wide “N” Knot Second Skin 7 1 Yes 44 26 59 53 1 No 4 4 2 0 2 Yes 54 21 37 55 2 No 0 6 5 0 3 Yes 29 NA NA NA 3 No 14 NA NA NA 14 1 Yes 23 14 48 38 1 No 9 3 2 1 2 Yes 33 9 25 42 2 No 1 5 2 0 3 Yes 22 NA NA NA 3 No 1 NA NA NA 21 1 Yes 9 7 30 24 1 No 7 1 2 3 2 Yes 14 3 8 25 2 No 2 3 6 5 3 Yes 7 NA NA NA 3 No 5 NA NA NA 28 1 Yes 2 1 11 16 1 No 5 2 8 2 2 Yes 7 0 0 15 2 No 2 3 9 6 3 Yes 3 NA NA NA 3 No 2 NA NA NA Necropsy Suture present 0 1 1 0 Suture not present 16 21 19 4 NA = Not Applicable. At Site 2 on Day 14 at 12°C, the Wide “N” treatment group had the fewest functional sutures (71.4%), followed the Wide “N” Knot (72.5%), 6-Point (92.6%), and the Second Skin (100%) treatment groups (N = 178, χ2 = 28.49, P < 0.0001; Table 3.4). At 17°C, the Wide “N” group had the fewest functional sutures (64.3%), followed by the Wide “N” Knot (92.6%), 6-Point (97.1%), and Second Skin (100%) treatment groups (N = 117, χ2 = 16.83, P = 0.0008; Table 3.5). 3.5 Functional Suture At Site 2 on Day 14 at 12°C, the Wide “N” treatment group had the fewest functional sutures (71.4%), followed the Wide “N” Knot (72.5%), 6-Point (92.6%), and the Second Skin (100%) treatment groups (N = 178, χ2 = 28.49, P < 0.0001; Table 3.4). At 17°C, the Wide “N” group had the fewest functional sutures (64.3%), followed by the Wide “N” Knot (92.6%), 6-Point (97.1%), and Second Skin (100%) treatment groups (N = 117, χ2 = 16.83, P = 0.0008; Table 3.5). At Site 2 on Day 21 at 12°C, the Wide “N” treatment group had the fewest functional sutures (43.8%), followed by the Wide “N” Knot (66.7%), 6-Point (93.0%), and Second Skin (98.3%) treatment groups (N = 152, χ2 = 36.72, P < 0.001; Table 3.4). At 17°C, although not significantly different, the 3.9 Wide “N” treatment group had the fewest functional sutures (50.0%), followed by the Wide “N” Knot (57.1%), Second Skin (83.3%), and 6-Point (87.5%) treatment groups (N = 66, χ2 = 6.58, P = 0.0866; Table 3.5). Wide “N” treatment group had the fewest functional sutures (50.0%), followed by the Wide “N” Knot (57.1%), Second Skin (83.3%), and 6-Point (87.5%) treatment groups (N = 66, χ2 = 6.58, P = 0.0866; Table 3.5). At Site 2 on Day 28 at 12°C, the Wide “N” Knot treatment group had the fewest functional sutures (76.9%), followed by the Wide “N” (83.3%), 6-Point (89.2%), and Second Skin (96.7%) treatment groups (N = 129, χ2 = 7.93, P = 0.0475; Table 3.4). At 17°C, the Wide “N” and Wide “N” Knots had no remaining functional sutures, followed by the Second Skin (71.4%) and 6-Point (77.8%) treatment groups (N = 42, χ2 = 23.47, P < 0.0001; Table 3.5). No statistical analyses were conducted on the third entry/exit point (Site 3) because the 6-Point treatment was the only treatment with three sites (Table 3.4 and Table 3.5). 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 4 (6.3%) 2 (3.0%) 1 (1.6%) 1 (1.5%) No 59 64 63 64 x̄ mm2 ± SD 0.02 ± 0.09 0.01 ± 0.06 0.00 ± 0.03 0.01 ± 0.06 14 Yes 28 (45.9%) 10 (15.9%) 10 (16.1%) 15 (23.8%) No 33 53 52 48 x̄ mm2 ± SD 0.29 ± 0.41 0.07 ± 0.21 0.10 ± 0.30 0.08 ± 0.17 21 Yes 20 (33.9%) 6 (9.8%) 18 (30.0%) 3 (4.9%) No 39 55 42 58 x̄ mm2 ± SD 0.36 ± 0.73 0.06 ± 0.21 0.19 ± 0.39 0.03 ± 0.15 28 Yes 15 (25.9%) 2 (3.3%) 11 (18.6%) 5 (8.3%) No 43 58 48 55 x̄ mm2 ± SD 0.55 ± 1.26 0.02 ± 0.10 0.22 ± 0.63 0.05 ± 0.22 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Figure 3.9. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. Data points only overlapped at 0.00 mm2. 3.6 Ulceration and Redness To determine whether suture pattern or type influenced tag loss or physiological stress, we examined the frequency of ulceration and redness occurrences and the total surface area by treatment (Figure 3.8). Simplifying the analysis to presence or absence of ulcerations, on Day 7 at 12°C there was no significant difference in the frequency (N= 258, χ2 = 2.97, P = 0.3962) or the total surface area of ulceration around the incision and/or the suture entry/exit sites (surface area measurements, mm2) between treatment groups (N = 258, F(3, 254) = 0.77, P = 0.5111; Table 3.6, Figure 3.9). At 17°C, there was no significant difference in the frequency (N = 247, χ2 = 1.14, P = 0.7670) or the total surface area of ulceration between treatment groups (N = 247, F(3, 243) = 1.0146, P = 0.3868 Table 3.7, Figure 3.10). Figure 3.8. Example of Ulceration and Redness Caused by Suture Tearing. The red circle (○) highlights redness not directly incorporated with ulceration. The two red squares (□) denote ulceration and redness that were separated using Image J. The circle and square do not denote the actual Image J patterns and measurements used for the final summations of total ulceration and redness. Figure 3.8. Example of Ulceration and Redness Caused by Suture Tearing. The red circle (○) highlights redness not directly incorporated with ulceration. The two red squares (□) denote ulceration and redness that were separated using Image J. The circle and square do not denote the actual Image J patterns and measurements used for the final summations of total ulceration and redness. 3.10 Table 3.6. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12°C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 4 (6.3%) 2 (3.0%) 1 (1.6%) 1 (1.5%) No 59 64 63 64 x̄ mm2 ± SD 0.02 ± 0.09 0.01 ± 0.06 0.00 ± 0.03 0.01 ± 0.06 14 Yes 28 (45.9%) 10 (15.9%) 10 (16.1%) 15 (23.8%) No 33 53 52 48 x̄ mm2 ± SD 0.29 ± 0.41 0.07 ± 0.21 0.10 ± 0.30 0.08 ± 0.17 21 Yes 20 (33.9%) 6 (9.8%) 18 (30.0%) 3 (4.9%) No 39 55 42 58 x̄ mm2 ± SD 0.36 ± 0.73 0.06 ± 0.21 0.19 ± 0.39 0.03 ± 0.15 28 Yes 15 (25.9%) 2 (3.3%) 11 (18.6%) 5 (8.3%) No 43 58 48 55 x̄ mm2 ± SD 0.55 ± 1.26 0.02 ± 0.10 0.22 ± 0.63 0.05 ± 0.22 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Figure 3.9. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. Data points only overlapped at 0.00 mm2. Table 3.6. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12°C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. Table 3.6. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12°C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. Table 3.6. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 12 C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 4 (6.3%) 2 (3.0%) 1 (1.6%) 1 (1.5%) No 59 64 63 64 x̄ mm2 ± SD 0.02 ± 0.09 0.01 ± 0.06 0.00 ± 0.03 0.01 ± 0.06 14 Yes 28 (45.9%) 10 (15.9%) 10 (16.1%) 15 (23.8%) No 33 53 52 48 x̄ mm2 ± SD 0.29 ± 0.41 0.07 ± 0.21 0.10 ± 0.30 0.08 ± 0.17 21 Yes 20 (33.9%) 6 (9.8%) 18 (30.0%) 3 (4.9%) No 39 55 42 58 x̄ mm2 ± SD 0.36 ± 0.73 0.06 ± 0.21 0.19 ± 0.39 0.03 ± 0.15 28 Yes 15 (25.9%) 2 (3.3%) 11 (18.6%) 5 (8.3%) No 43 58 48 55 x̄ mm2 ± SD 0.55 ± 1.26 0.02 ± 0.10 0.22 ± 0.63 0.05 ± 0.22 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Figure 3.9. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. Data points only overlapped at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 2 4 6 8 Figure 3.9. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. Data points only overlapped at 0.00 mm2. 3.11 Table 3.7. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 17°C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 15 (24.6%) 19 (31.1%) 21 (32.8%) 19 (30.6%) No 46 42 43 43 x̄ mm2 ± SD 0.21 ± 0.51 0.14 ± 0.24 0.62 ± 3.27 0.49 ± 1.14 14 Yes 28 (57.1%) 12 (24.5%) 34 (58.6%) 20 (37.7%) No 21 37 24 33 x̄ mm2 ± SD 1.41 ± 1.74 0.45 ± 1.04 0.85 ± 1.14 1.06 ± 2.23 21 Yes 17 (50.0%) 8 (21.6%) 24 (57.1%) 12 (30.8%) No 17 29 18 27 x̄ mm2 ± SD 1.86 ± 3.22 0.44 ± 1.32 0.92 ± 1.10 0.83 ± 1.87 28 Yes 9 (34.6%) 3 (9.7%) 15 (44.1%) 7 (22.6%) No 17 28 19 24 x̄ mm2 ± SD 1.51 ± 3.50 0.40 ± 1.71 0.81 ± 1.07 1.25 ± 2.93 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Figure 3.10. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. Table 3.7. Frequency and Mean Area of Ulceration for Each Treatment for Fish Held at 17°C. The total mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. mean and standard deviation of ulcerated area (mm2) are provided in the row for each day. 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 15 (24.6%) 19 (31.1%) 21 (32.8%) 19 (30.6%) No 46 42 43 43 x̄ mm2 ± SD 0.21 ± 0.51 0.14 ± 0.24 0.62 ± 3.27 0.49 ± 1.14 14 Yes 28 (57.1%) 12 (24.5%) 34 (58.6%) 20 (37.7%) No 21 37 24 33 x̄ mm2 ± SD 1.41 ± 1.74 0.45 ± 1.04 0.85 ± 1.14 1.06 ± 2.23 21 Yes 17 (50.0%) 8 (21.6%) 24 (57.1%) 12 (30.8%) No 17 29 18 27 x̄ mm2 ± SD 1.86 ± 3.22 0.44 ± 1.32 0.92 ± 1.10 0.83 ± 1.87 28 Yes 9 (34.6%) 3 (9.7%) 15 (44.1%) 7 (22.6%) No 17 28 19 24 x̄ mm2 ± SD 1.51 ± 3.50 0.40 ± 1.71 0.81 ± 1.07 1.25 ± 2.93 x̄ mm2 ± SD 1.51 ± 3.50 0.40 ± 1.71 0.81 ± 1.07 1.25 ± 2.93 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Figure 3.10. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Figure 3.10. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. 3.6 Ulceration and Redness Observation Day Ulceration 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 15 (24.6%) 19 (31.1%) 21 (32.8%) 19 (30.6%) No 46 42 43 43 x̄ mm2 ± SD 0.21 ± 0.51 0.14 ± 0.24 0.62 ± 3.27 0.49 ± 1.14 14 Yes 28 (57.1%) 12 (24.5%) 34 (58.6%) 20 (37.7%) No 21 37 24 33 x̄ mm2 ± SD 1.41 ± 1.74 0.45 ± 1.04 0.85 ± 1.14 1.06 ± 2.23 21 Yes 17 (50.0%) 8 (21.6%) 24 (57.1%) 12 (30.8%) No 17 29 18 27 x̄ mm2 ± SD 1.86 ± 3.22 0.44 ± 1.32 0.92 ± 1.10 0.83 ± 1.87 28 Yes 9 (34.6%) 3 (9.7%) 15 (44.1%) 7 (22.6%) No 17 28 19 24 x̄ mm2 ± SD 1.51 ± 3.50 0.40 ± 1.71 0.81 ± 1.07 1.25 ± 2.93 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Figure 3.10. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. 3.6 Ulceration and Redness Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Ulceration (mm2) 0 5 10 15 20 25 30 Figure 3.10. Total Ulceration (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. 3.12 On Day 14 at 12°C, the 6-Point treatment had significantly greater frequency of ulceration (N= 249, χ2 = 18.45, P = 0.0004), followed by the Second Skin, Wide “N” Knot, and Wide “N” treatments. The 6-Point treatment group also had significantly greater total ulceration surface area than the Wide “N”, Wide “N” Knot, and Second Skin treatment groups (N = 249, F(3, 245) = 7.66, P < 0.0001; Table 3.6, Figure 3.9). At 17°C, the Wide “N” Knot treatment group had significantly greater frequency of ulceration (N = 209, χ2 = 17.22, P = 0.0006), followed by the 6-Point, Second Skin, and Wide “N” treatment groups. The 6-Point treatment group had significantly greater total ulceration surface area than the Wide “N” treatment group (N = 209, F(3, 205) = 3.05, P = 0.0295; Table 3.7, Figure 3.10). On Day 21 at 12°C, the 6-Point treatment group had significantly greater frequency of ulceration (N = 241, χ2 = 25.82, P < 0.0001), followed by the Wide “N” Knot, Wide “N”, and Second Skin. The 6-Point treatment group also had significantly greater total ulceration than the Wide “N” and Second Skin treatment groups (N = 241, F(3, 237) = 7.52, P < 0.0001; ANOVA; Table 3.6, Figure 3.9). At 17°C, the Wide “N” Knot treatment group had significantly greater frequency of ulceration (N= 152, χ2 = 13.48, P = 0.0037), followed by the 6-Point, Second Skin, and Wide “N” treatment groups. The 6-Point treatment had significantly greater total ulceration surface area than the Wide “N” treatment group (N = 152, F(3, 148) = 3.20, P = 0.025; ANOVA; Table 3.7, Figure 3.10). 3.6 Ulceration and Redness On Day 28 at 12°C, the 6-Point treatment group had significantly greater frequency of ulceration (N = 237, χ2 = 16.27, P = 0.001), followed by the Wide “N” Knot, Second Skin, and Wide “N” treatment groups. The 6-Point treatment group also had significantly more total ulceration surface area than the Wide “N” treatment group (N = 237, F(3, 233) = 3.50, P = 0.0002; ANOVA; Table 3.6, Figure 3.9). At 17°C, the Wide “N” Knot treatment group had significantly greater frequency of ulceration (N= 122, χ2 = 11.34, P = 0.01), followed by the 6-Point, Second Skin, and Wide “N” treatment groups. However, total ulceration surface area was not different between treatment groups (N = 122, F(3, 233) = 1.20, P = 0.3112; ANOVA; Table 3.7, Figure 3.10). Table 3.8. Frequency and Mean Area of Redness for Each Treatment at 12°C Observation Day Redness 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 28 (44.4%) 20 (30.3%) 30 (46.9%) 13 (20.0%) No 35 46 34 52 x̄ mm2 ± SD 0.60 ± 1.34 0.24 ± 0.54 0.49 ± 1.53 0.10 ± 0.38 14 Yes 20 (32.9%) 14 (22.2%) 13 (30.0%) 8 (12.7%) No 41 49 49 55 x̄ mm2 ± SD 0.16 ± 0.34 0.08 ± 0.21 0.14 ± 0.35 0.04 ± 0.13 21 Yes 16 (27.1%) 5 (8.2%) 6 (10.0%) 6 (9.8%) No 43 56 54 55 x̄ mm2 ± SD 0.12 ± 0.39 0.04 ± 0.16 0.05 ± 0.21 0.04 ± 0.18 28 Yes 8 (13.8%) 4 (6.7%) 7 (11.9%) 3 (5.1%) No 50 56 52 57 x̄ mm2 ± SD 0.04 ± 0.12 0.02 ± 0.11 0.05 ± 0.25 0.02 ± 0.09 Table 3.8. Frequency and Mean Area of Redness for Each Treatment at 12°C 3.13 3.13 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Figure 3.11. Total Redness (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. 3.6 Ulceration and Redness Data points only overlapped at 0.00 mm2. Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 Figure 3.11. Total Redness (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 12°C. Data points only overlapped at 0.00 mm2. Table 3.9. Frequency and Mean Area of Redness for Each Treatment at 17°C Observation Day Redness 6-Point Wide “N” Wide “N” Knot Second Skin 7 Yes 28 (45.9%) 17 (27.9%) 25 (39.1%) 26 (41.9%) No 33 44 39 36 x̄ mm2 ± SD 0.34 ± 0.71 0.17 ± 0.48 0.24 ± 0.52 0.66 ± 1.30 14 Yes 9 (18.4%) 4 (8.2%) 14 (24.1%) 27 (50.9%) No 40 45 44 26 x̄ mm2 ± SD 0.12 ± 0.34 0.09 ± 0.50 0.21 ± 0.69 1.72 ± 3.43 21 Yes 5 (14.7%) 3 (8.1%) 9 (21.4%) 6 (15.4%) No 29 34 33 33 x̄ mm2 ± SD 0.09 ± 0.24 0.07 ± 0.30 0.12 ± 0.36 0.41 ± 1.36 28 Yes 3 (11.5%) 0 (0%) 3 (8.8%) 5 (16.1%) No 23 31 31 26 x̄ mm2 ± SD 0.08 ± 0.27 0.0 0.07 ± 0.31 0.07 ± 0.27 Table 3.9. Frequency and Mean Area of Redness for Each Treatment at 17°C 3.14 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Day 21 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Figure 3.12. Total Redness (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. 3.6 Ulceration and Redness Day 14 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Day 7 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Day 28 6 Point Wide "N" Wide "N" Knot Second Skin Total Redness (mm2) 0 2 4 6 8 10 12 14 16 18 Figure 3.12. Total Redness (mm2) by Treatment for 7, 14, 21, and 28 Days Post-Surgery at 17°C. Data points only overlapped at 0.00 mm2. When simplifying the analysis to the presence or absence of redness for each fish, on Day 7 at 12°C the Wide “N” Knot treatment group had significantly greater frequency of fish with redness, followed by the 6-Point, Wide “N”, and the Second Skin treatment groups (N = 258, χ2 = 13.89, P = 0.0031). When comparing total redness surface area (mm2) around the incision and/or the suture entry/exits sites, the 6-Point treatment group had significantly greater redness than the Second Skin (N = 258, F(3, 254) = 2.96, P = 0.0330; Table 3.8, Figure 3.11). At 17°C, there was no significant difference in frequency of redness between the treatment groups (N = 248, χ2 = 4.74, P = 0.1920). However, the Second Skin treatment group had significantly greater total redness surface area than the Wide “N” and Wide “N” Knot treatment groups (N= 248, F(3, 244) = 4.36, P = 0.0052; Table 3.9, Figure 3.12). On Day 14 at 12°C, there was no significant difference in the frequency of redness between the treatment groups (N = 249, χ2 = 7.39, P = 0.0604). However, total redness surface area significantly varied between treatments (N = 249, F(3.245) = 2.74, P = 0.0442; Table 3.8, Figure 3.11), but post hoc tests revealed that total redness surface area was similar for the treatment groups (all P > 0.05). At 17°C, the Second Skin group had significantly greater frequency of fish with redness followed by the Wide “N” Knot, 6-Point and Wide “N” treatment groups (N = 209, χ2 = 26.81, P < 0.0001). The Second Skin treatment group also had significantly greater total redness surface area when compared to the Wide “N”, Wide “N” Knot, and 6-Point treatment groups (N = 209, F(3, 205) = 10.31, P < 0.0001; Table 3.9, Figure 3.12). 3.6 Ulceration and Redness 3.15 On Day 21 at 12°C, the 6-Point treatment group had significantly greater frequency of fish with redness, followed by the Wide “N” Knot, Second Skin, and Wide “N” treatment groups (N = 241, χ2 = 10.70, P = 0.0135). There was no significant difference in the total surface area of redness between the treatment groups (N = 241, F(3, 237) = 1.50, P = 0.2154; Table 3.8, Figure 3.11). At 17°C, there was no difference in the frequency (N = 152, χ2 = 2.84, P = 0.4162) or total surface area of redness between treatment groups (N = 152, F(3, 148) = 1.80, P = 0.1490; Table 3.9, Figure 3.12). On Day 28 at 12°C, there was no significant difference in the frequency (N = 237, χ2 = 3.75, P = 0.2896) or total surface area of redness between treatment groups (N = 237, F(3, 233) = 0.64, P = 0.5869; Table 3.8, Figure 3.11). At 17°C, there was no significant difference in the frequency (N = 122, χ2 = 7.63, P = 0.0543) or total surface area of redness between treatment groups (N = 122, F(3, 118) = 0.71, P = 0.5485; Table 3.9, Figure 3.12). 3.7 Transmitter Bulge The effects of suture patterns and fish size were examined for their influence on tag bulging. The occurrence of transmitter bulging was low in this study and only one fish in the Wide “N” treatment group held at 12°C had transmitter bulging (5.36 mm2) by Day 28. 3.8 Performance Index Each characteristic of interest was ranked (1 = best, 4 = worst) to assist with recommendations based on the performance of each pattern (Table 3.10 and Table 3.11). The lowest average score indicates the best overall suture performance. At 12°C the Second Skin treatment performed better overall, followed by Wide “N” Knot, 6-Point, and Wide “N”. At 17°C, the Wide “N” treatment performed better overall, followed by Wide “N” Knot, 6-Point and Second Skin. 3.16 Table 3.10. Performance Index Based on Rank of Each Measured Treatment Observation at 12°C (1 = best, 4 = worst). The treatment with the lowest overall score is considered to have the overall best performance. Treatment Measured Observation 6-Point Wide “N” Wide “N” Knot Second Skin Mortality 3 3 3 1 AT/PITs Dropped 2.5 2.5 2.5 2.5 Percentage of Fish with Gaping, day 7 2 4 3 1 Percentage of Fish with Gaping, day 14 2.5 4 2.5 1 Percentage of Fish with Gaping, day 21 2 2 2 4 Percentage of Fish with Gaping, day 28 2 2 2 4 Functional Suture, site 1, day 7 4 4 1 1 Functional Suture, site 1, day 14 4 3 1.5 1.5 Functional Suture, site 1, day 21 4 4 1 1 Functional Suture, site 1, day 28 4 1.5 3 1.5 Functional Suture, site 2, day 7 1 4 4 1 Functional Suture, site 2, day 14 2.5 2.5 4 1 Functional Suture, site 2, day 21 2 4 3 1 Functional Suture, site 2, day 28 2 3 4 1 Area of Ulceration, day 7 3 3 1 3 Area of Ulceration, day 14 4 2 2 2 Area of Ulceration, day 21 4 1.5 3 1.5 Area of Ulceration, day 28 4 1.5 3 1.5 Area of Redness, day 7 4 2 2 2 Area of Redness, day 14 1 3 2 4 Area of Redness, day 21 1.5 3.5 1.5 3.5 Area of Redness, day 28 1 4 2 3 Tag Bulge 2 4 2 2 Average 2.70 2.96 2.39 1.96 Table 3.10. Performance Index Based on Rank of Each Measured Treatment Observation at 12°C (1 = best, 4 = worst). The treatment with the lowest overall score is considered to have the overall best performance. 3.17 Table 3.11. Performance Index Based on Rank of Each Measured Treatment Observation at 17°C (1 = best, 4 = worst). 4.0 Discussion The objective of this study was to assess the performance of the bi-directional knotless tissue-closure device in relation to MonocrylTM monofilament with the simple interrupted suture pattern, which is the currently accepted technique for wound closure in juvenile salmon. SYC were implanted with JSATS ATs and PITs, and the incisions were closed using one of four suture patterns: three patterns using the knotless suture material and one suture pattern using MonocrylTM monofilament. We examined seven categorical factors including survivorship, tag loss, presence of gaping, functional suture, ulceration, redness, and tag bulging. Finally, we ranked frequency of occurrence for each factor by treatment to determine a performance index of rank (1 to 4). Generally, the elevated temperature of 17°C greatly affected all factors analyzed, thus likely indicating multiplicative stress from the surgeries and the long- term holding. The categorical factors examined significantly varied among suture pattern and treatment types and were expressed differently between the two temperature treatments. Overall, in 12°C the performance index indicated that the Second Skin performed better than the other treatments, although not consistently superior across the examined factors. For the 17°C group, performance index indicated that the Wide “N” overall performed better than the other treatments, although not consistently superior across the examined factors. For purposes of biotelemetry, in 17°C, the Second Skin treatment had the greatest gaping (days 21 and 28) and tag loss (overall), which may be of a concern when tracking juvenile Chinook in warmer water. The bi-directional knotless suture material performance varied with the patterns examined throughout the study. In 12°C and 17°C groups, the 6-Point pattern performed consistently maintained tension across the wound more evenly than the other patterns thereby minimizing incision openness and increasing tag retention. With exception of one fish in the 12°C group, this pattern had similar incision openness on Day 0, 7, and 14 to the Second Skin, yet provided better apposition and less openness than Second Skin treatment group on days 21 and 28. However, the bi-directional knotless suture 6-Point pattern performance was at the cost of increased trauma (i.e., ulceration) as the suture moved through the tissue once the barbs become loose. As expected with salmon, the elevated temperature treatment, 17°C, had higher mortality indicating health or stress issues. 3.8 Performance Index The treatment with the lowest overall score is considered to have the overall best performance. Treatment Measured Observation 6-Point Wide “N” Wide “N” Knot Second Skin Mortality 4 1.5 1.5 3 AT/PITs Dropped 1.5 3 1.5 4 Percentage of Fish with Gaping, day 7 1 4 3 2 Percentage of Fish with Gaping, day 14 3.5 1.5 1.5 3.5 Percentage of Fish with Gaping, day 21 1.5 3 1.5 4 Percentage of Fish with Gaping, day 28 1.5 3 1.5 4 Functional Suture, site 1, day 7 3.5 3.5 2 1 Functional Suture, site 1, day 14 4 3 2 1 Functional Suture, site 1, day 21 4 1 2 3 Functional Suture, site 1, day 28 4 1.5 3 1.5 Functional Suture, site 2, day 7 1 4 4 1 Functional Suture, site 2, day 14 2 4 3 1 Functional Suture, site 2, day 21 1 2 4 3 Functional Suture, site 2, day 28 1.5 1.5 4 3 Area of Ulceration, day 7 2 1 4 3 Area of Ulceration, day 14 4 1 2 3 Area of Ulceration, day 21 4 1 2.5 2.5 Area of Ulceration, day 28 4 1 2 3 Area of Redness, day 7 3 1 2 4 Area of Redness, day 14 2 1 3 4 Area of Redness, day 21 1.5 1.5 3 4 Area of Redness, day 28 4 1 2.5 2.5 Tag Bulge 2 4 2 2 Mortality 4 1.5 1.5 3 AT/PITs Dropped 1.5 3 1.5 4 Percentage of Fish with Gaping, day 7 1 4 3 2 Average 2.63 2.13 2.50 2.74 3.18 4.0 Discussion SYC have shown a thermal preference for 12.9°C (Sauter 1996), thus based on physiological tolerance polygons (Brett 1995), mortality in the 12°C (3%) group is likely related either to pre-existing condition or the stress of the surgery. Accordingly, the majority of mortalities occurred within 1 day of surgery indicating that the surgery itself was likely too stressful (e.g., anesthetic-related, surgeon generated injury). However, at 17°C, the overall experimental mortality was relatively high (48.5%) and occurred throughout the range of the observation period. Because the 17°C-related mortalities were elevated across the treatment groups (excluding the Control group), it is possible that thermal stress and concomitant surgical stress resulted in the observed short- and long-term elevated mortality rate. Likewise, Panther et al. (2011) observed no mortality in yearling, hatchery-raised Chinook salmon held at 12°C, whereas there was up to a 20% mortality rate for fish held at 20°C when testing three different incision locations. Similar to this study, Panther et al. (2011) found that the control group (no surgery) had the lowest rate of morality (~11%) in the 20°C treatment group. Therefore, it is possible that the high mortality rate is a response to thermal and surgical stress rather than a suture treatment group. 4.1 Similar to the observed rate of mortality variation between temperature groups, elevated temperature increased the rate of dropped tags. Fish held at 12°C did not experience tag loss, but one fish in the Wide “N” treatment group had an AT pushing through the incision (5.36 mm2) by Day 28. In contrast, fish held at 17°C experienced a relatively greater (8.1%) tag loss; a total of 10 tags were dropped by Day 28. Similarly, Knights and Lasee (1996) and Bunnell and Isely (1999) also found that fish held at higher temperatures experienced significantly higher tag loss. Tissue remodeling and reduced growth have been attributed to cellular stress and higher temperature-related metabolic costs in Atlantic (Salmo salar) and Chinook salmon (Larsson et al. 2012; Jerrett et al. 1998), which may be partially the reason for increased damage from sutures and observed dropped tags in the 17°C exposure. Tag retention increases with the incision margins staying approximated as the sutures remain functional (i.e., maintain proper tension and remain knotted; modified Deters et al. 2010). 4.0 Discussion In theory, the 6-Point treatment should have greater tag retention because of the more uniform tension across the incision (relative to the increased number of entry and exit sites). The presence of the suture staying functional in the middle site (#2, see Figure 2.1) likely added to its effectiveness in retaining tags. When two simple interrupted sutures are used, the sutures are placed equidistant along the incision. For example, 7-mm incisions (most common length) have three sections of 2.3 mm of incision length that is anchored on the ends. This distance, 2.3 mm, is greater than the diameter of the PIT, 2.1 mm OD, allowing for the PIT tags to be dropped more easily. If the suture tension too tight or too loose and/or knots to large, the suture begins to tear at the entry exit points, it allows the gaping to increase and thus providing the opening for AT tags to drop more readily. This is likely a factor as to why the Second Skin treatment group had the highest suture retention and lowest tag retention). Conversely, the bi-directional knotless suture using the tested patterns allowed for a greater rate of tag retention likely due to the correct tension held across the incision, in particular, the middle of the incision for the first 7 days. It is possible that tag retention may be more related to proper suture tension across the incision to better approximate wound margins, instead of suture retention (i.e., sutures that remain must have correct tension) during the first few stages of wound healing. This may be even more important when salmon are exposed to elevated temperatures where there are indications of cellular stress (i.e., increase in heat shock proteins, oxidative stress response, and down regulation in genes involved with ion transport; Jefferies et al. 2012), and resultant tissue texture changes allow for greater tissue tearing and increased infection potential (Larsson et al. 2012; Jerrett et al. 1998). In addition, animals exposed to rapid pressure changes may also find the increase surface area across the incision advantageous. The bi-directional knotless tissue-closure device outperformed the currently used MonocrylTM monofilament with the simple interrupted suture pattern with regards to incision openness. In humans, bi- directional knotless sutures decreased openness by providing a more uniformly distributed tension across the wound rather than at specific sites (Sadick et al. 1994; Shermak et al. 2009). 4.0 Discussion This is consistent with our results for fish held at 12°C where, by Day 14, tissue had healed to the point where no openness was recorded. Greenburg (2010) found that the greatest degree of openness is due to unequal tension burdens being placed on the knots rather than on the length of the suture line. This tension gradient across the wound may subtly interfere with uniform healing and remodeling. Incision openness may affect mortality rates because the coelomic cavity of the fish would be exposed to the water, thereby increasing ion regulatory stress and exposure to bacteria. Fish held at 17°C experienced more health and stress issues, which delayed healing. By Day 14, with the exception of Second Skin treatment group, the wounds in fish held at 12°C had healed and no openness was recorded. Only one fish in the Wide “N” treatment group held at 12°C had a transmitter 4.2 bulging (5.36 mm2) by Day 28. However, by Day 28 at 17°C, all treatment groups had fish with incision openness; the greatest average openness occurred in the Second Skin treatment group (range = 0 to 7.59 mm2). Low temperatures likely delayed the appearance of tissue necrosis, macrophage response, and clearance of bacteria and necrotic muscle tissue (Anderson and Roberts 2006). These results are similar to those of Deters et al. (2010) who also found that juvenile Chinook salmon wounds healed quicker in cooler (12°C) rather than in warmer (17°C) water temperatures. Although the Second Skin treatment had the best overall functional suture performance, all treatments had issues with suture functionality. Fish held at 12°C had greater suture retention (73.5 to 98.3%) by Day 28 at Sites 1 and 2, whereas fish held at 17°C by Day 28 had two treatments (Wide “N” and Wide “N” Knot treatments) with no remaining functional sutures at Site 2 and an overall suture retention of 0 to 77.8% at Sites 1 and 2. Walsh et al. (2000) found that by 60 days at warm temperatures (mean = 25.5°C), more than 50% of the absorbable monofilament sutures used on hybrid striped bass were expelled, whereas even by 120 days post-surgery, fish held at cold temperatures (mean = 15°C) had expelled less than 25% of sutures. Deters et al. 4.0 Discussion (2010) also found suture loss was lower after 14 days in juvenile Chinook salmon held at 17°C (36%) than in fish held at 12°C (18%) when testing seven different sutures types. Similarly, Panther et al. (2010) found that suture retention in juvenile Chinook salmon is greater in lower temperatures. Bi-directional knotless sutures tend to be more rigid than traditional monofilament used in the Second Skin treatment; this likely contributes to the sutures working themselves loose on the ends and losing the desired suture pattern in the fish. Ulceration and redness occurred in all treatment groups on all examination days. By Day 28, fish held at 12°C and 17°C the 6-Point treatment group, at both 12 and 17°C, had significantly greater ulcerated area. This result was contrary to the purpose of the barbed suture, which was to distribute tension across the incision more evenly and minimize tissue tearing. Wagner et al. (2000) and Deters et al. (2010) found tissue trauma (number of entry/exit point) and skin-to-suture contact increases irritation. Ulceration was increased when the bi-directional knotless sutures were present but were no longer functional, creating drag and increased irritation. The “tearing” of tissue observed was related to 1) the drag created by the suture hanging out of the fish (Figure 4.1A); 2) tissue bunching resulting from the barbs moving during the swimming action of the fish (Figure 3.8, Figure 4.1B); 3) the barbs tearing the tissue immediately around the entry/exit points, eventually causing the suture to fall out of the fish (Figure 4.1C); and 4) the sutures tearing towards the incision causing the suture to fall out of the fish (Figure 4.1D). The question remains whether bi-directional knotless tissue-closure devices are as effective as or more effective than traditional sutures for incision closure in juvenile Chinook salmon. As temperatures increase, suture treatment effects were diluted due to increased health or stress issues. In cooler water Second Skin is the recommended approach (2 simple sutures); while in 17°C the Wide “N” is the recommended suture. However, both the Second Skin and Wide “N” treatments had more tags dropped than with the 6-Point treatment. Based on the suture retention and suture rigidity, bi-directional knotless sutures would likely be more suitable for use with large adult fish and/or fish with large scales. Several surgery factors should be considered prior to use in field conditions. 4.0 Discussion Tissue type or tissue consistency when exposed to thermal stress and suture geometry can influence retention/loss of the bi-directional knotless tissue-closure device (Ingle and King 2010; Jefferies et al. 2012). When the sutures are embedded in tissue there are two primary modes of failure—peeling or bending of the barb. Peeling occurs when the barb pulls away from the suture; bending occurs when the barb pulls back without breaking off. Bent barbs remain intact attached to the suture, but will eventually release from the 4.3 surrounding tissue (Ingle and King 2010). A more flexible suture, barb geometry, or even number of barbs per suture may be required for better anchoring in juvenile Chinook salmon tissue. Figure 4.1. Photos Taken from Day 7 Response Examinations that Show Ulceration and Redness Often Associated with the Barbed Suture Using the Wide “N” and 6-Point Suture Patterns. A) Wide “N” pattern in SYC where the suture has slipped out of the fish creating drag. B) 6-Point suture pattern where the suture is tightening, tearing the tissue towards the incision. C) Wide “N” pattern where the suture has slipped out or pulled into the fish leaving a torn or rubbed area associated with entry and exit points. D) Second Skin pattern where the suture has torn the tissue towards the incision. A B C D B A A A B A C D D C Figure 4.1. Photos Taken from Day 7 Response Examinations that Show Ulceration and Redness Often Associated with the Barbed Suture Using the Wide “N” and 6-Point Suture Patterns. A) Wide “N” pattern in SYC where the suture has slipped out of the fish creating drag. B) 6-Point suture pattern where the suture is tightening, tearing the tissue towards the incision. C) Wide “N” pattern where the suture has slipped out or pulled into the fish leaving a torn or rubbed area associated with entry and exit points. D) Second Skin pattern where the suture has torn the tissue towards the incision. Figure 4.1. Photos Taken from Day 7 Response Examinations that Show Ulceration and Redness Often Associated with the Barbed Suture Using the Wide “N” and 6-Point Suture Patterns. A) Wide “N” pattern in SYC where the suture has slipped out of the fish creating drag. B) 6-Point suture pattern where the suture is tightening, tearing the tissue towards the incision. 4.0 Discussion C) Wide “N” pattern where the suture has slipped out or pulled into the fish leaving a torn or rubbed area associated with entry and exit points. D) Second Skin pattern where the suture has torn the tissue towards the incision. 4.4 4.4 5.0 References Anderson CD and RJ Roberts. 1975. “A comparison of the effects of temperature on wound healing in a tropical and a temperate teleost.” Journal of Fish Biology 7:173–182. Angiotech 2011. http://www.angioedupro.com/Quill/index.php?ID=Photos. Accessed March 15, 2011. Bauer C and G Loupal. 2007. “Common carp tissue reactions to surgically implanted radio tags with external antennas.” Journal of Fish Biology 70(1):292–297. Brett JR. 1995. “Energetics.” In Physiological Ecology of Pacific Salmon, C Groot, L Margolis, and WC Clarke (eds.), pp. 3-68, University of British Columbia Press, Vancouver. 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Coutant CC and RR Whitney. 2000. 5.0 References “Fish behavior in relation to passage through hydropower turbines: A review.” Transactions of the American Fisheries Society 129(2):351–380. 5.1 Deters KA, RS Brown, KM Carter, JW Boyd, MB Eppard, and AG Seaburg. 2010. “Performance assessment of suture type, water temperature, and surgeon skill in juvenile Chinook salmon surgically implanted with acoustic transmitters.” Transactions of the American Fisheries Society 139(3):888–899. Faber DM, MA Weiland, R Moursund, TJ Carlson, N Adams, and D Rhondorf. 2001. Evaluation of the Fish Passage Effectiveness of the Bonneville I Prototype Surface Collector Using Three-Dimensional Ultrasonic Fish Tracking. PNNL-13526, prepared for the U.S. Army Corps of Engineers, Portland, Oregon, by Pacific Northwest National Laboratory, Richland, Washington. Fontenot DK and DL Neiffer. 2004. “Wound management in teleost fish: Biology of the healing process, evaluation, and treatment.” The Veterinary Clinics: Exotic Animal Practice 7(1):57–86. Frost DA, RL McComas, and BP Sandford. 2010. “The effects of a surgically implanted microacoustic tag on growth and survival in subyearling fall Chinook salmon.” Transactions of the American Fisheries Society 139(4):1192–1197. Gheorghiu C, J Hanna, JW Smith, DS Smith, and MP Wilkie. 2010. “Encapsulation and migration of PIT tags implanted in brown trout (Salmo trutta L.).” Aquaculture 298(3–4):350–353. Greenburg JA and RM Clark. 2009. “Advances in suture material for obstetric and gynecologic surgery.” Reviews in Obstetrics and Gynecology 2(3):146–158. Greenburg JA. 2010. “The use of barbed sutures in obstetrics and gynecology.” Reviews in Obstetrics and Gynecology 3(3):82–91. Harms CA. 2005. “Surgery in fish research: Common procedures and postoperative care.” Lab Animal 34(1):28–34. Ingle NP and MW King. 2010. “Optimizing the tissue anchoring performance of barbed sutures in skin and tendon tissues.” Journal of Biomechanics 43(2):302–309. Jefferies KM, SG Hinch, T Sierocinski, TD Clark, EJ Eliason, MR Donaldson, S Li, P Pavlidis, and KM Miller. 2012. “Consequences of high temperatures and premature mortality on the transcriptome and blood physiology of wild adult sockeye salmon (Oncorhynchus nerka).” Ecology and Evolution 2(7):1747–1764. Jepsen N, A Koed, EB Thorstad, and E Baras. 2002. “Surgical implantation of telemetry transmitters in fish: How much have we learned?” Hydrobiologia 483(1–3):239–248. Jerrett AR, AJ Holland, and SE Cleaver. 1998. “Rigor contractions in “rested” and “partially exercised” Chinook salmon white muscle as affected by temperature.” Journal of Food Science 63(1):53–56. Knights BC and BA Lasee. 1996. “Effects of implanted transmitters on adult bluegills at two temperatures.” Transactions of the American Fisheries Society 125(3):440–449. LaCroix GL, D Knox, and P McCurdy. 2004. 5.0 References “Effects of implanted dummy acoustic transmitters on juvenile Atlantic salmon.” Transactions of the American Fisheries Society 133(1):211–220. 5.2 Larsson T, T Mørkøre, K Kolstad, T-K Østbye, S Afanasyev, and A Krasnov. 2012. “Gene expression profiling of soft and firm Atlantic salmon filet.” PLoS One 7(6):e39219, 1–9. Leaper DL. 2010. “Surgical-site infection.” British Journal of Surgery 97(11):1601–1602. Leaper DL. 2010. “Surgical-site infection.” British Journal of Surgery 97(11):1601–1602. Leung JC, GL Ruff, MW King, and PP Dattilo, Jr. 2003. “Barbed, bidirectional surgical sutures.” International Conference and Exhibition on Healthcare and Medical Textiles, Conference Proceedings, July 8–9, Bolton, United Kingdom. Lin PH, MK Hirko, JA von Fraunhofer, and HP Greisler. 1996. “Wound healing and inflammatory responses to biomaterials.” In Wound Closure Biomaterials and Devices, CC Chu, JA von Fraunhofer, and HP Greisler (eds), pp. 7–24, CRP Press, Ithaca, New York. McComas RL, D Frost, SG Smith, JW Ferguson, TJ Carlson, and T Aboellail. 2005. A Study to Estimate Salmonid Survival Through the Columbia River Estuary Using Acoustic Tags, 2002. Report to the U.S. Army Corps of Engineers, Portland, Oregon. Contract #E86910060, National Oceanic and Atmospheric Administration- National Marine Fisheries Service, Northwest Fisheries Science Center, Seattle, Washington. Muir WD, SG Smith, JG Williams, and BP Sandford. 2001. “Survival of juvenile salmonids passing through bypass systems, turbines, and spillways with and without flow detector at Snake River Dams.” North American Journal of Fisheries Management 21(1):135–146. Mulcahy DM. 2003. “Surgical implantation of transmitters into fish.” ILAR Journal 44(4):295–306. Panther JL, RS Brown, G Gaulke, KA Deters, CM Woodley, and MB Eppard. 2010. Influence of Incision Location on Transmitter Loss, Healing, Survival, Growth, and Suture Retention of Juvenile Chinook Salmon. PNNL-19192, Pacific Northwest National Laboratory, Richland, Washington. Ploskey GR, MA Weiland, JS Hughes, SR Zimmerman, RE Durham, ES Fischer, J Kim, RL Townsend, JR Skalski, RA Buchanan, and RL McComas. 2008. Survival of Juvenile Chinook Salmon Passing the Bonneville Dam Spillway in 2007. PNNL-18113, prepared for U.S. Army Corps of Engineers, Portland District, Portland, Oregon, by Pacific Northwest National Laboratory, Richland, Washington. Rombough PJ. 2007. “Ontogenetic changes in the toxicity and efficacy of the anesthetic MS-222 (tricaine methanesulfonate) in zebrafish (Danio rerio) larvae.” Comparative Biochemistry and Physiology – Part A: Molecular & Integrative Physiology 148(2):463–469. Sadick NS, DL Damelio, and C Weinstein. 1994. 5.0 References “The modified buried vertical mattress suture – A new technique of buried absorbable wound closure associated with excellent cosmesis for wounds under tension.” Journal of Dermatologic Surgery & Oncology 20(11):735–739. Sauter ST. 1996. “Thermal Preference of Spring and Fall Chinook Salmon (Oncorhynchus tshawytscha) During Smoltification.” Master’s Thesis, Portland State University, Portland Oregon. 164 pp. Scruton DA, CJ Pennell, CE Bourgeois, RF Goosney, TR Porter, and KD Clarke. 2007. “Assessment of a retrofitted downstream fish bypass system for wild Atlantic salmon (Salmo salar) smolts and kelts at a hydroelectric facility on the Exploits River, Newfoundland, Canada.” Hydrobiologia 582(1):155–169. 5.3 Shermak MA, J Mallalieu, and D Change. 2009. “Barbed suture impact on wound closure in body contouring surgery.” Plastic and Reconstructive Surgery 126(5):1735–1741. Skalski JR. 1998. “Estimating season-wide survival rates of outmigrating salmon smolt in the Snake River, Washington.” Canadian Journal of Fisheries and Aquatic Sciences 55(3):761–769. Skalski JR, R Townsend, J Lady, AE Giorgi, JR Stevenson, and RD McDonald. 2002. “Estimating route-specific passage and survival probabilities at a hydroelectric project from smolt radio telemetry studies.” Canadian Journal of Fisheries and Aquatic Sciences 59(8):1385–1393. Stephenson JR, AJ Gingerich, RS Brown, BD Pflugrath, Z Deng, TJ Carlson, MJ Langeslay, ML Ahmann, RL Johnson, and AG Seaburg. 2010. “Assessing barotrauma in neutrally and negatively buoyant juvenile salmonids exposed to simulated hydro-turbine passage using a mobile aquatic barotrauma laboratory.” Fisheries Research 106(3):271–278. Summerfelt RC and LS Smith. 1990. “Anesthesia, surgery, and related techniques.” In Methods for Fish Biology, CB Schreck and PB Moyle (eds), pp. 213–263, American Fisheries Society, Bethesda, Maryland. van Rijssel EJ, R Brand, C Admiraal, I Smit, and JB Trimbos. 1989. “Tissue reaction and surgical knots: The effect of suture size, knot configuration, and knot volume.” Obstetrics and Gynecology 74(1):64–68. Wagner GN, ED Stevens, and P Byrne. 2000. “Effects of suture type and patterns on surgical wound healing in rainbow trout.” Transactions of the American Fisheries Society 129(5):1196–1205. Walsh MG, KA Bjorgo, and JJ Isely. 2000. “Effects of implantation method and temperature on mortality and loss of simulated transmitters in hybrid striped bass.” Transactions of the American Fisheries Society 129:539–544. Weiland MA, GR Ploskey, JS Hughes, Z Deng, T Fu, TJ Monter, GE Johnson, F Khan, MC Wilberding, AW Cushing, SR Zimmerman, DM Faber, RE Durham, RL Townsend, JR Skalski, J Kim, ES Fischer, and MM Meyer. 2009. OFFSITE M. Brad Eppard U.S. Army Corps of Engineers P.O. Box 2946 333 SW 1st Avenue, Robert Duncan Plaza Portland, OR 97208-2946 M. Brad Eppard U.S. Army Corps of Engineers P.O. Box 2946 333 SW 1st Avenue, Robert Duncan Plaza Portland, OR 97208-2946 AJ Bryson KA Wagner 5.0 References Acoustic Telemetry Evaluation of Juvenile Salmonid Passage and Survival at John Day Dam with Emphasis on the Prototype Surface Flow Outlet, 2008. PNNL-18890, Pacific Northwest National Laboratory, Richland, Washington. 5.4 5.4 PNNL-21986 PNNL-21986 Distribution No. of No. of Copies Copies 1 OFFSITE M. Brad Eppard U.S. Army Corps of Engineers P.O. Box 2946 333 SW 1st Avenue, Robert Duncan Plaza Portland, OR 97208-2946 ONSITE 3 Pacific Northwest National Laboratory AJ Bryson SEQUIM KA Wagner K6-75 CM Woodley SEQUIM No. of Copies No. of Copies
https://openalex.org/W3022663263	https://europepmc.org/articles/pmc7281964?pdf=render	English	null	Red Blood Cell Folate Likely Overestimated in Australian National Survey: Implications for Neural Tube Defect Risk	Nutrients	2,020	cc-by	4,051	Received: 24 March 2020; Accepted: 28 April 2020; Published: 1 May 2020 Abstract: In 2009, the Australian government mandated the addition of folic acid to bread ﬂour to reduce the incidence of neural tube defects (NTD)-aﬀected pregnancies. In 2011–2012, the Australian Health Measures Survey (AHMS) reported a mean red blood cell (RBC) folate in women of reproductive age (16–44 y) of 1647 nmol/L. Over 99% of women had an RBC folate ≥906 nmol/L, a concentration consistent with a very low risk of NTDs if a woman became pregnant. However, RBC folate was measured using an immunoassay, which is not a recommended method due to questionable accuracy. The microbiological assay is the preferred method for RBC folate measurement. To determine whether the immunoassay method may have led to spurious conclusions about the folate status of Australian women, we collected fasting blood samples from 74 healthy non-pregnant, non-lactating women (18–44 y) and measured RBC folate using both the immunoassay and microbiological methods. Mean RBC folate (95% conﬁdence interval) concentration measured with the immunoassay method was 1735 (1666, 1804) nmol/L compared with 942 (887, 1012) nmol/L using the microbiological method. No woman had an RBC folate < 906 nmol/L using the immunoassay method, whereas 46% of women had an RBC folate < 906 nmol/L using the microbiological method. The NTD risk was estimated to be 0.06% using the immunoassay method and 0.14% using the microbiological method. RBC folate using AHMS survey may have underestimated NTD risk in Australian women. Keywords: red cell folate; microbiological assay; immunoassay; folate measurement; neural tube defects Commentary Red Blood Cell Folate Likely Overestimated in Australian National Survey: Implications for Neural Tube Defect Risk Green 2,3,* 1 School of Agriculture, Food and Wine, Faculty of Sciences, University of Adelaide, Adelaide 5005, South Australia, Australia; Shannon.Hunt@sahmri.com 1 School of Agriculture, Food and Wine, Faculty of Sciences, University of Adelaide, Adelaide 5005, South Australia, Australia; Shannon.Hunt@sahmri.com 2 Women and Kids Theme, South Australian Health and Medical Research Institute, Adelaide 5000, South Australia, Australia; Merryn.Netting@sahmri.com (M.J.N.); Thomas.Sullivan@sahmri.com (T.R.S.); karen.best@sahmri.com (K.P.B.); Maria.Makrides@sahmri.com (M.M.); Bev.Muhlhausler@csiro.au (B.S.M.) 3 Adelaide Medical School, University of Adelaide, Adelaide 5005, South Australia, Australia 4 School of Public Health, University of Adelaide, Adelaide 5005, South Australia, Australia 5 Department of Human Nutrition, University of Otago, Dunedin 9016, New Zealand; lisa.houghton@otago.ac.nz 2 Women and Kids Theme, South Australian Health and Medical Research Institute, Adelaide 5000, South Australia, Australia; Merryn.Netting@sahmri.com (M.J.N.); Thomas.Sullivan@sahmri.com (T.R.S.); karen.best@sahmri.com (K.P.B.); Maria.Makrides@sahmri.com (M.M.); Bev.Muhlhausler@csiro.au (B.S.M.) 2 Women and Kids Theme, South Australian Health and Medical Research Institute, Adelaide 5000, South Australia, Australia; Merryn.Netting@sahmri.com (M.J.N.); Thomas.Sullivan@sahmri.com (T.R.S.); karen.best@sahmri.com (K.P.B.); Maria.Makrides@sahmri.com (M.M.); Bev.Muhlhausler@csiro.au (B.S.M.) 3 Adelaide Medical School, University of Adelaide, Adelaide 5005, South Australia, Australia 4 School of Public Health, University of Adelaide, Adelaide 5005, South Australia, Australia 2 Women and Kids Theme, South Australian Health and Medical Research Institute, Adelaide 5000, South Australia, Australia; Merryn.Netting@sahmri.com (M.J.N.); Thomas.Sullivan@sahmri.com (T.R.S.); karen.best@sahmri.com (K.P.B.); Maria.Makrides@sahmri.com (M.M.); Bev.Muhlhausler@csiro.au (B.S.M.) 3 Adelaide Medical School, University of Adelaide, Adelaide 5005, South Australia, Australia 4 School of Public Health, University of Adelaide, Adelaide 5005, South Australia, Australia 5 Department of Human Nutrition, University of Otago, Dunedin 9016, New Zealand; lisa.houghton@otago.ac.nz karen.best@sahmri.com (K.P.B.); Maria.Makrides@sahmri.com (M.M.); Bev.Muhlhausler@csiro.au (B.S.M.) 3 Adelaide Medical School, University of Adelaide, Adelaide 5005, South Australia, Australia 4 School of Public Health, University of Adelaide, Adelaide 5005, South Australia, Australia 5 Department of Human Nutrition University of Otago Dunedin 9016 New Zealand; Adelaide Medical School, University of Adelaide, Adelaide 5005, South Australia, Australia 4 School of Public Health, University of Adelaide, Adelaide 5005, South Australia, Australia 5 D f H N i i U i i f O D di 9016 N Z l d g g 6 Nutrition and Health, Health and Biosecurity Business Unit, Commonwealth Scientiﬁc and Industrial Research Organisation, Adelaide 5000, South Australia, Australia * C d ti @ h i T l 61 452 448 438 6 Nutrition and Health, Health and Biosecurity Business Unit, Commonwealth Scientiﬁc and Industrial * Correspondence: tim.green@sahmri.com; Tel.: +61-452-448-438 Received: 24 March 2020; Accepted: 28 April 2020; Published: 1 May 2020 Received: 24 March 2020; Accepted: 28 April 2020; Published: 1 May 2020 Commentary Red Blood Cell Folate Likely Overestimated in Australian National Survey: Implications for Neural Tube Defect Risk Shannon E. Hunt 1,2, Merryn J. Netting 2,3 , Thomas R. Sullivan 2,4, Karen P. Best 2,3, Lisa A. Houghton 5, Maria Makrides 2,3 , Beverly S. Muhlhausler 2,6 and Tim J. Green 2,3,* Shannon E. Hunt 1,2, Merryn J. Netting 2,3 , Thomas R. Sullivan 2,4, Karen P. Best 2,3, Lisa A. Houghton 5, Maria Makrides 2,3 , Beverly S. Muhlhausler 2,6 and Tim J. nutrients nutrients nutrients nutrients 1. Introduction Neural tube defects (NTD), such as spina biﬁda and anencephaly, are caused by the failure of the neural tube to close normally, at around 28 days post-conception. In controlled trials, it has been shown that folic acid taken prior to conception and early pregnancy reduces the incidence of NTDs by up to 80% [1,2]. In 2005 the Australian National Health and Medical Research Council recommended that all Nutrients 2020, 12, 1283; doi:10.3390/nu12051283 www.mdpi.com/journal/nutrients 2 of 6 Nutrients 2020, 12, 1283 women planning a pregnancy take 400 ug of folic acid daily prior to conception and until the end of the ﬁrst trimester to prevent NTDs Around 40% of pregnancies are unplanned in Australia [3], and because NTDs occur before many women know they are pregnant, more than 80 countries have mandated the addition of folic acid to food staples, typically wheat ﬂour. In 2009, the Australian Food Regulation Ministerial Council mandated the fortiﬁcation of bread ﬂour with folic acid [4]. This increased folic acid intakes of the population and reduced the incidence of NTDs, especially among teenagers and Indigenous women [5]. To achieve a maximal reduction in NTDs with folic acid fortiﬁcation, it is generally accepted that achieving a red blood cell (RBC) folate ≥906 nmol/L among women of reproductive age is desirable [6,7]. In the nationally representative 2011-2012 Australian Health Measures Survey (AHMS) the mean RBC folate in women of reproductive age (16–44 y) was 1647 nmol/L (relative standard error of 0.8%) with only 1% of sample concentrations < 906 nmol/L, suggesting that women were well protected against NTDs if they were to become pregnant [8]. The AHMS RBC folate concentrations are much higher than the mean concentration of 1057 nmol/L reported for women in the US National Health and Examination Survey, where more foods are fortiﬁed with folic acid and supplement use is higher [9]. The microbiological assay is considered the gold standard for RBC folate measurement, especially in population-based studies [6,10], and was the method used in the US national survey [9]. However, in the AHMS, RBC folate was measured by an immunoassay using a folate-binding protein on an automated clinical analyser. Although immunoassays are suitable for measuring plasma/serum folate where the predominant form of folate is 5 methyltetrahydrofolate, their accuracy for RBC folate measurement has been reported to be poor [10–13]. 1. Introduction This lack of accuracy has been attributed to matrix eﬀects in red cells as well as diﬀerent binding aﬃnities of the folate-binding protein for the various forms of folate found in red blood cells. Our aim in this paper was to investigate whether the immunoassay used in the AHMS led to erroneous conclusions about the folate status of Australian women following folic acid fortiﬁcation and calculated risk of an NTD aﬀected pregnancy. Here, we compare RBC folate concentrations measured in blood samples collected from women using the immunoassay (as used in the AHMS survey) and the gold standard microbiological assay method. 2.1. Participants Seventy-four healthy female volunteers were recruited through newspaper advertisements, posters, social media and leaﬂet distribution within Adelaide, South Australia. Women were eligible if they were not pregnant or breastfeeding, and/or had not taken folic acid containing supplements in the four months prior. Ethical approval was obtained from the University of Adelaide Human Research Ethics Committee HREC-2016-151 and women gave written informed consent. 2.3. Measurement of Red Cell Folate Immunoassay: Frozen whole blood samples were sent to SA Pathology (Adelaide) for whole blood folate determination using an Elecsys®Folate RBC kit (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) on a Roche Modular E 801 Immunology Analyzer (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) [14]. A detailed explanation of the principle of the assay is available online. This method was identical to that used in the AHMS. Microbiological assay: Whole blood lysates and plasma samples were sent on dry ice to The University of Otago, Dunedin, New Zealand for folate determination using the microbiological assay [15,16]. Brieﬂy, the microbiological assay was conducted using a 96-well plate with chloramphenicol-resistant Lactobacillus rhamnosus (ATCC 27773) (American Type Culture Collection, Manassas, VA, USA) and 5-methyltetrahydrofolate calibrator (Merck and Cie, Schaﬀhausen, Switzerland). External and internal controls were included on each microplate. Intra- and inter-assay variation was less than 10% for plasma folate and 14% for red cell folate. 2.2. Procedures Women attended a morning clinic after fasting since midnight. Venipuncture blood samples were collected into two evacuated containers containing EDTA (Becton Dickinson PTY, Macquarie Park, NSW). One tube was sent within 3 hours to a commercial lab for a full blood count, including hematocrit using an automated hematology analyzer (Clinpath, Adelaide, SA, Australia). For the immunoassay, whole blood was aliquoted in two cryovials. For the microbiological assay, whole blood was diluted 1:10 in 1% ascorbic acid (Sigma Aldrich, St Louis, MO, USA) incubated for 30 minutes at 37 ◦C and then aliquoted. The remaining whole blood was centrifuged at 3000 g for 10 minutes at 4 ◦C and the resulting plasma aliquoted. All samples were stored at -80 ◦C until analyzed. Nutrients 2020, 12, 1283 3 of 6 2.4. Data Analyses Assuming a standard deviation of 358 nmol/L for the immunoassay method [7], a sample size of 74 women allows for mean RBC folate concentration to be estimated with a suﬃciently narrow 95% conﬁdence interval (width of approximately ± 80 nmol/L). Red blood cell folate was determined by the equation used in the AHMS which corrects whole blood folate for hematocrit. This assumes the amount of folate present in plasma is negligible. RBC folate concentrations were found to be approximately normally distributed for both the immunoassay and the microbiological method. The association between the two measurement methods was assessed using linear regression, with agreement quantiﬁed using a Bland–Altman plot. Predicted NTD risk based on RBC folate was then calculated for each method using the equation in Daly et al. [7]. All statistical calculations were performed using Stata 15.0 (StataCorp, College Station, Texas, USA). 4. Discussion We have shown that RBC folate concentrations among women of reproductive age in Australia may have been overestimated in the AHMS survey. The use of immunoassays to measure RBC folate have been particularly problematic because assays, particularly calibrators, are not harmonized among manufacturers, they have high lot-to-lot variation, and are of questionable accuracy. Although immunoassays are suitable for serum folate where folate is present primarily as 5-methyltetrahydrofolate, the folate binding protein used in these assays has diﬀerent aﬃnities for the many forms of folate present in in red blood cells. Colapinto et al. [11] undertook a method comparison to allow for adjustment of RBC folate concentrations in the Canadian Health Measures Survey measured using an Immulite 2000 immunoassay (Siemens Canada Limited) with the US National Health and Nutrition Examination Survey using the microbiological assay. They reported a mean percent diﬀerence of 24% between the microbiological assay and immunoassay methods (95% limits of agreement -26% to 75%) and a correlation of 0.67 between methods. This ﬁnding compares to the absolute mean diﬀerence of 793 nmol/L (95% limits of agreement 198 to 1388 nmol/L) and weaker correlation of 0.40 in our study. To our knowledge, only one other study compared the immunoassay used in the Elecsys®Folate RBC kit to other immunoassays. Golding placed himself on a folate deﬁcient diet and measured RBC folate weekly until megaloblastic anemia appeared. RBC folate measured using the AHMS system consistently gave much higher concentrations than other immunoassay methods as well as the microbiological method [12]. Another important ﬁnding was that the diﬀerence in folate values obtained using the two methods was not merely systematic but also random, meaning that the folate values estimated in the AHMS could not be reliably converted to equivalent values that would be obtained using the microbiological assay. The present study has a number of strengths including the measurement of folate by the methods in the same participant blood sample, removing any intra-individual variation in folate concentrations. Secondly, the immunoassay used, the Elecsys®Folate RBC kit, is identical to that used in the AHMS. Finally, we have conﬁdence in the values obtained from the microbiological assay. The microbiologic assay used by the University of Otago was compared against target values from samples provided by the US Centre for Disease Control in a round robin comparison [10]. 3. Results The median (inter-quartile range) age of the women was 27 (23, 34) years, 92% were of European ethnicity, 78% had an undergraduate degree or higher, and 66% had a normal BMI (18.5–24.9 kg/m2). Mean RBC folate (95% conﬁdence interval) concentration measured with immunoassay method was almost twice that of the microbiological method; 1735 (1666, 1804) compared with 942 (887, 1012) nmol/L, respectively. The mean diﬀerence between the two methods was 793 nmol/L (724, 862); p < 0.001), indicating a high degree of systematic error. There was a poor correlation between RBC folate measured using the two methods (R2 = 0.16; p < 0.001) (Figure 1A). The Bland–Altman plot also showed a poor level of agreement between the two methods (Figure 1B), with the immunoassay method expected to produce concentrations between 198 and 1388 nmol/L larger than the microbiological method for 95% of women (equivalent to the mean diﬀerence ± 2 standard deviations). The large width of the 95% limits of agreement indicates a high degree of random error. Using the immunoassay method, no women had an RBC folate < 906, whereas 34 women (46%) had an RBC folate < 906 nmol/L using the microbiological method. 4 of 6 Nutrients 2020, 12, 1283 Figure 1. Comparison of red blood cell folate concentrations measured in concurrent venous blood samples using microbiological [chloramphenicol-resistant L. rhamnosus (ATCC 27773 or NCIB 10463) assay and erythrocyte folate measured by the protein-binding assay (Roche Modular E 801 Immunology Analyzer) (A) by regression; (B) and by a Bland–Altman Plot (diﬀerence on the y-axis is the immunoassay – microbiological RBC folate concentrations, with the shaded area corresponding to a 95% conﬁdence interval for the mean diﬀerence). Figure 1. Comparison of red blood cell folate concentrations measured in concurrent venous blood samples using microbiological [chloramphenicol-resistant L. rhamnosus (ATCC 27773 or NCIB 10463) assay and erythrocyte folate measured by the protein-binding assay (Roche Modular E 801 Immunology Analyzer) (A) by regression; (B) and by a Bland–Altman Plot (diﬀerence on the y-axis is the immunoassay – microbiological RBC folate concentrations, with the shaded area corresponding to a 95% conﬁdence interval for the mean diﬀerence). 4. Discussion Values produced by Otago were 4.2% ± 9.6% higher for RBC folate than the US Centre for Disease Control target value. There are limitations to our study. Although attaining a red blood cell folate concentration of 906 nmol/L is a threshold recommended by WHO and others, it does not represent a threshold for the maximum prevention of neural tube defects. There is debate around the optimal cutoﬀfor NTD 5 of 6 Nutrients 2020, 12, 1283 prevention. An RBC folate concentration of 906 nmol/L was the lower bound of the upper quintile of red blood cell folate in the Irish case–control study [7]. We stress that the model for the relationship between RBC folate and NTD risk is continuous and have provided NTD risk estimates using both the immunoassay and microbiological methods (that diﬀer substantially). Any increase in RBC folate would be expected to decrease NTD risk. However, we accept that our estimates of NTD risk based on RBC folate should be interpreted with caution as they are based on data from a diﬀerent population [7]. Another important consideration is that we used 5-methyltetrahydrofolate as a calibrator, which is consistent with current recommendations, instead of folic acid as was used in earlier studies. It has been suggested that 5-methyltetrahydrofolate gives RBC folate concentrations from the microbiological method that are ~20% lower [17]. As such a lower cutoﬀof > 748 nmol/L for RBC folate has been suggested for NTD protection when 5-methyltetrahydrofolate is used as the calibrator. Krider et al., using data from two studies in China, a large population-based study (n = 247831) and a dose response trial (n = 1194), estimated 822 nmol/L (calibrator adjusted) as a cutoﬀ[18]. In our sample, 27% and 34% of women still had RBC folate concentrations below 748 and 822nmol/L, respectively. Importantly, adjusting RBC folate measurements according to the calibrator used would not change conclusions about the high degree of systematic and random error of the immunological method. We are not suggesting that our sample is representative of the general Australian population, or that folic acid fortiﬁcation has been ineﬀective in Australia. Clearly folic acid intakes have increased and rates of NTDs have decreased, especially among vulnerable population subgroups in vulnerable populations, as a result of the fortiﬁcation [5]. 4. Discussion Our mean RBC folate concentrations of 942 nmol/L are more consistent with the 1060 nmol/L reported in population-based US data (National Health and Nutrition Examination Survey 2008–2010) using the microbiological method [9]. 5. Conclusions In conclusion, there is an urgent need to assess the impact of folic acid fortiﬁcation on RBC folate using accurate methods not only in women of reproductive age but also in non-target populations who are being exposed to folic acid with no known beneﬁt such as children, men, and older people. Given that most clinical laboratories in Australia measure RBC folate using immunoassays, caution is warranted when interpreting these results. Author Contributions: Conceptualization, S.E.H., T.J.G., M.J.N. and B.S.M.; methodology, L.A.H.; data collection, S.E.H.; formal analysis, T.R.S.; writing–original draft preparation, S.E.H. and T.J.G.; K.P.B. and M.M.—review of scientiﬁc content; writing—review and editing, all co-authors. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding Conﬂicts of Interest: M.M. reports grants from National Health and Medical Research Council during the conduct of the study; she has also received honoraria from Nestle and Fonterra for memberships of Scientiﬁc Advisory Board, which have been paid to her institution, outside the submitted work. B.S.M. has received honoraria for serving on the Nestle Nutrition Institute Advisory board and for giving presentations for BASF, which have been paid to her institution, outside the submitted work. T.J.G. reports grants from the International Development Research Centre, Nutrition International, and the Nestle Nutrition Institute; he has also received honoraria from Fonterra for membership on a Scientiﬁc Advisory Board that has been paid to his institution, outside the submitted work. 1. 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https://openalex.org/W2070430432	https://europepmc.org/articles/pmc2952798?pdf=render	English	null	The Association between the Availability of Sugar-Sweetened Beverage in School Vending Machines and Its Consumption among Adolescents in California: A Propensity Score Matching Approach	Journal of environmental and public health	2,010	cc-by	3,112	Hindawi Publishing Corporation Journal of Environmental and Public Health Volume 2010, Article ID 735613, 5 pages doi:10.1155/2010/735613 Hindawi Publishing Corporation Journal of Environmental and Public Health Volume 2010, Article ID 735613, 5 pages doi:10.1155/2010/735613 Hindawi Publishing Corporation Journal of Environmental and Public Health Volume 2010, Article ID 735613, 5 pages doi:10.1155/2010/735613 Correspondence should be addressed to Lu Shi, lushi.pku@gmail.com Received 10 June 2010; Revised 31 August 2010; Accepted 31 August 2010 Academic Editor: David Vlahov Copyright © 2010 Lu Shi. 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. There is controversy over to what degree banning sugar-sweetened beverage (SSB) sales at schools could decrease the SSB intake. This paper uses the adolescent sample of 2005 California Health Interview Survey to estimate the association between the availability of SSB from school vending machines and the amount of SSB consumption. Propensity score stratiﬁcation and kernel- based propensity score matching are used to address the selection bias issue in cross-sectional data. Propensity score stratiﬁcation shows that adolescents who had access to SSB through their school vending machines consumed 0.170 more drinks of SSB than those who did not (P < .05). Kernel-based propensity score matching shows the SSB consumption diﬀerence to be 0.158 on the prior day (P < .05). This paper strengthens the evidence for the association between SSB availability via school vending machines and the actual SSB consumption, while future studies are needed to explore changes in other beverages after SSB becomes less available. 1. Introduction This paper uses population survey data to examine the magnitude of the association between the availability of sugar-sweetened beverage from schools’ vending machines and the amount of SSB consumption among California’s adolescents, while controlling for sociodemographic and behavioral confounders [10]. Speciﬁcally, by estimating how much the availability of SSB through school vending machines independently predicts the SSB consumption, this study provides a benchmark for future evaluation of SSB interventions among younger populations. Health researchers and public health activists found school the environment to be an important determinant of eating and drinking behaviors among children [1, 2]. Competitive foods, that is, foods and beverages sold from vending machines, school stores, and so forth, remain a prevalent health risk for school-age children [3–7]. In recent years, eﬀorts have been focused on taxing sugar elements in the sugar-sweetened beverage (SSB) or sugar itself. For example, California bill SB677, a law passed in 2003, bans soda from elementary and middle schools and limits soda sales in high schools during school hours. However, there remains considerable controversy over how much the increase in the consumption of SSB has contributed to the increase in childhood obesity and how much limiting the SSB sales at schools could decrease the soda intake and the body weight [8, 9]. Lu Shi Department of Health Services, UCLA School of Public Health, 650 Charles E. Young Drive S., 61-253 CHS, Los Angeles, CA 90095, USA Journal of Environmental and Public Health Journal of Environmental and Public Health Journal of Environmental and Public Health 2 Table 1: Descriptive statistics about variables used in the propensity score matching. Variables Descriptive statistics (mean or frequency distribution) Outcome variable Number of SSBs on the prior day 1.09 (1.45) Exposure variable Self-reported availability of sugar-sweetened beverage at school vending machines Yes 2285 (57.4%) No 1698 (42.6%) Predictors Age (mean) 14.4 (.026) Gender (female) 1979 (49.1%) Parental education Less than high school (referent group) 184 (21.0%) High school only 180 (20.3%) Some college 189 (21.3%) Graduated from college 334 (37.7%) Race/ethnicity White (referent group) 70 (7.9%) African American 176 (4.4%) Latino 823 (20.7%) Asian 321 (8.1%) School type (public schools) 3505 (87.0%) Household income (at or above federal poverty line) 3490 (87.6%) N = 3983 Table 1: Descriptive statistics about variables used in the propensity score matching. Table 2: Probit regression predicting the propensity scores of sugar-sweetened beverage’s availability at school vending machines. Predictor Probit coeﬃcient Gender (female = 1) 0.06 Age 2 .33∗∗∗ Age square −0.07∗∗∗ Parental education Less than high school (referent group) High school only −0.03 Some college 0.07 Graduated from college −0.11 Race/ethnicity White (referent group) African American 0.02 Latino 1.02 Asian and other 0.98 Household income (above federal poverty line = 1) 0.05 School type (attending public school = 1) 0.16∗∗ N = 3983 Coeﬃcient signiﬁcant at 10%∗; 5%∗∗; 1%∗∗∗. Table 2: Probit regression predicting the propensity scores of sugar-sweetened beverage’s availability at school vending machines. Predictor Probit coeﬃcient Gender (female = 1) 0.06 Age 2 .33∗∗∗ Age square −0.07∗∗∗ Parental education Less than high school (referent group) High school only −0.03 Some college 0.07 Graduated from college −0.11 Race/ethnicity White (referent group) African American 0.02 Latino 1.02 Asian and other 0.98 Household income (above federal poverty line = 1) 0.05 School type (attending public school = 1) 0.16∗∗ N = 3983 Coeﬃcient signiﬁcant at 10%∗; 5%∗∗; 1%∗∗∗. g p g p p Predictor Gender (female = 1) Age Age square Parental education Less than high school (referent group) High school only Some college Graduated from college Race/ethnicity White (referent group) African American Latino Asian and other Household income (above federal poverty line = 1) School type (attending public school = 1) N = 3983 Coeﬃcient signiﬁcant at 10%∗; 5%∗∗; 1%∗∗∗. 2. Method The dataset used in this study is the adolescent sample of 2005 California Health Interview Survey (CHIS). CHIS is a biennial population health survey based on telephone interviews, and its adolescent sample is conducted with Coeﬃcient signiﬁcant at 10%∗; 5%∗∗; 1%∗∗∗. Journal of Environmental and Public Health Journal of Environmental and Public Health 3 Table 3: Blockwise t-tests of the mean diﬀerence of propensity score between the exposure group and the control group. Block Mean of control group Mean of exposure group P value of the t-tests [0, 0.2) 0.19 0.19 .71 [0.2,0.4) 0.27 0.26 .23 [0.4, 0.5) 0.44 0.45 .20 [0.5,0.6) 0.57 0.57 .20 [0.6,0.625) 0.61 0.61 .11 [0.625,0.65) 0.64 0.64 .73 [0.65,0.7) 0.68 0.68 .60 [0.7,0.8) 0.74 0.74 .08 [0.8,1) 0.81 0.81 .57 N = 3983 se t-tests of the mean diﬀerence of propensity score between the exposure group and the control group. reported that their schools had SSB available through the vending machines, and 1698 said that their schools did not have SSB through the vending machines. Table 1 lists the descriptive characteristics of the 3983 adolescents who gave a yes or no answer to the SSB availability question. The predictors were then used in a probit regression (Table 2) to produce a propensity score that represents the predicted probability of being exposed to an SSB-selling vending machine at school. The entire sample was then divided into nine blocks according to diﬀerent propensity score values, and t-tests were run within each block to check if exposure and control cases were similar to each other in all confounding variables. These t-tests show that the diﬀerences in predictors between the two groups were not signiﬁcant, which means that the propensity score used here successfully created a control group comparable to the exposure group. Table 3 shows the means of propensity scores in the exposure group and the control group for each block, while Table 4 shows the means of SSB consumption in the exposure group and the control group for each block. context, the predicted probability of attending a school that has SSB through vending machines (the propensity score) is estimated through a logistic regression. Each individual in the exposure group is then compared with control group members that have a close propensity score, and their diﬀerences in the outcome variable (in our case, the number of SSB the adolescent had on the prior day) are summed to give an overall diﬀerence, which indicates whether the exposure variable (SSB availability through schools’ vending machines) is signiﬁcantly associated with the outcome variable. 4. Conclusion With a population-representative large sample, this study strengthened the evidence for the association between SSB availability via school vending machines and the actual SSB consumption. The use of propensity score matching, a method designed to address the selection bias, further showed that the SSB availability at school vending machines and the SSB consumption have an independent and unam- biguous association. Recent evidence shows that both the SSB consumption and the childhood obesity declined after California’s ban on soda sales at schools in 2003 [15], and this study helps us understand a possible mechanism behind these phenomena. Journal of Environmental and Public Health In this analysis, only 42.6% of the sample are control cases (i.e., the adolescent’s school does not have soda in its vending machines), which means that propensity score matching methods like nearest neighbor and radius matching could mean throwing away a lot of observations and increasing the variance of the estimator [13]. Thus, we use matching methods that make use of all observations in implementing the propensity score matching: propensity score stratiﬁcation and kernel-based matching [14]. Strat- iﬁcation matching, as implemented in this study, stratiﬁes the sample into ﬁve strata such that within each stratum, treated and control units have the same average propensity score. The average treatment eﬀect is calculated by averaging the between-group outcome diﬀerences over the ﬁve strata. Kernel-based matching, on the other hand, compares each exposure case with a weighted sum of all control cases, with the weights inversely related to the propensity score diﬀerence between the exposure case and the control case. These two matching methods were implemented by the user-written commands of atts and attk in STATA 10, while the propensity score was computed by user-written STATA program of pscore.ado. The predictors we use in the probit regression include the adolescent’s gender, age (and a quadratic term of age squared), race/ethnicity, parental education, household income level (at or above the federal poverty level), and whether the adolescent attended a public school. Table 5 shows the results of the two propensity score- matched comparisons. Propensity score stratiﬁcation shows that adolescents who had access to SSB through their school vending machines consumed 0.181 more drinks of SSB than those who did not (P < .05). Kernel-based propensity score matching shows the SSB consumption diﬀerence to be 0.159 on the prior day (P < .05). Journal of Environmental and Public Health To address the selection bias that might occur with cross-sectional survey data, this study uses propensity score matching [12] to create a control group (adolescents whose school did not have SSB available through vending machines) that is similar to the exposure group (adolescents whose school did have SSB available through vending machines) in all observed confounding predictors of SSB intake. In this adolescents living in sampled households [11]. The 2005 CHIS adolescent sample asked the respondent whether his or her school has SSB available at vending machines, which is the key independent variable of this study. The survey also asked the respondent how many servings of SSB he or she had on the prior day, which is used in this study as the outcome variable. Journal of Environmental and Public Health 3. Results Of all 4029 adolescents who responded to the 2005 CHIS survey, 46 gave no answer or said “don’t know,” 2285 Journal of Environmental and Public Health 4 Table 4: Blockwise t-tests of the Mean Diﬀerence of Sugar-sweetened Beverage Consumption on the Prior Day between the Exposure Group and the Control Group. Diﬀerence of Sugar-sweetened Beverage Consumption on the Prior Day between the Exposure Group Table 4: Blockwise t-tests of the Mean Diﬀerence of Sugar-sweetened Beverage Consumption on the Prior Day between the Exposure Group and the Control Group. Block Mean of control group Mean of exposure group Mean diﬀerence (standard error) [0, 0.2) 0.32 1.11 −0.79 (0.24) [0.2,0.4) 0.87 1.09 −0.22 (0.09) [0.4, 0.5) 1.16 1.10 0.06 (0.14) [0.5,0.6) 0.95 1.25 −0.11 (0.14) [0.6,0.625) 0.97 1.26 −0.29 (0.23) [0.625,0.65) 0.85 1.00 −0.15 (0.18) [0.65,0.7) 1.04 1.18 −0.14 (0.18) [0.7,0.8) 1.02 1.21 −0.20 (0.09) [0.8,1) 1.00 .9 0.10 (0.64) N = 3983 Mean diﬀerence (standard error) Table 5: The association between SSB Availability at School Vending Machines and SSB Consumption with propensity score stratiﬁcation and kernel-based matching. Number of Exposed Number of control Proportional diﬀerence using matched sample t-value of the diﬀerence Stratiﬁcation 2285 1698 0.181 (.046) 3.972 Kernel-based matching 2285 1698 0.159 (.059) 2.668 able 5: The association between SSB Availability at School Vending Machines and SSB Consumption with pro nd kernel-based matching. een SSB Availability at School Vending Machines and SSB Consumption with propensity score stratiﬁcation Ludwig et al. [16] estimated from a longitudinal sample of younger adolescents that for each additional serving of consumed SSB, both body mass index (BMI) (mean 0.24 kg/m2; P = .03) and frequency of obesity (odds ratio 1.60; P = .02) increased, after being adjusted for anthropometric, demographic, dietary, and lifestyle vari- ables. If one additional serving of SSB per day increases the odds of being obese by 60%, then our estimated eﬀect of SSB availability through school vending machines on daily SSB consumption, 0.181 or 0.159 serving, is not an ignorable factor in childhood obesity prevention. Our descriptive analysis showed that the average consumption of SSB on the prior day was 1.09 serving, which means that on average the exposure to SSB from school vending machines could account for around one sixth of the daily SSB consumption among adolescents aged 12–17. 3. Results If this might seem like a larger eﬀect than what was shown by earlier studies of elementary school students (e.g., Fernandes [6]), this might be due to the fact that adolescents are more likely to buy beverage from school vending machines than children under 12. Thus, banning SSB sales at schools has a larger eﬀect among adolescents than among younger children. much bigger than what we have witnessed from children and adolescent samples. This study is limited in that the estimation was done in a cross-sectional dataset. Even though the propensity score matching method helps deal with the selection bias issue, it will be ideal if we can work with longitudinal datasets covering SSB consumption before and after the soda ban at schools. Moreover, as children and adolescents might replace their SSB intake with other kinds of beverage after a restriction on their access to SSB, further studies are also needed to examine what could happen to consumption of other kinds of beverage (juice, milk, water, coﬀee, etc.) after those SSB bans at schools. Journal of Environmental and Public Health 5 junior and senior high schools: a needs assessment,” Journal of the American Dietetic Association, vol. 100, no. 6, pp. 701–703, 2000. [6] K. W. Cullen, J. Eagan, T. Baranowski, E. Owens, and C. de Moor, “Eﬀect of a la carte and snack bar foods at school on children’s lunchtime intake of fruits and vegetables,” Journal of the American Dietetic Association, vol. 100, no. 12, pp. 1482– 1486, 2000. [7] M. M. Fernandes, “The eﬀect of soft drink availability in elementary schools on consumption,” Journal of the American Dietetic Association, vol. 108, no. 9, pp. 1445–1452, 2008. [8] D. B. Johnson, B. Bruemmer, A. E. Lund, C. C. Evens, and C. M. Mar, “Impact of school district sugar-sweetened beverage policies on student beverage exposure and consumption in middle schools,” Journal of Adolescent Health, vol. 45, no. 3, pp. S30–S37, 2009. [9] J. M. Fletcher, D. Frisvold, and N. Teﬀt, “Taxing soft drinks and restricting access to vending machines to curb child obesity,” Health Aﬀairs, vol. 29, no. 5, pp. 1059–1066, 2010. [10] A. Drewnowski and S. E. Specter, “Poverty and obesity: the role of energy density and energy costs,” American Journal of Clinical Nutrition, vol. 79, no. 1, pp. 6–16, 2004. [11] N. A. Ponce, S. A. Lavarreda, W. Yen, E. R. Brown, C. DiSogra, and D. E. Satter, “The California health interview survey 2001: translation of a major survey for California’s multiethnic population,” Public Health Reports, vol. 119, no. 4, pp. 388– 395, 2004. [12] P. R. Rosenbaum and D. B. Rubin, “The central role of the propensity score in observational studies for causal eﬀects,” Biometrika, vol. 70, no. 1, pp. 41–55, 1983. [13] M. Caliendo and S. Kopeinig, “Some practical guidance for the implementation of propensity score matching,” IZA discus- sion paper no. 1588, 2005, http://ssrn.com/abstract=721907 . [14] S. Becker and A. Ichino, “Estimation of average treatment eﬀects based on propensity scores,” The Stata Journal, vol. 2, pp. 358–377, 2002. [15] L. Shi and J. Van Meijgaard, “Substantial decline in sugar- sweetened beverage consumption among California’s children and adolescents,” International Journal of General Medicine, vol. 3, pp. 221–224, 2010. [16] D. S. Ludwig, K. E. Peterson, and S. L. Gortmaker, “Rela- tion between consumption of sugar-sweetened drinks and childhood obesity: a prospective, observational analysis,” The Lancet, vol. 357, no. 9255, pp. 505–508, 2001. [17] D. J. Whalen, J. S. Silk, M. References [1] Committee on Food Marketing and the Diets of Children and Youth, Food Marketing to Children and Youth: Threat or Opportunity? National Academies Press, Washington, DC, USA, 2006. [2] Alliance for a Healthier Generation: At School, May 2010, http://www.healthiergeneration.org/schools.aspx . [3] S. M. Lee, C. R. Burgeson, J. E. Fulton, and C. G. Spain, “Physical education and physical activity: results from the school health policies and programs study 2006,” Journal of School Health, vol. 77, no. 8, pp. 435–463, 2007. The broader signiﬁcance of reducing children’s exposure to SSB lies beyond childhood obesity prevention. Some of the SSBs could cause mental disorders among children and adolescents via their caﬀeine component [17], and SSB consumption is also associated with dental caries among children [18]. Moreover, as adolescence is a time when taste preference formation takes place [19], the SSB availability total eﬀect on a cohort’s adulthood obesity might actually be [4] S. A. French, M. Story, J. A. Fulkerson, and A. F. Gerlach, “Food environment in secondary schools: a la carte, vending machines, and food policies and practices,” American Journal of Public Health, vol. 93, no. 7, pp. 1161–1167, 2003. f [5] L. Harnack, P. Snyder, M. Story, R. Holliday, L. Lytle, and D. Neumark-Sztainer, “Availability of a la carte food items in 5 Journal of Environmental and Public Health Journal of Environmental and Public Health Semel et al., “Caﬀeine consumption, sleep, and aﬀect in the natural environments of depressed youth and healthy controls,” Journal of Pediatric Psychology, vol. 33, no. 4, pp. 358–367, 2008. [18] J. L. Kolker, Y. Yuan, B. A. Burt et al., “Dental caries and dietary patterns in low-income African American children,” Pediatric Dentistry, vol. 29, no. 6, pp. 457–464, 2007. y [19] C. T. Nu, P. MacLeod, and J. Barthelemy, “Eﬀects of age and gender on adolescents’ food habits and preferences,” Food Quality and Preference, vol. 7, no. 3-4, pp. 251–262, 1996.
https://openalex.org/W2395637433	https://europepmc.org/articles/pmc4568521?pdf=render	English	null	Criteria for Applying the Lucas-Washburn Law	Scientific reports	2,015	cc-by	5,233	Criteria for Applying the Lucas- Washburn Law Kewen Li1,2, Danfeng Zhang1, Huiyuan Bian1, Chao Meng1 & Yanan Yang1 received: 28 January 2015 accepted: 17 August 2015 Published: 14 September 2015 Spontaneous imbibition happens in many natural and chemical engineering processes in which the mean advancing front usually follows Lucas-Washburn’s law. However it has been found that the scaling law does not apply in many cases. There have been few criteria to determine under what conditions the Washburn law works. The effect of gravity on spontaneous imbibition in porous media was investigated both theoretically and experimentally. The mathematical model derived analytically was used to calculate the imbibition rates in porous media with different permeabilities. The results demonstrated that the effect of gravity on spontaneous imbibition was governed by the hydraulic conductivity of the porous media (permeability of the imbibition systems). The criteria for applying the Lucas-Washburn law have been proposed. The effect of gravity becomes more apparent with the increase in permeability or with the decrease in CGR number (the ratio of capillary pressure to gravity forces) and may be ignored when the CGR number is less than a specific value ⁎ Ncg ≅ 3.0. The effect of gravity on imbibition in porous media can be modeled theoretically. It may not be necessary to conduct spontaneous imbibition experiments horizontally in order to exclude the effect of gravity, as has been done previously. Spontaneous imbibition describes the phenomenon of a wetting phase fluid invading into a porous medium and displacing a non-wetting resident phase at a constant external pressure. This process exists in many industries and plays an important role in numerous natural, chemical, and commercial pro- cesses1–25. Spontaneous imbibition is of great significance not only for its fundamental aspects but also for its technological applications like liquid delivery in nano materials (liquid imbibition into nanotubes), fil- tration (liquid imbibition into porous material), construction (water penetration into concrete or cement pastes), printing processes (ink penetration in paper or coating of paper), irrigation (displacement of gas by water), and oil recovery (displacement of gas or oil by a different liquid)4–19. y y p g yf q It has long been known that the mean advancing front follows Washburn’s law, < h> ~ t1/2 in many cases1. Later on, similar models have been reported in different forms11–14. Actually the behavior of < h> ~ t1/2 has been even earlier reported by Lucas et al.25. 1China University of Geosciences (Beijing), 29 Xueyuan Road, Beijing 100083, China. 2Stanford University, 367 Panama St., CA 94305, USA. Correspondence and requests for materials should be addressed to K.L. (email: likewen@cugb.edu.cn) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 received: 28 January 2015 accepted: 17 August 2015 Published: 14 September 2015 www.nature.com/scientificreports/ simulations of nanotubes imbibing oil at an oil/vapor interface at 298 K. Supple and Quirke4 found that the imbibition into nanotubes did not obey the macroscopic Lucas-Washburn equation and was very rapid via complex imbibition dynamics. The penetration length was a linear function of time instead of time to the one-half power as foreseen by Lucas-Washburn’s law. simulations of nanotubes imbibing oil at an oil/vapor interface at 298 K. Supple and Quirke4 found that the imbibition into nanotubes did not obey the macroscopic Lucas-Washburn equation and was very rapid via complex imbibition dynamics. The penetration length was a linear function of time instead of time to the one-half power as foreseen by Lucas-Washburn’s law. p y Miranda et al.6 investigated the behavior of the spontaneous imbibition interface (ink-paper) as a function of time and paper orientation. The results showed that the dynamics of the rough interface depend on the orientation of the paper fibers. However the mechanism behind this observation has not been studied. The results reported by Miranda et al.6 indicated that the ink-paper interface do not move according to the Lucas-Washburn law. g According to the literature22–24, three characteristic regimes may be distinguished for spontane- ous imbibition in a single cylindrical tube. Regime I is the inertial regime that is the very beginning of the invasion process, where the liquid invades in a rather ballistic manner into the pore space. In this regime the height of the front scales linearly with the elapsed time t. The duration of this time can be estimated by the scaling model22. Regime II is the Lucas-Washburn regime with the “classic” square-root-of-time-behavior (Lucas-Washburn-law), where capillary and viscous forces prevail. Regime III is the regime that is usually the very late period of spontaneous imbibition where gravitational forces significantly affect the dynamics. gi yf y Note that both capillarity and gravity forces exist in spontaneous imbibition, although capillarity forces dominate in some cases. Clearly, gravity is not included in Lucas-Washburn’s law, and may be one of the reasons why Lucas-Washburn’s law does not work in some cases11–13. Researchers have tried hard to perform horizontal spontaneous imbibition tests5. Even so, few experi- ments have been conducted in horizontal directions in order to avoid the effect of gravity on spontaneous imbibition5. One can see that it is of great significance to investigate the effect of gravity on spontaneous imbibition. www.nature.com/scientificreports/ However little is known about at what conditions gravity force could be ignored.hi g y g In this paper, the criteria for applying the Lucas-Washburn law have been developed. The specific value of CGR number ⁎ Ncg was determined to analyze when the effect of gravity force on spontaneous imbibition can be neglected. It has been proved in this article that the effect of gravity on imbibition in porous media can be considered mathematically. Therefore, it may be unnecessary to conduct spontane- ous imbibition experiments horizontally in order to exclude the effect of gravity, as done in the past5. Criteria for Applying the Lucas- Washburn Law Therefore, the behavior of < h> ~ t1/2 was termed as Lucas-Washburn’s law in the following parts.h There is widespread literature on the wetting dynamics of homogeneous and structured surfaces1–16. Soriano et al.5 reported experiments on spontaneous imbibition of a viscous fluid by a model porous medium in the absence of gravity. They concluded that the average position of the interface satisfied Lucas-Washburn’s law. Scaling of the interface fluctuations suggested a dynamic exponent of about 3.0, indicative of global dynamics driven by capillary forces.ll g y y p y Recently Xue et al.21 showed for aqueous electrolyte imbibition in nanoporous gold that the fluid flow could be reversibly switched on and off through electric potential control of the solid–liquid interfacial tension. They found that Lucas-Washburn’s law also works in most of these special imbibition processes.h could be reversibly switched on and off through electric potential control of the solid liquid interfacial tension. They found that Lucas-Washburn’s law also works in most of these special imbibition processes. The results from the literature show that Lucas-Washburn-like scaling is not always observed in many spontaneous imbibition experiments because of a lot of factors4,5,11–13. These include non-Newtonian character of the liquid such as ink, the change in the property of the porous media (for example, swell- ing of the paper fibers during imbibition). Supple and Quirke4 have carried out molecular dynamics h y The results from the literature show that Lucas-Washburn-like scaling is not always observed in many spontaneous imbibition experiments because of a lot of factors4,5,11–13. These include non-Newtonian character of the liquid such as ink, the change in the property of the porous media (for example, swell- ing of the paper fibers during imbibition). Supple and Quirke4 have carried out molecular dynamics 1 www.nature.com/scientificreports/ Theoretical Background g Lucas-Washburn-like models are frequently utilized to investigate spontaneous imbibition. However these equations may not be applied to the cases where gravity is considered. To this end, a linear cor- relation between the spontaneous imbibition rate and the reciprocal of the cumulative imbibition or recovery, with gravity forces included, was used13: = − ( ) q a R b 1 1 w 0 0 ( ) 1 where R is the recovery in the unit of pore volume. Note that recovery is the ratio of water imbibed to the pore volume of the porous medium (core sample), directly proportional to imbibition front height, < h> , in many cases. qw is the volumetric spontaneous imbibition rate of the wetting phase and is equal to dR/dt, directly proportional to the derivative of h to imbibition time, dh/dt. y p p a0 and b0 are two constants associated with capillary and gravity forces, respectively, and are expressed as follows: ( ) = − ( ) ⁎ ⁎ a AM S S L P 2 e wf wi c 0 ( ) 2 where A is the cross-sectional area of the rock sample, Swf is the water saturation (volume of water divided by the total pore volume of the porous medium) behind the imbibition front, Swi is the initial wetting-phase (water in this study) saturation in the core sample, ⁎ Pc is the capillary pressure at Swf. L is the length of the core sample. where A is the cross-sectional area of the rock sample, Swf is the water saturation (volume of water divided by the total pore volume of the porous medium) behind the imbibition front, Swi is the initial wetting-phase (water in this study) saturation in the core sample, ⁎ Pc is the capillary pressure at Swf. L is the length of the core sample. ρ = ∆ ( ) ⁎ b AM g 3 e 0 ( ) 3 here Δρ is the density difference between the wetting phase and the nonwetting phase (= ρw − ρnw), g is the gravity constant, ⁎ Me is defined as the global mobility of the two phases. ere Δρ is the density difference between the wetting phase and the nonwetting phase (= ρw ρnw), g is he gravity constant, ⁎ Me is defined as the global mobility of the two phases. www.nature.com/scientificreports/ www.nature.com/scientificreports/ where ⁎ Mw is the wetting phase mobility at Swf and ⁎ Mnw the nonwetting phase mobility at 1−Swf. The wetting and nonwetting phase mobilities are expressed as follows: where ⁎ Mw is the wetting phase mobility at Swf and ⁎ Mnw the nonwetting phase mobility at 1−Swf. The wetting and nonwetting phase mobilities are expressed as follows: The μ = ( ) ⁎ ⁎ M k 5 w w w μ = ( ) ⁎ ⁎ M k 6 nw nw nw μ = ( ) ⁎ ⁎ M k 5 w w w ( ) 5 μ = ( ) ⁎ ⁎ M k 6 nw nw nw ( ) 6 where k* w and ⁎ knw are the effective permeabilities of the wetting and nonwetting phases at Swf and 1−Swf respectively, μw and μnw are the viscosities of the wetting and nonwetting phases.l where k* w and ⁎ knw are the effective permeabilities of the wetting and nonwetting phases at Swf and 1−Swf respectively, μw and μnw are the viscosities of the wetting and nonwetting phases.l In the case of gas-liquid two phase flow, equations (1) and (4) can be reduced because the gas mobility is much greater than the liquid mobility11,12. For gas-liquid two phase flow, the expression of the rela- tionship between imbibition rate and the reciprocal of recovery stays the same as equation (1) but those of a0 and b0 are simplified as follows: ( ) μ = − ( ) ⁎ ⁎ a Ak S S L P 7 w wf wi w c 0 ( ) 7 μ ρ = Δ ( ) ⁎ b Ak g 8 w w 0 ( ) 8 ased on equations (7) and (8), capillary pressure can be calculated: ( ) ρ = − Δ ( ) ⁎ P S S a b gL 1 9 c wf wi 0 0 ( ) 9 According to equation (10), the effective water permeability at the water saturation of Swf can be computed as follows: μ ρ = Δ ( ) ⁎ k A g b 10 w w 0 ( ) 10 The values of a0 and b0 in equations (9) and (10) can be determined from the plot of imbibition rate and the reciprocal of the gas recovery. Swf can be measured during the water imbibition tests. Where a and b are constants related to a0 and b0. Where a and b are constants related to a0 and b0. www.nature.com/scientificreports/ Therefore, one can infer both capillary pressure and effective water permeability from the experimental data of spontaneous water imbibition using equations (9) and (10). A great challenge in characterizing spontaneous imbibition behavior in gas-liquid systems was to calculate the effective water permeability kw and capillary pressure Pc separately. The method (eqs. (9) and (10)) described here may provide a solution to this problem and is especially useful for the porous media with very low permeability such as shale rocks.i y p y Equations (1) and (6) have been used and verified in many cases, including the prediction of oil production11–13, scaling of experimental data17,18. Hognesen et al. (2004) analyzed the conditions of the applicability of equation (1). They reported that whether and when one could expect the parameter “c” in equation (1) to be constant. Hognesen et al. (2004) also showed that the data have a realistic error scatter around the regression curves and the regions where equation (1) is not valid are identified.i g g qi For simplification and generalization, equation (1) can also be expressed as follows: = − ( ) dh dt ah b 1 11 ( ) 11 Theoretical Background 1/2 g y ei g y p Obviously equation (1) is reduced to Lucas-Washburn-like model, < R> ∼ t1/2, if gravity force is ignored.l i Obviously equation (1) is reduced to Lucas-Washburn-like model, < R> ∼ t1/2, if gravity force is ignored.l g For co-current spontaneous imbibition (the flowing direction of the wetting phase is the same as that of the nonwetting phase), the expression of the effective mobility is represented as follows: = − ( ) ⁎ ⁎ ⁎ ⁎ ⁎ M M M M M 4 e w nw w nw ( ) 4 Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 2 Results In this Paper, we studied the effect of gravity on spontaneous imbibition rate in porous media with dif- ferent properties using equation (1) and experimental data. According to equation (1), the effect of gravity on spontaneous imbibition was governed by the ratio of capillary pressure to gravity forces, or the CGR number ( ρ = /Δ ⁎ N P gh cg c ) of the imbibition systems. Note that Ncg is a constant. This is because gravity is constant and the capillary pressure is the specific one, ⁎ Pc , which is also constant (see Fig. 1a)11.h g y yi c g The rock (or porous media) with greater permeability generally has smaller capillary forces, the vice versa. Therefore the effect of gravity increases with the increase in permeability or decreases with the increase in capillary number. Theoretically (foreseen by eq. (1)) gravity forces may be ignored when the Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 3 www.nature.com/scientificreports/ Figure 1. Relationship between water imbibed and time (model results), (a) Schematic of the modeled spontaneous imbibition tests; (b) Model data of spontaneous imbibition. Figure 1. Relationship between water imbibed and time (model results), (a) Schematic of the modeled spontaneous imbibition tests; (b) Model data of spontaneous imbibition. Figure 2. Relationship between water imbibed and the square root of time (model results). Figure 2. Relationship between water imbibed and the square root of time (model results). permeability or CGR number is less than a specific value ( ⁎ Ncg). This prediction will be tested in the following section. Note that the value of ⁎ Ncg has not been reported before.h permeability or CGR number is less than a specific value ( ⁎ Ncg). This prediction will be tested in the following section. Note that the value of ⁎ Ncg has not been reported before.h g Theoretical calculations using equation (1) were based on spontaneous water imbibition into dry (gas-saturated) rocks at a temperature of 20 °C to obtain the specific value of Ncg. Rock samples were assumed to position vertically and were contacted with water at the bottom (see Fig. 1a). Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Relationship between water imbibition rate and the reciprocal of recovery (model results). Figure 3. Relationship between water imbibition rate and the reciprocal of recovery (model results). g p p y ( Figure 4. Schematic of apparatus for spontaneous imbibition tests11. Figure 5. Relationship between water imbibed and the square root of time in different rocks. Figure 4. Schematic of apparatus for spontaneous imbibition tests11. Figure 4. Schematic of apparatus for spontaneous imbibition tests11. Figure 5. Relationship between water imbibed and the square root of time in different rocks. Figure 5. Relationship between water imbibed and the square root of time in different rocks. recovery is shown in Fig. 3. All of the relationships are linear for the rocks with different permeabilities as expected. h b d d h d f h l l d l11i In the above section, it is demonstrated using the data from the analytical model11 verified previously that Lucas-Washburn’s law does not work in the cases in which the gravity force is dominating ( ⁎ Ncg < 3.0). We will verify the above model phenomenon experimentally in the following section. To this end, spon- taneous water imbibition experiments were conducted in different types of dry rocks positioned verti- cally11,12. The schematic of the apparatus for conducting spontaneous imbibition tests is shown in Fig. 4. The rock sample was hung under a balance which had an accuracy of 0.01 g and a range from 0 to 1600 g. The water imbibed into the core sample was recorded in time by the balance using an under-weighing method and the real-time data were measured continuously by a computer through an RS-232 interface. The purpose of using the under-weighing method was to reduce the experimental error caused by water evaporation. Air was used as the gas phase and distilled water as the liquid phase. The experimental details can be found more in the refs 11–13. Figure 5 shows the relationship between water imbibed and the square root of time in rocks with different permeability, ranging from 0.56 (the greywacke) to over 25700 md (the glass-bead pack). The fired and natural Berea sandstone core samples had permeabilities of 1200 and 804 md respectively. Results The values of the rock and fluid property are listed as follows: the diameter of rock (D) was 3.40 cm for calculating A, the length was 29.5 cm, porosity of the rock (φ) was 0.386, Swf = 0.575, Swi = 0.0, the surface tension of water and contact angle used to calculate capillary pressure were 72.696 dyne/cm and zero respectively, the viscosity of water was 1.0 cp, the relative permeability of water was 0.614, and the permeability of rock samples ranged from 1000 to 100000 md. It is assumed in this study that the length of the rock sample is less than the height corresponding to ⁎ Pc , that is, L < h* (see Fig. 1a).h c The theoretical data of water spontaneous imbibition into gas-saturated rocks were calculated with the above parameters using equation (1) for core samples with different permeabilities. The results are plotted in Fig. 1b as the relationship between water imbibed and time.if g One can see the significant effect of permeability or CGR number on spontaneous imbibition, as shown in Fig. 1b. However it is almost impossible to tell the effect of gravity. For this reason, Lucas-Washburn-like model was used to process the theoretical data. Figure 2 shows the relationship between water imbibed and time to the one-half power using data from Fig. 1. All of the relationships should be linear if Lucas-Washburn’s law applies. One can see that the relationships are not linear once the permeability is greater or the value of 1/Ncg is less than a specific value (gravity forces become dominating).f It is known that the effect of gravity is greater in the core samples with greater permeabilities or cap- illary number, Ncg. But the critical value of Ncg, ⁎ Ncg, is not known for possibly ignoring gravity force. According to the results shown in Fig. 2, the value of ⁎ Ncg may be equal to 3.0 in the cases studied. This implies that the effect of gravity force on spontaneous imbibition may be neglected and Lucas-Washburn model works if the value of Ncg is greater than 3.0 approximately, and vice versa.hf cg g pp y The same set of data shown in Fig. 2 was used but plotted in a different way by using equation (1) in which gravity force is included. The relationship between the imbibition rate and the reciprocal of Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 4 www.nature.com/scientificreports/ It is obvious that the recovery (directly proportional to imbibition front height) in the glass-bead pack with a high permeability of about 25700 md does not scale with the square root of time, as predicted Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 5 www.nature.com/scientificreports/ Figure 6. Relationship between water imbibition rate and the reciprocal of recovery in different rocks. Figure 6. Relationship between water imbibition rate and the reciprocal of recovery in different rocks. by Lucas-Washburn’s law. One can see from all of the experimental results, similar to the model data as shown in Fig. 2, that Lucas-Washburn’s law does not apply in cases where the permeability is greater than a specific value.f by Lucas-Washburn’s law. One can see from all of the experimental results, similar to the model data as shown in Fig. 2, that Lucas-Washburn’s law does not apply in cases where the permeability is greater than a specific value.f i To test equation (1), the experimental data shown in Fig. 5 are plotted in a different way as imbibition rate vs. 1/R and the results are demonstrated in Fig. 6. Remarkably all of the relationships between water imbibition rate and the reciprocal of recovery in very different rocks are linear, as predicted by equation (1). This demonstrates that gravity forces may be well considered in equation (1). The model (eq. (1)) is derived from physical principles and matches the experimental data of spontaneous water imbibition very well with great values of regression coefficient (R2). Note that Lucas-Washburn’s law cannot match the spontaneous imbibition data in the cases in which gravity forces dominate.hf g y The above experimental data proved that equation (1) could consider the effect of gravity satisfacto- rily. On the other hand, both the model and experimental data demonstrate that equation (1) can match the spontaneous imbibition data in both cases where Lucas-Washburn’s law works and does not work, which implies equation (1) is more general in terms of characterizing spontaneous imbibition in porous media. y f fl p 3. Dullien, F. A. L. Porous media: fluid transport and pore structure 2nd edn. (Academic Press, 1991). Discussion It is well-known that Lucas-Washburn’s law has been extensively utilized in many processes happened in nature and industries for about one hundred years. The importance of Lucas-Washburn’s law or Lucas-Washburn-like scaling is out of question. However it does not apply in many cases4,5,11–13 as stated previously. It has been determined in this paper that one of the reasons is the ignorance of gravity in Lucas-Washburn’s law. Also investigated was how and when gravity should be considered.hi In summary, the criteria for using the Lucas-Washburn law has been proposed as follows. The specific value of CGR number ⁎ Ncg was determined to be about 3.0. The effect of gravity force on spontaneous imbibition may be ignored and Lucas-Washburn’s law applies when ⁎ Ncg > 3.0; the effect of gravity force cannot be neglected and Equation (1) may be utilized when ⁎ Ncg < 3.0.h g y cg The model and phenomenon described in this paper are of both fundamental and applied inter- est; important parameters such as effective water permeability, for example, may be inferred using the imbibition test data. With this model, one may easily conduct spontaneous imbibition experiments in a vertical direction, which obviates the need for difficult horizontal experiments that exclude the effect of gravity. The data indicate that the effect of gravity on imbibition can be considered theoretically and the model described here extended Lucas-Washburn’s law, which has been widely used in many industries and research areas for almost a century.hh y The number of examples for applying the results in this article is limited. There are many more areas and cases, including nanostructured materials26,27, where liquid imbibition happens and the ideas pro- posed in this article may be useful. Note that the imbibition rates may be much faster at nanoscale than those in porous media with conventional sizes4,7. 5. Soriano, J. et al. Anomalous roughening of viscous fluid fronts in spontaneous imbibition. J. Phys. Rev. L. 95, 104501 (2005). 6. Miranda, A. M., Menezes-Sobrinho, I. L. & Couto, M. S. Spontaneous imbibition experiment in newspaper sheets. J. Phys. Rev L. 104, 086101 (2010).hll 1. Washburn, E. W. The dynamics of capillary flow. J. Phys. Rev. 17, 273–283 (1921).l l 4. Supple, S. & Quirke, N. Rapid imbibition of fluids in carbon nanotubes. J. Phys. Rev. L. 90, 214501 (2003). 8. Whitby, M. & Quirke, N. Fluid flow in carbon nanotubes and nanopipes. J. Nature Nanotech. 2, 87–94 (2007). 7. Whitby, M., Cagnon, L., Thanou, M. & Quirke, N. Enhanced fluid flow through nanoscale carbon pipes. J. Nano Le 2632–2637 (2008).l Author Contributions K.L. developed the main ideas and revised the main manuscript text, D.Z. and H.B. wrote the main manuscript text, C.M. and Y.Y. prepared all of the figures. All authors discussed the results and reviewed the manuscript. www.nature.com/scientificreports/ de Gennes, P. G., Brochard-Wyart, F. & Quere, D. Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves. Chapter 5 (Springer, 2004). 22. de Gennes, P. G., Brochard-Wyart, F. & Quere, D. Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves. Chap (Springer, 2004). 23. Quere, D. Inertial capillarity. Europhys. Lett. 39, 533–538 (199 p y p y ( ) 24. Huber, P. Soft matter in hard confinement: phase transition thermodynamics, structure, texture, diffusion and flow in nanopo media J Phys Condens Matter 27 103102 (2015) p y p y 24. Huber, P. Soft matter in hard confinement: phase transition thermodynamics, structure, texture, diffusion and flow in nanoporous media. J. Phys Condens Matter. 27, 103102 (2015). ti media. J. Phys Condens Matter. 27, 103102 (2015). y 25. Lucas, R. On the time law of the capillary rise of liquids. Kolloid-Z. 23, 15 (1918). 25. Lucas, R. On the time law of the capillary rise of liquids. p y q 26. Ariga, K. et al. Layer-by-layer nanoarchitectonics: invention, innovation, and evolution. Chem. Lett. 43, 36–68 (2014). 26. Ariga, K. et al. Layer-by-layer nanoarchitectonics: invention, innovation, and evolution. Chem. Lett. 43, 36–68 (2014). 27 G D & S hü h F S h i f ili id Ch S R 43 313 344 (2014) . Ariga, K. et al. Layer-by-layer nanoarchitectonics: invention, inn 6. Ariga, K. et al. Layer-by-layer nanoarchitectonics: invention, innovation, and evolution. Chem. Lett. 43, 36– 7. Gu, D. & Schüth, F. Synthesis of non-siliceous mesoporous oxides. Chem. Soc. Rev. 43, 313–344 (2014). 26. Ariga, K. et al. Layer-by-layer nanoarchitectonics: invention, innovation, and evolution. Chem. Lett. 43, 36– 27. Gu, D. & Schüth, F. Synthesis of non-siliceous mesoporous oxides. Chem. Soc. Rev. 43, 313–344 (2014). Acknowledgementsh g This research was conducted partially with financial support from the National Natural Science Foundation of China for Key Program under Grant 51034004 and Beijing Key Laboratory for Geological Appraisal and Development of Unconventional Natural Gas (School of Energy Resources, China University of Geosciences, Beijing), the contribution of which is gratefully acknowledged. Scientific Reports \| 5:14085 \| DOI: 10.1038/srep14085 References W hb E pp pl y 5. Soriano, J. et al. Anomalous roughening of viscous fluid fronts in spontaneous imbibition. J. Phys. Rev. L. 95, 104501 (2005). 7. Whitby, M., Cagnon, L., Thanou, M. & Quirke, N. Enhanced fluid flow through nanoscale carbon pipes. J. Nano Lett. 8, 2632–2637 (2008).l 8. Whitby, M. & Quirke, N. Fluid flow in carbon nanotubes and nanopipes. J. 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Li, K. & Horne, R. N. Generalized scaling approach for spontaneous imbibition: an analytical model. J. SPEREE. 9, 251–258 (2006). 4. Handy, L. L. Determination of effective capillary pressures for porous media from inhibition data. J. Petroleum Transaction AIME. 219, 75–80 (1960).i 15. Nurkamelia & Arihara, N. Analysis of spontaneous capillary imbibition for improved oil recovery. SPE Asia Pacific Oil and Gas Conference and Exhibition, 18–20 October, Perth, Australia (2004). 6. Babadagli, T., Hatiboglu, C. U. & Hamida, T. Evaluation of matrix-fracture transfer functions for counter-current capillary imbibition. J. Transp. Porous Med. 80, 17–56 (2009). p 7. Tavassoli, Z., Zimmerman, R. W. & Blunt, M. J. Analytic analysis for oil recovery during counter-current imbibition in strongly water-wet systems. J. Transp. Porous Med. 58, 173–189 (2005). y p 18. Hognesen, E. J., Standnes, D. C. & Austad, T. Scaling spontaneous imbibition of aqueous surfactant solution into prefere oil-wet carbonates. J. Energy Fuels. 18, 1665–1675 (2004). gy 19. Schechter, D. S., Zhou, D. & Orr, Jr. F. M. Low IFT drainage and imbibition. J. Sci. and Eng. 11, 283–300 (1994). gy 19. Schechter, D. S., Zhou, D. & Orr, Jr. F. M. Low IFT drainage 20. Cai, J. C., Yu, B. M., Zou, M. Q. & Mei, M. F. Fractal characterization of spontaneous co-current imbibition in porous me Energy Fuels. 24, 1860–1867 (2010). gy 21. Xue, Y. et al. Switchable imbibition in nanoporous gold. J. Nat Commun. 5, 4237 (2014). gy 21. Xue, Y. et al. Switchable imbibition in nanoporous gold. J. Na 22. Competing financial interest Competing financial interests: The authors declare no competing financial interests. How to cite this article: Li, K. et al. Criteria for Applying the Lucas-Washburn Law. Sci. Rep. 5, 14085; doi: 10.1038/srep14085 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W3204827382	https://atheneadigital.net/article/download/v21-3-pineiro-polo/2967-pdf-es, https://ddd.uab.cat/pub/athdig/athdig_a2021v21n3/athdig_a2021v21n3pe2967.pdf	es		El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico	Athenea digital	2,021	cc-by	8,828	Athenea Digital - 21(3): e2967 (noviembre 2021) -ENSAYOS- ISSN: 1578-8946 EL SUMAK KAWSAY ANDINO COMO RESISTENCIA SOCIOPOLÍTICA Y COMO DESAFÍO EPISTÉMICO THE ANDEAN SUMAK KAWSAY AS SOCIOPOLITICAL RESISTANCE AND AS AN EPISTEMIC CHALLENGE Eleder Piñeiro Aguiar; Jorge Polo Blanco* * Universidade da Coruña; **Escuela Superior Politécnica del Litoral; elederpa1983@gmail.com; polo@espol.edu.ec Historia editorial Resumen Recibido: 30-06-2020 Desde comienzos del siglo XXI, pero principalmente a partir de 2006, en la República del Ecuador se dejó sentir a nivel sociopolítico una significativa influencia indígena, que se manifestó en textos constitucionales, planes estatales de desarrollo y políticas públicas. Analizaremos ciertos insumos que han ofrecido los pueblos originarios en el país andino y que han supuesto elementos de resistencia frente a la hegemonía neoliberal y al desarrollismo extractivista. En concreto, examinaremos cómo se han ido construyendo discursos y prácticas en torno al Buen Vivir (Sumak Kawsay) y cómo se articula semejante proceso con lógicas más globales de avanzada del indigenismo en la región latinoamericana. Comprobaremos qué significa, política y epistémicamente, la apuesta que el Ecuador ha hecho por dotar al país de derechos de la naturaleza y por definirse, por primera vez en su historia, como plurinacional y pluriétnico. Aceptado: 06-05-2021 Publicado: 06-10-2021 Palabras clave Buen Vivir; Desarrollo comunitario; Ecuador; Grupo étnico; Sumak Kawsay Abstract Keywords Buen Vivir; Community development; Ecuador; Ethnic group; Sumak Kawsay Since the beginning of the 21st century, but mainly as of 2006, in the Republic of Ecuador a significant indigenous influence was felt at the socio-political level, which was manifested in constitutional texts, state development plans and public policies. We will analyze certain inputs that the indigenous peoples have offered in the Andean country and that have represented elements of resistance against neoliberal hegemony and extractive developmentalism. Specifically, we will examine how discourses and practices have been built around Good Living (Sumak Kawsay) and how such process is articulated with more advanced global logics of indigenism in the Latin American region. We will verify what it means, politically and epistemically, the commitment that Ecuador has made to endow the country with the rights of nature and to define itself, for the first time in its history, as plurinational and multiethnic. Piñeiro Aguiar, Eleder & Polo Blanco, Jorge (2021). El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico. Athenea Digital, 21(3), e2967. https://doi.org/10.5565/rev/athenea.2967 El Sumak Kawsay (Buen Vivir) como discurso contrahegemónico El Buen Vivir, tal y como fue teorizado por la intelectualidad indígena (y no indígena), resultó ser una construcción discursiva que, partiendo de algunos elementos referidos a los modos de vida de las nacionalidades y comunidades indígenas (principalmente andinos, pero también amazónicos), buscó propuestas alternativas al desarrollismo occidental (Ruiseco, 2009). Se buscaba propiciar algún tipo de restitución de la “comunión” entre naturaleza y seres humanos (Hidalgo et al., 2014; Maldonado, 2014; Yam- 1 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico para, 2011). El Buen Vivir ha sido un discurso axiológico y político al mismo tiempo, dado que trató de erigir los fundamentos de una nueva “ética” y de un nuevo orden socioeconómico (Guzmán y Polo Blanco, 2017). Planteó, de igual modo, un proyecto transformador que pretendía fundamentarse en una epistemología distinta que, por decirlo sucintamente, promoviese valores como la igualdad, la equidad, la reciprocidad, la no-competitividad y la solidaridad, todo ello dentro de un marco de crecimiento económico sustentable que, en sus versiones más radicalmente biocéntricas —esto es, alejadas del “antropocentrismo occidental”— contemplaba incluso la concesión de derechos intrínsecos a la naturaleza. Esto último quedó plasmado en la Constitución de Montecristi, vigente en la República del Ecuador desde 2008. Desde tales coordenadas, se concebía la participación de los seres humanos en un conjunto vital de carácter cósmico, no organizado ontológicamente por la escisión cultura/naturaleza, típicamente occidental (Descola, 2005/2012). Porque lo que se pretende, desde esa concepción de la vida, es estar instalado en una íntima “relacionalidad” —o armonía— con la naturaleza. Fernando Huanacuni (2010, p. 7) ha remarcado lo incorrecto de establecer alguna suerte de equivalencia entre el Sumak Kawsay del ámbito kichwa (en Ecuador) o el Sumaq Qamaña en el ámbito aymara (en Bolivia), y el bienestar “occidental” (las comillas son necesarias, dado el de este término en contraposición a “lo indígena”). Colocar como equivalentes dichos términos sería un reduccionismo y no reflejaría toda la complejidad que el término tiene para los mencionados pueblos “originarios”. Los vocablos sumak y sumaq podrían traducirse como “plenitud”, “sublime”, “excelente”, “magnífico”, “hermoso”. Kawsay y qamaña, por su parte, podrían equivaler a “vivir”, “convivir”, “estar siendo”, “ser estando” o, sencillamente, “vida”. Esa vida en plenitud no significaría vivir “mejor”, dado que esto último conllevaría una graduación jerárquica, algo que nos avocaría a juegos de suma cero, a competencias y a rivalidades. El “Vivir Bien” andino estaría mal avenido con la competitividad, el lujo, la opulencia o el derroche consumista (Huanacuna, 2010, p. 22). Es cierto que algunos autores han sostenido que la noción de Buen Vivir no es más que un neologismo ideológico construido ad hoc, resultando muy difícil encontrar alusiones a esa idea o noción en la abundantísima literatura etnográfica disponible sobre comunidades andinas anterior al cambio de milenio. La literatura política surgida en torno al Sumak Kawsay, tejida en torno a una proliferación de “discursos pachamamistas”, carecería de una contextualización adecuada, y por ende no se hallaría fundamentada en información empírica rigurosa (Viola, 2014). Por otro lado, se ha de considerar que en muchas ocasiones el indigenismo y el “discurso indianista antimodernidad” se han retroalimentado con los procesos de globalización, que promueven ese turismo de occidentales ávidos de primitivismo; “blancos” que se muestran impacientes 2 Eleder Piñeiro Aguiar por experimentar esa adrenalina mística que creen hallar en el “contacto” con una presunta alteridad cultural (Gascón, 2009). Algo que, paradójicamente, también sirve para que las comunidades indígenas sobrevivan, así sea pagando el precio de una irreversible aculturación (Ullán de la Rosa, 2000); diversas fórmulas de turismo indigenista o “turismo comunitario” que, definitivamente, conllevan una cierta mercantilización de la propia identidad cultural (Comaroff y Comaroff, 2011). Sin olvidar una paradoja recurrente: el discurso indigenista no ha sido encabezado en demasiadas ocasiones por los propios indígenas, sino por las élites criollas (Quijano, 1995). Pero, más allá de estas críticas, lo cierto es que esta noción ha sido pragmáticamente movilizadora. Además de su eventual preexistencia histórica y etnográfica, lo determinante es que la noción Buen Vivir se fue convirtiendo en un verdadero catalizador político que, finalmente, fue incluso capaz de poner en pie una cierta contrahe gemonía. Es más, podemos aseverar que el Buen Vivir llegó a convertirse en política de Estado, no solo porque atravesaba todo el corpus del nuevo texto constitucional ecuatoriano, algo valioso e inédito en sí mismo, sino porque en cierto modo (aunque con notables limitaciones y contradicciones) llegó a constituirse en una suerte de brújula que podía o pretendía orientar algunas de las más decisivas políticas públicas (Manosalvas, 2014). Sin olvidar que esa dimensión política, como ya hemos señalado, hallaba su razón de ser en ciertos elementos éticos extraídos de la propia cosmovisión o modus vivendi de los pueblos indígenas (Molina, 2015, p. 145). Asumiendo que no existe un consenso cerrado a la hora de establecer una definición de qué es, qué implica y qué significa el Sumak Kawsay indígena, lo cierto es que se evidencia un conflicto entre la cosmovisión indígena y los patrones nacionales-estatales-capitalistas. Genealógicamente, el Buen Vivir emerge en el altiplano boliviano como respuesta a un “desmantelamiento de lógicas comunitarias” (Huertas y Urquidi, 2015, p. 86). Los pueblos originarios, en ese contexto, se hallaban enfrentados al go bierno nacional y a poderes internacionales, interesados en introducirles ideas exógenas. Gobiernos y transnacionales, blandiendo como arma arrojadiza las nociones de “progreso” y “desarrollo”, acusaban a estos pueblos de mostrar un recalcitrante carácter ineficiente, arcaico y antiutilitario. Frente a esto, diversas resistencias indígenas expusieron sus identidades, cosmovisiones y derechos en torno al ayllu: organizaciones basadas en la reciprocidad, la complementariedad, las relaciones de parentesco y la búsqueda de una vida armónica. Ahí entraría en juego la “casa cósmica andina” (Yampara, 2011, p. 241), una cohabitación armoniosa entre lo humano, lo espiritual y la naturaleza. Catherine Walsh (2009, p. 14), al referirse a la introducción político-estatal de estas cosmovisiones indígenas señalaba que “este período, probablemente más que cual- 3 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico quier otro, se distingue por los procesos de repensar y refundar el Estado, la sociedad y el país, dejando atrás el modelo neoliberal y haciendo que las diferencias étnicas, culturales y coloniales se visibilicen y guíen el debate”. Se habría dado, así, un doble proceso de cambio: desde la unicidad a la pluralidad y, al mismo tiempo, desde la mono culturalidad a la pluriculturalidad. Dicha transformación habría sido uno de los aspectos más significativos y relevantes de aquel proceso constituyente ecuatoriano que cristalizó en la mencionada Constitución de Montecristi (2008), toda vez que se pusieron “sobre el tapete cuestiones referidas al carácter racista, racializado y excluyente de las sociedades actuales” (Walsh, 2009, p. 14). La transformación jurídico-política que supuso la aprobación de la mencionada Constitución, y la subsiguiente implementación del “Plan Nacional del Buen Vivir”, supuso en los planos normativo e ideológico una crítica al modelo social y civilizatorio de la modernidad eurocéntrica capitalista. Todo ello ejecutado con el aporte indígena y sus reivindicaciones históricas, con la promoción de nuevos derechos y cosmovisiones alternativas; y, finalmente, con la configuración de un Estado verdaderamente plurinacional y pluricultural que trataba de recuperar el papel redistribuidor del Estado frente a las incansables ofensivas neoliberales (Peña y Lillo y Polo Blanco, 2018). Este proceso constituyente supuso un momento de efervescencia enmarcado en esa “primavera política” que se vivió en algunas naciones de América Latina. Un momento disruptivo de revoluciones políticas y “epistemológicas” construidas (principalmente en Ecuador y Bolivia) desde la visión de los pueblos originarios; procesos cimentados en historias milenarias y en presentes participativos, que buscaban una alternativa civilizatoria de largo alcance, superando hasta cierto punto la visión monocultural criollo-mestiza. Los nuevos textos constitucionales, tanto en Ecuador como en Bolivia, tuvieron mucha trascendencia histórica, por sus contenidos radicalmente innovadores y por el hecho crucial (cargado de simbolismo) de que algunas de sus orientaciones provenían de aquellos pueblos “originarios” que habían sido obliterados de forma sistemática. Asimismo, es de destacar que el Buen Vivir, que David Cortez (2011) ha denominado “herramienta movilizadora”, se presenta teleológicamente por cuanto es una meta a cumplir por el conjunto de la población, algo que se manifiesta en la constante referencia a su concepción constructivista (Piñeiro, 2016, p. 56) a lo largo de los planes de desarrollo del país (SENPLADES, 2013). Boaventura de Sousa Santos (2012) se refirió a los procesos de Bolivia y Ecuador (iniciados a mediados de los 2000) como “constitucionalismo transformador”. Pero lo que subyace a esta propuesta es algo todavía más profundo, a saber, una crítica a todas las crisis y contradicciones provenientes de un capitalismo hiperconsumista, individualista y explotador, lo que para Fernando Huanacuni (2010, p. 6) supone “una crisis de vida” ante la que diferen- 4 Eleder Piñeiro Aguiar tes comunidades campesinas e indígenas han presentan propuestas alternativas, siendo el Buen Vivir del mundo andino una de ellas. Debe comprenderse el profundo carácter crítico que conlleva hablar en términos de Buen Vivir, como contrapuesto a otros modelos de desarrollo, lo cual engarza con otras corrientes teóricas críticas occidentales y no occidentales. Pero lo más destacable es que dicha propuesta crítica se haga desde sectores históricamente invisibilizados y subalternizados provenientes principalmente de áreas rurales, de comunidades no citadinas y, sobre todo, del mundo indígena. El simbolismo de la propuesta sea quizá su primer logro, puesto que con ella han adquirido un carácter protagónico actores sociales que, desde hacía siglos, permanecían silenciosamente marginados en un rincón de la Historia. Rompiendo con su mutismo secular (aunque, bien es cierto, este mutismo jamás fue absoluto), estas comunidades humanas se transformaron en sujetos colectivos políticamente activos. El Buen Vivir, como discurso movilizador, insufló dignidad en estos grupos por demasiado tiempo ignorados; un discurso desde el cual ofrecer, como mínimo, resistencia y resiliencia. Pero todo ello, además, cristalizó en conquistas positivas, logrando introducir en la Constitución aspectos como el derecho al agua, la soberanía alimenticia, la vivienda, la salud, la educación, la energía, el ambiente salu dable y los derechos de la naturaleza, entre otros (Gudynas y Acosta, 2011, p. 107). En contra de lo que pensaba John Holloway (2002, p. 33), parece que sí es crucial la cuestión de quién detenta el poder, más allá de las quimeras poéticas de un mundo despojado de todo poder. Modificar el “sentido común de época”, por usar términos de Antonio Gramsci, resultará crucial a la hora de cortocircuitar la “normalidad” atornillada en la subjetividad de los dominados. Porque cualquier régimen de poder (incluidos el neoliberalismo y el neocolonialismo) nos introduce siempre en una determinada visión del mundo, entendiendo por tal un conglomerado más o menos articulado de imágenes, nociones, metáforas y valores desde los cuales sentimos y pensamos la realidad. El pensador italiano nos hablaba de la necesidad perentoria de incidir en el “sentido común”, o en el “sentir popular”, como prerrequisito de todo proceso político que pretendiera transformar lo existente. Impactar en ese horizonte es crucial, si es que se desea dar una batalla contrahegemónica mínimamente exitosa. La “reforma intelectual y moral” (Gramsci, 1999, pp. 117-120), resultará un prerrequisito casi ineludible de todo proyecto político transformador que pretenda tener algún éxito. Pues bien, a nuestro modo de ver, el Buen Vivir andino ha supuesto un ejemplo perfecto de contra hegemonía en este sentido gramsciano, toda vez que resultó ser un discurso capaz de incidir de manera transformadora en el “sentido común de época” de ese Ecuador de inicios del siglo XXI. 5 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico Confrontando las ideas de “modernización” y “desarrollo” La construcción de los Estados iberoamericanos, monoculturales hasta la médula, presuponía la existencia de un componente atávico refractario al progreso, una rémora cultural que obstaculizaba de manera persistente los impulsos modernizadores: el mundo indígena. Pero, por otro lado, los análisis de tipo marxista-leninista y maoísta que proliferaron a partir de los años sesenta del siglo XX, vinculados al surgimiento de movimientos guerrilleros insurgentes, también participaron de ese imaginario de colonialidad. ¿Por qué? Porque desde dichos movimientos se categorizó o clasificó a los pueblos indígenas como “campesinos pobres”, un discurso que también ignoraba y oscurecía su componente étnico específico; además, eran tratados en muchas ocasiones como elementos “reaccionarios” e incluso “contrarrevolucionarios” (Bonfil Batalla, 1977). Las aspiraciones de estos pueblos debían ser formuladas en el lenguaje economicista de la lucha de clases; y es por ello que tenían que ser clasificados como “campesinos pobres”, pero jamás debían vehicular su lucha a través de una vindicación comunitaria que aludiese a su identidad étnica o “nacional”. También aquí su idiosincrasia quedaba completamente subsumida en una exterioridad asimiladora. El marxismo, por ende, estaba operando con esquemas ostensiblemente “modernizadores” y eurocéntricos (Lander, 2006, pp. 232-233; Polo Blanco, 2018a). Es digna de recordarse la excepcionalidad de José Carlos Mariátegui (1928/1994), que siempre reconoció la especificidad irreductible de la “cuestión amerindia”. Las “reivindicaciones étnicas” no podían quedar arrinconadas o recubiertas por la lucha de clases. Ciertos marxismos (atravesados por la filosofía hegeliana de la historia) carecían de los instrumentos teóricos necesarios para aprehender en su especificidad la realidad histórico-cultural de los llamados “pueblos originarios”. Las consecuencias políticas de lo anterior se manifestarían en una radical incomprensión, por parte de muchos comunistas europeos, de la especificad no subsumible del “problema” indígena. El colombiano Orlando Fals Borda (2007), precisamente con la intención de huir de aquellos esquemas eurocéntricos, ha puesto en juego su noción de “socialismo raizal”, explorado así las vías que puedan conducir a un “socialismo nuestro” que enraíce en otros ethos culturales no europeos o no occidentales. Desde el mundo andino se han planteado vías de impugnación contrahegemónica sustancialmente diferentes a las realizadas por el pensamiento crítico occidental. “Lo que verdaderamente distingue las luchas indígenas de las restantes luchas sociales en el continente americano es el hecho de reivindicar una precedencia histórica y una autonomía cultural que desafían todo el edificio jurídico y político del Estado moderno colonial” (Sousa Santos, 2012, p. 14). La población afrodescendiente construyó sus pro6 Eleder Piñeiro Aguiar pias impugnaciones, desde luego. La revolución negra de los esclavos haitianos (James, 1938/2003; Trouillot, 1995) conllevaba unas ideas humanistas más profundas y radicales que las evidenciadas en las revoluciones europeas burguesas, por cuanto ampliaba el concepto de ciudadanía y de libertad, atravesando incluso el concepto de raza, para mostrar la complicidad de los ideales pretendidamente ilustrados con el régimen esclavista. Allí, en aquella isla caribeña sublevada y ensangrentada a partir de 1791, quedó escenificada toda la tragedia política de la modernidad. Las excepciones micro, si bien no siempre se hallan todo lo conectadas que deberían, sí pueden marcar hitos o promover quiebras significativas (abriendo líneas de fuga) en las tendencias estructurales y homogeneizadoras del “sistema-mundo capitalista/patriarcal occidentalocéntrico/ cristianocéntrico moderno/colonial” (Grosfoguel, 2011). No reconocer lo diverso tiene que ver, en el plano ideológico-social, con construir un pensamiento monolítico; en un plano estatal-nacional, con asimilar la soberanía a la unidad; y en un plano ontológico-epistémico, con una organización colonial y pretendidamente universal del mundo. En conjunto, de lo que trata el no reconocimiento de la diversidad es de naturalizar múltiples relaciones de subalternidad, desigualdad, subordinación y discriminación. Y ese no reconocimiento, entre otros factores, se produce por la imposibilidad de pensar en alternativas socioeconómicas, dándose por sentado que el modelo capitalista neoliberal es el único viable o que los postulados de la economía ortodoxa responden al orden natural de las cosas (Peabody et al., 1977). Desnaturalizar ese supuesto orden es una de las tareas que se propuso el mencionado Plan Nacional del Buen Vivir en el Ecuador, durante los mandatos del presidente Correa, por cuanto no se debía caer en el chantaje de una determinada idea de progreso (Acosta, 2015, p. 26). Tampoco debía admitirse un estadio evolutivo presuntamente definitivo e inmejorable, en el que se hubiera llegado al “fin de la historia” (Fukuyama, 1995), dadas las repercusiones que el orden actual de las cosas conlleva para millones de personas: migrantes empobrecidos, desplazados o expulsados de las matrices hegemónicas del sistema. Al fin y al cabo, “lo que se observa en el mundo es un “mal desarrollo” generalizado, existente inclusive en los países considerados como desarrollados” (Gudynas y Acosta, 2013, p. 103). Además de todo lo mencionado, hemos de contemplar asimismo el plano ético e ideológico, como veremos. Lo que tampoco podemos ignorar es que este proceso andino tiene como horizonte trayectorias más largas a nivel geopolítico, centradas grosso modo en las últimas siete décadas y en torno al apoyo o desafección de la idea de pro greso. Dicho término proviene de un discurso del presidente estadounidense Harry S. Truman, en 1949, quien confirmaba la idea de mundos asimétricos y jerarquizados en escalas de poder (no solo económicas, si bien principalmente) (1949/s. f.). Desde enton- 7 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico ces, la idea de “desarrollo/progreso” ha seguido marcando las agendas globales, las intervenciones en países, las búsquedas de materias primas y energías o la sustracción de recursos humanos y naturales (De la Cuadra, 2015, p. 7). El “discurso del desarrollo”, como bien señaló Arturo Escobar (2007), se apoderó en la posguerra mundial de los imaginarios políticos occidentales (europeos y norteamericanos), pero también de los imaginarios operantes en todas aquellas naciones (neo)colonizadas que, de tal modo, asumían acríticamente buena parte de las definiciones y los diagnósticos que sobre ellas construían las potencias industriales. Se desplegó y enseñoreó una racionalidad desarrollista que fue esencialmente etnocéntrica y tecnocrática. El “desarrollo” se convirtió, finalmente, en un fetiche y en un poderoso régimen de representación que contribuyó a transmutar a dos tercios de la humanidad en “Tercer Mundo”; y operó, igualmente, como un cuerpo discursivo capaz de componer estrategias de acción macroeconómica destinadas a la estricta aplicación de determinados ajustes que transformarían (“modernizarían”) dichos países en un sentido muy determinado y, ciertamente, nunca desfavorable a los intereses de las grandes potencias occidentales. El Buen Vivir, en ese sentido, ha pretendido ser una contrarréplica a esas ideas hegemónicas de progreso y desarrollo (Acosta, 2011; Dávalos, 2011). Su inserción en la Constitución ecuatoriana sugiere el diseño de una ciudadanía que subvierta una secular situación que otorgaba a las poblaciones originarias y afroamericanas el papel de “salvajes”, “primitivos” o “bárbaros” (Polo Blanco, 2018b). Desde las coordenadas del Buen Vivir se han sugerido también formas diferentes y diversas de pensar las relaciones de poder, nuevas maneras de abordar las problemáticas de la identidad-alteridad al interior de un territorio y, en definitiva, ha pretendido generar una inédita conciencia ciudadana que pueda ir zafándose de ciertos elementos de racialización y colonialidad. Y lo cierto es que desde tal marco han surgido formas inéditas de entender la identidad de pueblos, nacionalidades, minorías, poblaciones y sujetos, más en consonancia con el respeto a la diversidad y avanzando por la senda de la interculturalidad (en el terreno educativo, por ejemplo), quebrando la homogeneización cultural (y administrativo-estatal) o superando el monismo jurídico. La Constitución ecuatoriana de 2008 reconoce explícitamente el “pluralismo jurídico” y el derecho de las comunidades indígenas a tener sus propias formas de aplicación de justicia, en algunos supuestos (Salgado, 2002). El Buen Vivir entroncaría en cierto modo con los presupuestos de la Teoría de la Dependencia (Dos Santos, 1978), incluso con propuestas “occidentales” que sostienen modelos económicos basados en “la gente primero” (Sen y Kliksberg, 2007). Algunos de los nombres que se le han ido dando al “desarrollo”, y que sirven tanto para mostrar las críticas que se han venido desplegando contra dicho concepto (pero reafirmando, en realidad, la vigencia del mismo, pues solamente lo matizan o edulcoran), son las si- 8 Eleder Piñeiro Aguiar guientes: “desarrollo humano” (Delval, 1994), “desarrollo sostenible” (Xercavins, 2005), “desarrollo participativo” (Geilfus, 2002), “desarrollo con identidad” (Deruyttere, 2001), “etnodesarrollo” (Palenzuela, 2009), “desarrollo comunitario” (Gutiérrez, 2013), “desarrollo local” (Vázquez Barquero, 1988) y algunos otros. Todos ellos, empero, no ponen en cuestión la esencia misma del concepto “desarrollo”; no lo impugnan holísticamente. En ese sentido, también el Buen Vivir reconoce que el desarrollo y progreso de unos países se produjo gracias al subdesarrollo y al atraso de otros. Es por ello que el reforzamiento del Estado y la recuperación de soberanía están en el eje central de los enunciados del Sumak Kawsay. No se despreocupa, en ese sentido, de las cuestiones geopolíticas y geoeconómicas. Muy entrelazados al discurso del Buen Vivir aparecen los conceptos de raza, género, clase y etnia. Pero también se trata, en último término, de en una reivindicación permanente de los elementos culturales de poblaciones históricamente denostadas o invisibilizadas. En ese sentido, la idea de mestizaje quizás no resulta del todo suficiente, puesto que más bien ha continuado reproduciéndose lo que Silvia Rivera Cusicanqui (2010) ha analizado como una “matriz colonial” del mestizaje, asentada en el expolio y en la dominación de los pueblos afros e indígenas, siendo el concepto (idea-imaginario) de raza el axioma fundamental. Porque “es evidente que la historia del capitalismo está intensamente racializada y generizada” (Harvey, 2014, p. 23). Es por ello que la crítica realizada desde poblaciones originarias pretende romper con dicha colonialidad del ser, del saber y del poder (Quijano, 2000, p. 342), huyendo de un entendimiento folclorizado del mestizaje. Sea como fuere, la sola circunstancia de que el Buen Vivir haya sabido colocar en la mesa del debate político dichas cuestiones ya ha de ser considerado un avance real en la superación de las matrices de blanqueamiento/blanquitud que, desde la época colonial, han troquelado los imaginarios hegemónicos en esta región del mundo. Una mirada alternativa desde los movimientos indígenas El mundo indígena andino, se ha de reconocer, fue capaz de lanzar una cierta impugnación de los modelos de desarrollo impuestos desde las potencias centrales del “sistema-mundo moderno/colonial” (Mignolo, 2003). Con su discurso y con su praxis supieron impulsar y dar dirección, siquiera sea parcialmente, a ciertos procesos sociopolíticos de signo emancipador. Ya en 1922 se había publicado El indio ecuatoriano, de Pío Jaramillo Alvarado, obra en la que se fundamentaba la construcción del indigenismo ecuatoriano y en la que se ofrecía una imagen del “indio” vindicando su papel activo en la construcción nacional, de manera similar a lo realizado en México, en fechas si - 9 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico milares (Botero, 2013, p. 8), por antropólogos como Manuel Gamio o por el filósofo José Vasconcelos en su ensayo La raza cósmica. La “asimilación” del “indio” por medio del sistema educativo, y el tema de la propiedad de las tierras, fueron los principales problemas analizados desde el indigenismo ecuatoriano. En el terreno de la literatura, el quiteño Jorge Icaza había publicado en 1934 la novela Huasipungo, una durísima y descarnada descripción de la terrible situación de sometimiento experimentada por las comunidades indígenas en los latifundios ecuatorianos. La falta de integración en lo nacional, que atravesó durante décadas las políticas públicas que afectaban a los pueblos originarios en el Ecuador, en cierto sentido continuaron hasta épocas recientes. Desde los años ochenta del siglo pasado, las crecientes protestas y movilizaciones indígenas pusieron de relieve un conjunto de jerarquías, desigualdades y discriminaciones de grandes capas de la población que, por sus características étnicas o “nacionales”, habían sido relegadas a un segundo plano. Dichas características específicas y diferenciales se hacían sentir en el plano del idioma, pero también en el de su espiritualidad, en sus costumbres y tradiciones. Esa identidad propia también fue valorizada mediante protestas y demandas; mediante luchas por recursos y por “derechos colectivos”. Debemos retrotraernos al año 1989, fecha en la que la Organización Internacional del Trabajo crea el famoso artículo 169 sobre los Derechos de los Pueblos Originarios (un artículo crucial que, casi treinta años después, la Constitución ecuatoriana de 2008 subscribe); a las marchas zapatistas de 1994, en donde se internacionaliza la protesta indígena y se viraliza a nivel global, gracias al uso de las nuevas tecnologías; al año 2007, cuando se crea la “Declaración de las Naciones Unidas sobre os Derechos de los Pueblos Indígenas”. En este proceso, sin embargo, una de las falencias más flagrantes que ha mostrado la academia ecuatoriana fue la de no trabajar más sistemáticamente con teorizaciones propias, puesto que en demasiadas ocasiones se asumieron conceptualizaciones indigenistas foráneas. En el Ecuador existen (o son reconocidas) 14 nacionalidades de pueblos originarios, y en torno a un millón y medio de ciudadanos que se reconocen como indígenas. En este contexto, importantes conflictos y tensiones han marcado la agenda política, siendo el mundo indígena un nuevo y relevante actor, consolidado desde hace casi cuarenta años, con diferentes aperturas y cerrazones a la negociación, y no con toda la homogeneidad interna que cabría esperar. En el caso ecuatoriano, “durante el gobierno de Rafael Correa se ha pasado de una mutua aceptación y defensa a un rechazo y condena del otro” (León Trujillo, 2010, p. 14). De hecho, desde diferentes medios indígenas se lanzó la acusación de traición, con respecto a las promesas previas a la toma del po der por parte de Rafael Correa (cuyo mandato fue de 2007 a 2017). Debe recordarse que el movimiento indígena se había integrado en la candidatura de Alianza País. La 10 Eleder Piñeiro Aguiar réplica oficialista consistió en señalar que el movimiento indígena era títere de partidos de la derecha. León Trujillo (2010) ha expuesto sucintamente los procesos de incorporación indígena al mapa político en las últimas décadas, desde las reivindicaciones por la igualdad hasta las más actuales de reconocimiento por la diferencia. Desde los años noventa del pasado siglo, diversos partidos de todo el espectro habían venido incorporando líderes y propuestas indígenas en sus programas, algo que muestra la histórica inclusión diferencial de los pueblos originarios en las agendas políticas nacionales. Organizaciones como la Confederación de Nacionalidades Indígenas del Ecuador (CONAIE) y el Consejo de Pueblos y Organizaciones Indígenas Evangélicos del Ecuador (FEINE) aumentaron la presencia indígena en el ámbito estatal, cristalizando en partido político Pachakutik. Previo a esto, ya en 1990, la CONAIE propuso un cambio en el primer artículo de la constitución para que el Ecuador se declarase pluricultural. Dicho partido y la Confederación apoyaron el programa transformador de Rafael Correa en 2006, acumulando desde entonces tanto tensiones como acuerdos. Porque hubo simbolismo indígena y visibilidad real, es verdad, pero también folklorización, esencialismo y denostación. Se paliaron discriminaciones históricas, pero también hubo criminalización de líderes indígenas y paternalismo estatal (Santillana, 2015). En la actualidad. los niveles de pobreza y desatención estatal en áreas rurales y hacia poblaciones afro, montubias e indígenas siguen siendo mucho más altos que en regiones urbanas y en poblaciones blancas y/o mestizas (Vera y Llanos-Escobar, 2016). Pero, a pesar de todas esas limitaciones conflictuales, lo cierto es que la asunción del Buen Vivir por parte de un partido-movimiento que terminó alcanzado el gobierno ha sido uno de los mayores logros del indigenismo políticamente organizado. La violencia estatal contra el movimiento indígena nunca desapareció del todo. Ya en 1974, en el proceso de otras protestas indígenas por la tenencia de tierras que llevaron al Estado ecuatoriano a elaborar una nueva Ley de Reforma Agraria, se produjo la muerte de un líder étnico (Botero, 2010, p. 1). O en el año del quinto centenario de la Conquista, cuando cientos de indígenas peregrinaron desde el Amazonas hacia Quito, en protesta por las injerencias petroleras en la región (Ortiz, 2002). Además, tras el terrible terremoto acaecido el 16 de abril, en la provincia amazónica de Morona-Santiago se produjeron violentas protestas indígenas contra la extracción minera, que desembocaron en el asesinato tanto de líderes indígenas como de policías (Meléndez y Moncagatta, 2017, p. 416). En este caso, se evidenció crudamente una contradicción entre los proclamados derechos colectivos de las naciones indígenas y las políticas neoextractivistas de las que no pudo zafarse el gobierno de Correa, a pesar de otorgar derechos intrínsecos a la naturaleza en el texto constitucional. En el Ecuador existen al menos doscientos dirigentes comunitarios enjuiciados, muchos de ellos con cargos de terro- 11 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico rismo o de sabotaje, por luchar en la defensa del territorio. No se puede ser un gobierno cuyos delineamientos estratégicos pretenden fundamentarse en el Buen Vivir y, al mismo tiempo, enviar a prisión a los que defienden en la práctica, precisamente, el Buen Vivir. Es una flagrante contradicción (Sousa Santos, 2012, pp. 34-35). Por otra parte, que el Buen Vivir recaiga, en cuanto a su propuesta de construc ción, en comunidades quechuas y aymaras de Bolivia y Ecuador no significa que dichas comunidades sean las únicas que tengan la exclusividad de estas propuestas. Mapuches, guaraníes, multitud de pueblos amazónicos o aborígenes australianos, entre muchos otros, tienen modos de vivir y pensar que podrían estar muy cercanos a estas propuestas. Y es que “el Buen Vivir no es patrimonio de ningún grupo o sector social en particular” (De la Cuadra, 2015, p. 8). Desde tales coordenadas se recalca la necesidad de reconocer que históricamente el mundo indígena ha resistido (solo hasta cierto punto) a la modernidad capitalista, no dejándose fagocitar totalmente por ella; y lo ha hecho de diversas maneras, más o menos violentas. Porque si la categoría de democra cia está en constante construcción, también lo está la categoría de ciudadano/a. Por lo tanto, invisibilizar, “blanquear” (Echeverría, 2007, 2010) o “asimilar” al indio —que han sido procesos y tácticas de los que se ha servido el capital a la hora de obtener más “rendimiento” de los cuerpos y de la naturaleza— han sido procedimientos criticados desde todos esos otros paradigmas ontológicos, epistemológicos y políticos propuestos desde las comunidades originarias andinas. Aun con todo ello, diversos pueblos y grupos étnicos han conseguido dar continuidad a ritos, mitos, tradiciones, formas culturales, cosmovisiones e identidades propias, siendo quizá uno de los hitos la continuidad a la justicia indígena al interior de las comunidades, pero también en conexión con los Estados envolventes. “La justicia indígena, al contrario de la plurinacionalidad, no es un proyecto, algo por construir, una novedad. Es una realidad que, reconocida o no por el Estado, ha formado y forma parte de la vida de las comunidades” (Sousa Santos, 2012, p. 16). Tener derecho al propio derecho, en suma, ha sido también uno de los elementos combativos del mundo indígena (Stavenhagen, 2010). Pero el debate no está tanto entre modernidad y tradición, pues además de que toda tradición (incluida la de la modernidad) es constantemente reinventada (Hobsbawm y Ranger, 1983/2002), lo cierto es que toda modernidad corre paralela a otras posibles. “Lo indígena” y “lo occidental”, permítasenos usar estas categorías hipostasiadas, tienen mecanismos diferentes de adaptación al cambio, los cuales en ocasiones interactúan y no necesariamente siempre de manera conflictiva. La justicia indígena no opta por un castigo individual, sino que se centra en el equilibrio y en la recuperación de la armonía colectiva. 12 Eleder Piñeiro Aguiar Considerando cuáles podrían ser los principales aportes del modelo social del Buen Vivir, debiera mencionarse su concepto de comunidad y su relación con la “convivialidad”, definida por Boaventura de Sousa Santos (2011) como “un tipo de encuentro cultural y político basado en intercambios tendencialmente iguales y en la autoridad compartida” (Grijalva, 2012, p. 71). Las cosmovisiones indígenas no destruyen la individualidad; más bien la incluyen de manera holística en el interior de la colectividad, buscando una situación de equilibrio entre el individuo y lo común. Ello implica comprender que todo está unido, integrado y es complementario. En la comunidad, no solo nos encontramos con una serie de individuos y estructuras-instituciones, sino que dicha comunidad es una unidad vital que debe ser vista de manera holística, y no solo como un territorio de convivencia de individuos yuxtapuestos. La relación y la interacción son claves para entender el equilibrio, la armonía y el respeto por la norma comunal. Tal comunitarismo permite un trabajo más local y localizado, en donde los aspec tos económicos y político-sociales no se diluyen. La convivialidad y la complementariedad son claves para organizar lo político y lo económico en el mundo indígena. Todo lo cual permitiría avanzar hacia la “recuperación” de cierta soberanía (Sousa Santos, 2012, p. 30). Dentro de esto, se percibe la naturaleza como un “ente vital” capaz de sentir, conocer y actuar, algo que colisiona con la visión que históricamente la modernidad occidental ha tenido de ella. Frente al mecanismo de subsunción de todas las formas de trabajo en el capital, emergen otras formas de reciprocidad tales como las planteadas en el marco del Sumak Kawsay andino, en donde la cooperación horizontal, la complementariedad y la solidaridad constituyen la base de las relaciones sociales, cósmicas, naturales e incluso económicas. Seres humanos, naturaleza y deidades conforman el ayllu, un grupo de parientes unidos más allá del linaje humano o parentesco de sangre. Aprender a criar y dejarse criar con respeto, empatía, reciprocidad y en goce son principios y prácticas primordiales en los Andes. (Gonzales, 2014, p. 128) El núcleo del asunto reside en el hecho de que en la cosmovisión indígena el dua lismo, lo teleológico y la historia lineal (ligada a las ideas de desarrollo y progreso) son contrarrestadas por un pensamiento-praxis que engloba en un carácter holístico las relaciones de las personas con el medio, la naturaleza y el mundo espiritual. Esto impli ca, entre otros aspectos, eliminar la visión expropiadora de la naturaleza, no contemplándola ya como un elemento externo, para proponer una lógica de empatía y comunión con ella; no verla como un objeto sobre el cual intervenir de manera ilimitadamente depredadora, sino como un lugar y un ser en el que vivir y con el que vivir. 13 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico El Buen Vivir es una alternativa orientada a tratar de rehacer la vida socioambiental a partir de la solidaridad humana y con la naturaleza, no solo en la actividad económica y productiva, sino en todas las dimensiones de la existencia social: el trabajo, el sexo, la autoridad colectiva, la subjetividad y la naturaleza. (Marañón, 2014, p. 41) El extractivismo no es solamente una práctica económica; es también una epistemología, una forma de entender el mundo (Grosfoguel, 2016). Convertir un hábitat en territorio/paisaje, reduciéndolo a “recurso natural”, va contra las lógicas indígenas, por cuanto éstas entienden a la tierra no solo como un lugar y un contexto para vivir, sino como un ente vivo con el que se está en comunión. Es decir, “para los indígenas, el territorio no es algo delimitado y menos sin vida, todo en su seno porta y hace posible la vida, todo se degrada y se transforma en ella y es mediante los mitos y los rituales que la tierra como territorio está siempre presente” (Molina, 2015, p. 148). La idea de reciprocidad con la madre naturaleza y de los seres entre sí es central en todo esto, pero “no se trata de idealizar o ideologizar la reciprocidad, pues es importante considerar que algunas formas de reciprocidad vertical conducen a la alienación y dependencia” (López Córdova, 2014, p. 105). La antropología ha prestado especial atención a este concepto, recalcando el triple carácter obligatorio de dar, recibir y devolver, considerando el carácter de “hecho social total” del don, así como la energía espiritual de la naturaleza y de las cosas, las cuales debían ser devueltas (Godbout y Caillé, 1992/1997). Lo importante de la reciprocidad son las personas, y no los objetos; ellas se relacionan con base en la confianza y a la solidaridad, creando vínculos que son reforzados por el intercambio y por la redis tribución, no por la ostentación y el consumo conspicuo. La tensión y la dislocación surgen cuando los intercambios del mercado interfieren con los valores de la reciprocidad, puesto que mientras la reciprocidad es una relación entre personas, el mercado es algo abstracto e impersonal. Es por esto que la propuesta del Buen Vivir critica dicho sistema de mercado, enfatizando la relacionalidad de la reciprocidad. “El sistema de intercambio lleva a la competencia y la acumulación privada, a la dominación y explotación entre los hombres y con la naturaleza, así como a la ruptura de los lazos sociales” (López Córdova, 2014, p. 112). Los detentadores del discurso del Buen Vivir han pretendido, con mayor o menor éxito, ser adversarios de los modelos socioeconómicos fundamentados en el individualismo posesivo y competitivo; adversarios de la racionalidad del homo oeconomicus (León, M., 2009 y 2010). 14 Eleder Piñeiro Aguiar Conclusiones El Buen Vivir, es cierto, no constituye una alteridad absoluta con respecto a la “racio nalidad occidental”. De hecho, interioriza dialógicamente algunos de los elementos más valiosamente emancipadores de dicha racionalidad. Pretende realizar una conjugación inédita, esto es: Trata de articular dos herencias culturales, expresadas en una nueva racionalidad liberadora y solidaria: por un lado, la razón histórica de la modernidad, con sus promesas de libertad, igualdad social y bienestar, y por otro, la razón «india» prehispánica, vinculada con la reciprocidad, la solidaridad social y el trabajo colectivo. (Marañón, 2014, p. 11, cursivas del original) En lo que a la parte pluriétnica del modelo se refiere, la inclusión del Buen Vivir como política estatal reivindica el papel de la interculturalidad y, por ende, una aproximación tanto política como epistémica al entendimiento integrador de la diversidad sociocultural y económica de los pueblos. Esto rompería con las categorías monolíticas de la modernidad occidental, en tanto que la unidad en torno a una soberanía, un territorio, una cultura y una población se irían difuminando en aras del reconocimiento de una pluralidad; y ello bajo los criterios de simetría, igualdad y equidad. La “mirada occidentalizada” y colonizada de un buen número de ecuatorianos, con respecto al mundo indígena, podría empezar de tal modo a deconstruirse (Botero, 2013, p. 2). El discurso del Buen Vivir contribuyó significativamente a quebrar, al menos en el plano ideológico, el secular proceso de “blanqueamiento” y homogeneización cultural. Lo emancipatorio del proyecto estriba en su carácter dialógico con la alteridad, y la puesta en juego de intereses colectivos más amplios que los basados en el crecimiento cortoplacista. Algunos de sus promulgadores consideran que el concepto de Sumak Kawsay contribuye al entendimiento de lo que es el “Bien Común de la Humanidad” (Houtart, 2011). Reconocer las diversas composiciones étnicas en un territorio, y su aporte a la política común, es uno de los principales avances. Interviene en prácticas racistas/racializadas y genera nuevas formas de entender la ciudadanía desde puntos de vista más plurales y heterogéneos. Tratando de no caer en esencialismos “buensalvajistas” (precaución no siempre adoptada por parte de estos teóricos y activistas), lo cierto es que desde el universo ideológico del Buen Vivir se construyó una subjetividad parcialmente decolonizada, o al menos esa fue la pretensión. Por otra parte, y a pesar de las críticas que se pueden realizar por sus enormes complicaciones de implementación, que conllevarían la necesidad no solamente de cambios jurídicos, económicos y administrativos, sino un giro radical de las mentalidades y de los valores. El Buen Vivir, al menos conceptualmente, se perfila como una 15 El Sumak Kawsay andino como resistencia sociopolítica y como desafío epistémico versión que supera los desarrollos “alternativos” e intenta ser una “alternativa al desarrollo” (Gudynas y Acosta, 2011, p. 109). No somos, en este sentido, tan optimistas como aquellos que puedan afirmar que el Buen Vivir rompe con los paradigmas coloniales, a raíz de su inclusión constitucional. La aplicación de políticas que caminen hacia el ecologismo, la interculturalidad, el reconocimiento, la lucha contra las matrices coloniales y el respeto de los seres vivos (humanos y no humanos) en armonía con su entorno, todo ello, es un terreno complejo que requiere mucho tiempo. No ha pasado todavía una generación que pueda percibir los cambios deseados, ni es lapso de tiempo suficiente para cambiar inercias seculares, llevando a la práctica concreta esos “deber ser” del Buen vivir que figuran en algunos planes de desarrollo y en el marco constitucional. Quizás el Buen Vivir no sea, a la postre, más que una lógica diferente para pensar en modelos civilizatorios distintos y alternativos. Y, en cualquier caso, a través de él han resurgido con fuerza el mundo indígena y las reivindicaciones históricas de todos esos pueblos originarios que fueron invisibilizados —aunque nunca acallados del todo— por el progreso modernizador occidental. Referencias Acosta, Alberto (2011). El buen vivir en el camino del post-desarrollo. Algunas reflexiones al andar. En Gabriela Weber (Ed.), Debates sobre cooperación y modelos de desarrollo. Perspectivas desde la sociedad civil en el Ecuador (pp. 6182). 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https://openalex.org/W2914884735	https://europepmc.org/articles/pmc6379230?pdf=render	English	null	Percutaneous endoscopic transforaminal discectomy precedes interlaminar discectomy in the efficacy and safety for lumbar disc herniation	Bioscience reports	2,019	cc-by	6,555	Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) We searched several databases from the times of their inception to 20 December 2018. Randomized controlled trials and cohort studies that compared percutaneous endoscopic transforaminal discectomy (PETD) with percutaneous endoscopic interlaminar discectomy (PEID) were identified. We used a random-effects model to calculate the relative risks (RRs) of, and standardized mean differences (SMDs) between the two techniques, with 95% con- fidence intervals (CIs). Twenty-six studies with 3294 patients were included in the final analysis. Compared with PEID, PETD reduced the short-term (SMD −0.68; 95% CI −1.01, −0.34; P=0.000) and long-term (SMD −0.47; 95% CI −0.82, −0.12; P=0.000) visual analog scale scores, blood loss (SMD −4.75; 95% CI −5.80, −3.71; P=0.000), duration of hospi- tal stay (SMD −1.86; 95% CI −2.36, −1.37; P=0.000), and length of incision (SMD −3.93; 95% CI −5.23, −2.62; P=0.000). However, PEID was associated with a lower recurrence rate (P=0.035) and a shorter operative time (P=0.014). PETD and PEID afforded compara- ble excellent- and good-quality data, long- and short-term Oswestry disability index (ODI) scores, and complication rates. PETD treated lumbar disc herniation (LDH) more effectively than PEID. Although PETD required a longer operative time, PETD was as safe as PEID, and was associated with less blood loss, a shorter hospital stay, and a shorter incision. PETD is the best option for patients with LDH. © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Received: 15 October 2018 Revised: 15 January 2019 Accepted: 25 January 2019 Percutaneous endoscopic transforaminal discectomy precedes interlaminar discectomy in the efﬁcacy and safety for lumbar disc herniation Peng Chen1, Yihe Hu1 and Zhanzhan Li2 1Department of Orthopedic, Xiangya Hospital, Central South University, Changsha, Hunan Province, China; 2Department of Oncology, X Changsha, Hunan Province, China Peng Chen1, Yihe Hu1 and Zhanzhan Li2 1Department of Orthopedic, Xiangya Hospital, Central South University, Changsha, Hunan Province, China; 2Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan Province, China Correspondence: Zhanzhan Li (liche4006@126.com) or Yihe Hu (yihehumed@126.com) ss article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Introduction Lumbar disc herniation (LDH) is common today, even in young individuals, and more often in males than females [1]. Most herniation sites are located at L5/S1 and L4/5. LDH is caused by degenerative changes in the intervertebral discs; external forces cause rupture of the annulus fibrosus, nuclear herniation, or compression of the cauda equina nerve roots, triggering tissue inflammation, edema, and poor micro- circulation, followed by low back pain, lower extremity sciatic radiating pain, and other disorders [2], in turn compromising the quality of life [3]. Therapeutic strategies include conservative and surgical treat- ments. Most patients benefit greatly from conservative treatments, such as intravenous and oral medica- tion administration, traction therapy, and manipulative rehabilitation, but some require surgery [4]. The surgical options include open discectomy, microdiscectomy, microendoscopic discectomy (MED), and percutaneous endoscopy lumbar discectomy (PELD) [5]. In recent years, with the rapid development of surgical techniques, minimally invasive spine surgery has become imperative. Compared with open dis- cectomy, minimally invasive surgery is associated with a shorter operative time, less blood loss, less muscle injury, and faster functional recovery [6–8]. PELD includes percutaneous endoscopic transforaminal dis- cectomy (PETD) and percutaneous endoscopic interlaminar discectomy (PEID). Some previous studies confirmed the therapeutic efficacy of PETD, but others did not [9,10]. PETD is rather difficult in patients 1 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 with high cristae iliacae and narrow foramina, especially at L5/S1. However, the L5/S1 interlaminar space is usually adequate [11]. Ruetten et al. [12] were the first to perform intervertebral disc discectomy and decompression by creating an intervertebral foramen in the vertebral canal between the upper and lower vertebral discs. Several studies have compared the efficacies of PETD and PEID in patients with LDH; the results were inconsistent. Hence, we comprehensively analyzed this topic. with high cristae iliacae and narrow foramina, especially at L5/S1. However, the L5/S1 interlaminar space is usually adequate [11]. Ruetten et al. [12] were the first to perform intervertebral disc discectomy and decompression by creating an intervertebral foramen in the vertebral canal between the upper and lower vertebral discs. Several studies have compared the efficacies of PETD and PEID in patients with LDH; the results were inconsistent. Hence, we comprehensively analyzed this topic. Data extraction and quality assessment We used a standard Excel sheet for data extraction. Two investigators (P.C. and Z.L.) independently extracted the following data: first author, publication year, mean patient age, study design (retrospective compared with prospec- tive), sample size, follow-up duration, and outcome measures. We sought to contact the authors when information was missing. Differences in opinion were resolved by discussion with the third investigator (Y.H.). g p y g All prospective and retrospective studies were evaluated using the Newcastle–Ottawa scale (which compares pa- tient selection, comparisons, and outcomes; maximal score 9). Studies with scores ≥7 were considered to be of high quality [13]. We used the Cochrane risk-of-bias tool to assess study quality [14]; the tool explores random sequencing, allocation concealment, blinding of participants and personnel, outcome assessments, outcome data completeness, selective reporting, and other biases. We scored each study as being at a low, high, or unclear risk of bias. Studies in which at least one key domain was considered to be at high risk of bias were regarded as high risk; other studies were considered to be of low or unclear risk. Materials and methods gy Two investigators (P.C. and Z.L.) independently searched the following databases from their inception to 20 July 2018: PubMed, Web of Science, Embase, China National Knowledge Infrastructure, and WanFang. The following search terms were used: ‘lumbar disc herniation’ OR ‘LDH’, ‘percutaneous endoscopic lumbar discectomy’ OR ‘percutaneous endoscopic transforaminal discectomy’ OR ‘PLED’ OR ‘PELD’, and ‘microendoscopic discectomy’ OR ‘percutaneous endoscopic interlaminar discectomy’. We restricted the languages to Chinese and English. We checked the reference lists of selected full-text and review articles to identify other potentially relevant works. Inclusion and exclusion criteria The inclusion criteria were: (i) examination of a population of patients with LDH, (ii) randomized controlled trial or retrospective study comparing the efficacies of PETD and PEID in terms of LDH treatment, and (iii) comparison of PETD and PEID interventions. The primary outcome requirements were: (i) at least one short-term or long-term visual analog scale (VAS) or Oswestry disability index (ODI) score, and (ii) excellent or good data quality. The sec- ondary outcomes were the (iii) complication rate, (iv) recurrence rate, (v) operative duration, (vi) amount of blood loss, (vii) length of incision, and (viii) length of hospital stay. Reviews, comments, duplicate and case reports, letters, and animal and experimental studies were excluded. Statistical analysis We used fixed- and random-effects models to evaluate pooled data [15]. We calculated relative risks (RRs) with 95% confidence intervals (CIs) for dichotomous data and standardized mean differences (SMDs) with 95% CIs for con- tinuous data. Within-study heterogeneity was assessed by calculating the I2 statistic and Cochran’s Q; when I2-values >50% and P-values <0.05 indicated significant heterogeneity, we employed the random-effects model [16]; we used the fixed-effects model otherwise. To evaluate heterogeneity further, we performed subgroup analyses of primary outcomes (VAS and ODI scores). Publication bias was assessed by visual inspection of a funnel plot and application of the Egger’s/Begg’s test [17,18]. All statistical analyses were performed with the aid of Stata ver. 14.0 (StataCorp LP) and RevMan ver. 5.3 (Nordic Cochrane Center) software; P-values <0.05 were considered to reflect significance. © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). tudy selection and characteristics Study selection and characteristics y Figure 1 shows the study selection flow. Our initial search returned 679 records; we found no additional text when exploring other possible sources. After removal of duplicates and scanning of titles and abstracts, we selected 71 full-text articles for further assessment. We excluded 45 of these articles. Finally, 26 studies were included in our qualitative and quantitative analyses (Supplementary Material S1). The general characteristics of the studies are listed 2 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Figure 1. Flow chart of study selection Figure 1. Flow chart of study selection in Table 1; all studies were published between 2009 and 2018. The mean ages of patients treated with PETD ranged from 33.1 to 69.2 years, and those of patients treated with PEID ranged from 36.8 to 68.9 years. Nine studies were retrospective and seventeen were prospective. The duration of follow-up ranged from 3 to 26 months. The types of disease included central, paracentral, and far-lateral disease, and disease of the intervertebral foramen. in Table 1; all studies were published between 2009 and 2018. The mean ages of patients treated with PETD ranged from 33.1 to 69.2 years, and those of patients treated with PEID ranged from 36.8 to 68.9 years. Nine studies were retrospective and seventeen were prospective. The duration of follow-up ranged from 3 to 26 months. The types of disease included central, paracentral, and far-lateral disease, and disease of the intervertebral foramen. Quality assessment We included 8 randomized controlled trials and 18 follow-up studies. Supplementary Material S2 lists the risks of bias and includes the bias graphs. Two studies were considered to exhibit high risks of bias because neither the study participants nor personnel were blinded. On the Newcastle–Ottawa scale, the mean score of observational studies was >7, indicating high quality. Pooled results The summarized results are presented in Table 2. Short- and long-term VAS scores Twelve articles provided short-term and eleven provided long-term VAS scores. Significant heterogeneity was appar- ent (I2 > 50%, P=0.000). The random-effects model was used to analyze both datasets. Meta-analysis showed that the short-term (SMD −0.68; 95% CI −1.01, −0.34; P=0.000; Figure 2 and long-term (SMD −0.47; 95% CI −0.82, −0.12; P=0.000; Figure 3) scores associated with PETD were significantly lower than those associated with PEID. 3 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Li 4 0 (CC BY) © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Figure 2. Comparison of short-term VAS between PETD and PEID Figure 3. Comparison of long-term VAS between PETD and PEID. © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Figure 2. Comparison of short-term VAS between PETD and PEID Figure 2. Comparison of short-term VAS between PETD and PEID Figure 3. Comparison of long-term VAS between PETD and PEID. Figure 3. Comparison of long-term VAS between PETD and PEID. 4 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Pooled results Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Table 1 Characteristics of included studies in the meta-analysis Author Publication year Age (PEID/PETD) Study design Sample size Follow-up time (months) Type PEID PETD Fang 2012 43.5/45.8 Retrospective 40 40 6 (1)(2)(3) Le 2014 37.2/38.4 Prospective 190 185 12 (1)(2)(3) Guan 2014 - Prospective 35 35 3 - Wu 2009 4.5/45.8 Prospective 30 30 6 (1)(2)(3) Wu 2015 38.5/41.3 Prospective 50 36 6 (1)(2)(3) Zhang 2015 43.2/41.5 Prospective 30 30 26 (3)(4) Zhang 2015 37.5/35.8 Retrospective 21 21 12 (1)(2)(4) Fu 2014 - Prospective 8 62 12 (1)(2)(3)(4) Zeng 2015 - Prospective 25 25 24 - Li 2013 38.3/43.3 Prospective 212 208 - - Li 2015 51.5/51.6 Retrospective 50 50 - - Yang 2015 48.4/48.0 Prospective 82 57 3 - Tang 2015 - Prospective 38 38 24 - Zhao 2012 39.4/43.2 Retrospective 245 261 - - Chen 2015 - Prospective 25 13 13.5 (3)(4) Mao 2015 37.5/37.8 Retrospective 35 30 12 - Yoon 2012 45.9/56.5 Retrospective 37 35 6 - Sinkemani 2015 44.2/41.5 Retrospective 50 36 14 - Liu 2012 - Prospective 25 13 13.5 (3)(4) Chen 2018 64.1/64.2 Prospective 137 136 12 (1)(2)(4) Chen 2018 40.7/40.2 Prospective 73 80 12 (1)(2)(3)(4) Huang 2018 32.1/32.3 Retrospective 52 50 6 (1)(2)(3)(4) Ding 2017 54.2/54.4 Prospective 40 40 3 (1)(2)(3)(4) Liu 2017 69.2/68.9 Prospective 25 25 3 (1)(2)(3)(4) Liu 2018 33.1/36.2 Prospective 63 60 24 (1)(2)(3)(4) Wu 2017 38.7/40.8 Retrospective 40 40 12 (1)(2)(3) (1) Central type, (2) Para central, (3) Intervertebral foramen, and (4) Far-lateral. Table 1 Characteristics of included studies in the meta-analysis (1) Central type, (2) Para central, (3) Intervertebral foramen, and (4) Far-lateral. Pooled results Table 2 Comparison of pooled parameters between percutaneous endoscopic lumbar, transforaminal discectomy and interlaminar discectomy Table 2 Comparison of pooled parameters between percutaneous endoscopic lumbar, transforaminal discectomy and interlaminar discectomy Parameters Number of study Pheterogeneity RR/SMD 95% CI P Egger Begg Short-term VAS 12 0.000 −0.68 −1.01, −0.34 0.000 0.012 0.002 Long-term VAS 11 0.000 −0.47 −0.82, −0.12 0.000 0.900 0.224 Short-term ODI 5 0.000 −0.06 −0.33, 0.22 0.691 0.306 0.951 Long-term ODI 7 0.000 −0.15 −0.36, 0.06 0.123 0.238 0.537 Excellent and good rate 13 0.015 1.02 0.97, 1.07 0.509 0.232 0.272 Complication rate 15 0.438 0.78 0.54, 1.13 0.185 0.149 0.400 Recurrence rate 11 0.128 1.90 1.04, 3.47 0.035 0.017 0.008 Duration of operation 18 0.000 0.70 0.14, 1.26 0.014 0.226 0.058 Blood loss 15 0.000 −4.75 −5.80, −3.71 0.000 0.273 0.235 Length of incision 8 0.000 −3.93 −5.23, −2.62 0.000 0.067 0.063 Duration of hospital stay 15 0.000 −1.86 −2.36, −1.37 0.000 0.081 0.038 Short-term and long-term ODI scores Five articles provided short-term ODI scores and seven provided long-term scores. We used a random-effects model for analysis because significant heterogeneity was in play. Neither the short- nor long-term ODI score differed signif- icantly between PETD and PEID (SMD −0.06; 95% CI −0.03, 0.22; P=0.691; Figure 4A; and SMD −0.15; 95% CI © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Short-term and long-term ODI scores Five articles provided short-term ODI scores and seven provided long-term scores. We used a random-effects model for analysis because significant heterogeneity was in play. Neither the short- nor long-term ODI score differed signif- icantly between PETD and PEID (SMD −0.06; 95% CI −0.03, 0.22; P=0.691; Figure 4A; and SMD −0.15; 95% CI © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). −0.36, 0.06; I = 0.123; Figure 4B, respectively). −0.36, 0.06; I = 0.123; Figure 4B, respectively). Excellent and good data The data from 13 studies were rated as excellent or good; the degree of heterogeneity was moderate (I2 = 51.8%, P=0.015). The random-effects model indicated that the excellent and good rates in the two groups were nearly iden- tical (RR = 1.02; 95% CI 0.97, 1.07; P=0.509; Figure 5A). Complication and recurrence rates p Complication rates were reported in 15 articles; the degree of heterogeneity was very low (I2 = 1.1%, P=0.438). A fixed-effects model revealed no significant between-group difference (RR = 0.78; 95% CI 0.54, 1.13; P=0.185; Figure 5B). Recurrence rates were reported in 11 articles; no heterogeneity was evident (I2 = 0.0%, P=0.128) and the data were analyzed using a fixed-effects model. The pooled results suggested that the recurrence rate after PETD was higher than that after PEID (RR = 1.90; 95% CI 1.04, 3.47; P=0.035; Figure 5C). Duration of operation and blood loss Random-effects models were used to analyze the duration of operation and blood loss because significant heterogene- ity was evident (I2 > 50.0%, P<0.05). Eighteen articles provided data on the operative duration and fifteen provided data on blood loss. Compared with PEID, PETD required a longer operative time (SMD 0.70; 95% CI 0.14, 1.26; P=0.014; Figure 6A), but was associated with less blood loss (SMD −4.75; 95% CI −5.80, −3.71; P=0.000; Figure 6B). Pooled results 5 ed parameters between percutaneous endoscopic lumbar, transforaminal discectomy and Table 2 Comparison of pooled parameters between percutaneous endoscopic lumbar, transforaminal discectomy and interlaminar discectomy Parameters Number of study Pheterogeneity RR/SMD 95% CI P Egger Begg Short-term VAS 12 0.000 −0.68 −1.01, −0.34 0.000 0.012 0.002 Long-term VAS 11 0.000 −0.47 −0.82, −0.12 0.000 0.900 0.224 Short-term ODI 5 0.000 −0.06 −0.33, 0.22 0.691 0.306 0.951 Long-term ODI 7 0.000 −0.15 −0.36, 0.06 0.123 0.238 0.537 Excellent and good rate 13 0.015 1.02 0.97, 1.07 0.509 0.232 0.272 Complication rate 15 0.438 0.78 0.54, 1.13 0.185 0.149 0.400 Recurrence rate 11 0.128 1.90 1.04, 3.47 0.035 0.017 0.008 Duration of operation 18 0.000 0.70 0.14, 1.26 0.014 0.226 0.058 Blood loss 15 0.000 −4.75 −5.80, −3.71 0.000 0.273 0.235 Length of incision 8 0.000 −3.93 −5.23, −2.62 0.000 0.067 0.063 Duration of hospital stay 15 0.000 −1.86 −2.36, −1.37 0.000 0.081 0.038 mparison of pooled parameters between percutaneous endoscopic lumbar, transforaminal dis discectomy Table 2 Comparison of pooled parameters between percutaneous endoscopic lumbar, interlaminar discectomy Short-term and long-term ODI scores © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). 5 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Figure 4. Forest plot for short-term and long-term ODI between PETD and PEID Comparison of short-term (A) and long-term (B) ODI between PETD and PEID. Figure 4. Forest plot for short-term and long-term ODI between PETD and PEID Comparison of short-term (A) and long-term (B) ODI between PETD and PEID. Figure 4. Forest plot for short-term and long-term ODI between PETD and PEID Comparison of short-term (A) and long-term (B) ODI between PETD and PEID. ion of operation and blood loss p Random-effects models were used to analyze the duration of operation and blood loss because significant heterogene- ity was evident (I2 > 50.0%, P<0.05). Eighteen articles provided data on the operative duration and fifteen provided data on blood loss. Compared with PEID, PETD required a longer operative time (SMD 0.70; 95% CI 0.14, 1.26; P=0.014; Figure 6A), but was associated with less blood loss (SMD −4.75; 95% CI −5.80, −3.71; P=0.000; Figure 6B). 6 6 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Figure 5. Forest plot for clinical outcomes Comparisons of clinical outcomes between PETD and PEID: (A) excellent and good rate; (B) complication rate; (C) recurrence and residue rate. Figure 5. Forest plot for clinical outcomes Figure 5. Forest plot for clinical outcomes Comparisons of clinical outcomes between PETD and PEID: (A) excellent and good rate; (B) complication rate; (C) recurrence and residue rate. Length of incision and duration of hospital stay The length of incision and duration of hospital stay were also evaluated. Eight articles provided data on the length of incision and fifteen provided data on the duration of hospital stay. Both indicators evidenced significant heterogeneity (I2 > 50.0%, P<0.0.5). Random-effects models indicated that PETD required a shorter incision (SMD −3.93; 95% CI −5.23, −2.62; P=0.000; Figure 6C) and a shorter hospital stay (SMD −1.86; 95% CI −2.36, −1.37; P=0.000; Figure 6D). Sensitivity analysis and publication bias y y p We subjected the operative durations reported in most (n=18) articles to sensitivity analysis; we omitted one study at a time. The pooled results ranged from 0.10 to 0.63 (Supplementary Material S3). All results were significant. The Egger’s/Begg’s test indicated that publication bias was not in play (P>0.05), except in terms of the short-term VAS score and the recurrence rate (Table 2). The funnel plot was slightly asymmetrical (Supplementary Material S4). 7 7 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Forest plot for symptoms ons of duration of operation (A), blood loss (B), length of incision (C), and length of hospital stay (D). Figure 6. Forest plot for symptoms Comparisons of duration of operation (A), blood loss (B), length of incision (C), and length of hospital stay (D). Figure 6. Forest plot for symptoms Figure 6. Forest plot for symptoms Comparisons of duration of operation (A), blood loss (B), length of incision (C), and length of hospital stay (D). g p y p Comparisons of duration of operation (A), blood loss (B), length of incision (C), and length of hospital stay (D Comparisons of duration of operation (A), blood loss (B), length of incision (C) © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Second, no more than five studies were included in certain comparisons. Blood loss, bed time after surgery, and duration of hospital stay were reported in only two articles; the veracity of the pooled results is thus debatable. Third, our study involved the analysis of more information. We evaluated the short- and long-term VAS and ODI scores, and the complication and recurrence rates. Finally, our study (with a larger sample) indicated that PETD significantly reduced the blood loss, operative duration, length of incision, and duration of hospital stay; such conclusions could not be drawn in the previous study [10]. p y We found that PETD significantly reduced the short- and long-term VAS scores. The short-term VAS score reflects not only made improvements in disc herniation, but also the extent of surgical trauma. The incision at the interverte- bral foramen was smaller (generally approximately 0.8 cm) in PETD than in PEID [19]. The PETD approach channel is expanded via blunt muscle separation, which damages tissue and muscle to lesser extents than the PEID approach [20]. Patients can feel nerve root pain during surgery. The long-term VAS scores further suggested that PETD caused less tissue injury than PEID. However, we found that PETD required a longer operative time. In general, longer spinal surgery times are associated with more complications and re-operations; surgical time is an important comparative parameter when selecting an approach. Most herniations were located at L5/S1 and L4/5, where the intervertebral disc spaces are wide; traditional surgery is not difficult. However, the anatomical structure renders it challenging to puncture and remove disc fragments via PETD, especially at L5/S1 [21,22]. Moreover, PEID is easier to surgically master; this approach uses elements of traditional surgery. We found no significant difference in complication rates, but PETD was associated with a higher recurrence rate than PEID. In terms of radiation exposure during surgery, a prospective study showed that a surgeon should perform no more than 291 procedures [23]. PETD is associated with more radiation exposure than PEID [24], reflecting the longer operative time caused by puncture difficulties, particularly in patients with high cristae iliacae, narrow foramina, or large facet joints. Radiation exposure increases with the operative time. We found no significant difference in the complication rates of the two groups, in contrast with previous reports [10]. Funding g The authors declare that there are no sources of funding to be acknowledged. A retrospective cohort study including 5338 patients showed that the adult spinal surgery time was associated with several postoperative complications, including (but not limited to) wound and pulmonary complications, ve- nous thromboembolism, the need for postoperative transfusion, length of hospital stay ≥5 days, sepsis, the need for re-operation, and unplanned re-admission [25]. We analyzed the recurrence rate, blood loss, and duration of hospital stay. These results also differed from previous findings. PETD was associated with a longer operative time, but less blood loss and a shorter hospital stay, than was PEID. We speculate that complications tend to increase with longer trauma duration; less trauma leads to fewer complications. However, the degree of heterogeneity amongst studies was high; the results may be unreliable. The principal strength of our study was that we adhered to the PRISMA checklist and the recommendations of the Cochrane collaboration [26]. We reviewed many studies with large samples. However, limitations remain. First, most included studies were retrospective in nature; only eight were randomized controlled trials (which yield higher quality evidence). Further work is required. Second, the degree of within-study heterogeneity was rather high for certain parameters; such heterogeneity was statistical and/or clinical, and may compromise the reliability of our pooled data. Third, the surgical approach was probably influenced by disease severity/type. However, our examination of a large sample may overcome these limitations. In conclusion, PETD more effectively treated LDH than PEID. The PETD operative time was longer than that of PEID, but the two techniques were equally safe. PETD was associated with less blood loss, a shorter hospital stay, and a smaller incision than PEID. PETD should thus be preferred when treating LDH. Randomized controlled trials with larger samples are required to confirm our findings. Acknowledgements We thank the colleagues of Department of Orthopedics, Xiangya Hospital, Central South University. Author contribution Z.L. and Y.H. conceived and designed the research. P.C. and Z.L. analyzed the data. Z.L. created all tables and figures. P.C. drafted the manuscript. Z.L. and Y.H. critically revised the manuscript. All authors read and approved the final manuscript. Discussion We comprehensively and systematically reviewed the literature and found that: (i) PETD resulted in lower short- and long-term VAS scores than PEID, despite the absence of a significant difference between PETD and PEID in terms of short- and long-term ODI scores and the numbers of studies of excellent and good quality; (ii) although the complication rates of PETD and PEID were similar, PEID was associated with significantly less recurrence; and (iii) compared with PEID, PETD required a longer operative time, but was associated with less blood loss, a shorter incision, and a shorter hospital stay. Overall, PETD was better and safer than PEID. y PELD has become a more popular treatment for LDH than open discectomy. A previous study assessed the efficacy of PELD using transforaminal and interlaminar approaches [10]. However, that study had several limitations. First, only nine studies involving 621 patients were included in the analysis; we included 26 studies with 3294 patients. 8 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution cense 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Competing interests p g The authors declare that there are no competing interests associated with the manuscript. 9 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Bioscience Reports (2019) 39 BSR20181866 https://doi.org/10.1042/BSR20181866 Abbreviations CI, confidence interval; LDH, lumbar disc herniation; ODI, Oswestry disability index; PEID, percutaneous endoscopic interlam- inar discectomy; PELD, percutaneous endoscopy lumbar discectomy; PETD, percutaneous endoscopic transforaminal discec- tomy; RR, relative risk; SMD, standardized mean difference; VAS, visual analog scale. References 1 Amin, R.M., Andrade, N.S. and Neuman, B.J. (2017) Lumbar disc herniation. Curr. Rev. Musculoskelet. 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W4200094612.txt	https://recima21.com.br/index.php/recima21/article/download/965/806	pt		CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA	Recima21	2,021	cc-by	3,745	v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA DRUG CONSUMPTION AND SCHOOL PERFORMANCE: NA INTEGRATIVE REVIEW Camila Victória Pereira da Silva1, Adeilson Pereira da Silva2, Clésia Oliveira Pachú3 e211965 https://doi.org/10.47820/recima21.v2i11.965 RESUMO O consumo de drogas se torna cada dia mais usual entre adolescentes que frequentam instituições de ensino e acabam por obstaculizar o rendimento escolar. De forma simultânea, acentuando a desvalorização da educação. Objetivo: Verificar correlação entre estudantes adolescentes que fazem uso de substâncias psicoativas e o declínio do desempenho escolar por meio de revisão integrativa. Metodologia: Estudo qualitativo descritivo versando acerca do consumo de substâncias psicoativas por adolescentes e o rendimento escolar realizado por intermédio de revisão integrativa da literatura científica. Como fonte de informações foram incluídos artigos indexados em bases de dados como a Biblioteca Virtual em Saúde (BVS), Scientific Electronic Library Online (Scielo) e PubMED, dos últimos 10 anos. Foram utilizados os descritores “Uso de álcool e outras drogas”; “Consumo de drogas”; “Rendimento escolar” e “Adolescentes”. Resultados: Da totalidade de doze artigos recuperados, quatro foram selecionados para o presente estudo por apresentarem dados acerca do consumo de drogas psicoativas e o rendimento escolar. De acordo com a análise realizada foi possível perceber a associação entre o baixo desempenho escolar de adolescentes que utilizam algum tipo de substância psicoativa. Conclusões: Aponta-se a imprescindibilidade de práticas educativas e preventivas ao uso de drogas em âmbito escolar, bem como o auxílio em rede, contactado pela escola, em casos de situação de uso já está em vigência. Desta maneira, faz-se necessário reforçar medidas e ações públicas já existentes, de modo a atenuar os desastrosos vieses que induzem o adolescente à drogadição. PALAVRAS-CHAVE: Consumo de drogas. Adolescentes. Rendimento escolar ABSTRACT The consumption drug is becoming more and more common among adolescentes who attend educational institutions and end up hurting school performance. Simultaneously, accentuating the devaluation of education. Objective: To verify the correlation between adolescent students who use psychoactive substances and the decline in school performance through an integrative review. Methodology: Qualitative descriptive study on the consumption of psychoactive substances by adolescents and school performance carried out through an integrative review of the scientific literature. As a source of information, articles indexed in databases such as the Virtual Health Library (VHL), Scientific Electronic Library Online (Scielo) and PubMED, from the last 10 years were included. The descriptors “Use of alcohol and other drugs” were used, “Drug use”, “School performance” and “Teenagers”. Results: Of the total of twelve articles retrieved, four were selected for the present study because they present data about the consumption of psychoactive drugs and school performance. According to the analysis performed, it was possible to perceive the association between the low school performance of adolescents who use some type of psychoactive substance. Conclusions: It points out the indispensability of educational and preventive practices to the use of drugs in the school environment, as well as the network assistance, contacted by the school, in cases of situation of use is already in effect. Thus, it is necessary to reinforce existing public measures and actions, in order to mitigate the disastrous biases that induce adolescents to drug addiction. KEYWORDS: Consumption the drug. Teens. School performance 1 Universidade Estadual da Paraíba - UEPB Universidade Estadual da Paraíba - UEPB 3 Universidade Estadual da Paraíba - UEPB 2 RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 1 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú INTRODUÇÃO O VI Levantamento Nacional do Uso de Drogas Psicotrópicas realizado com estudantes no ano de 2010 apresentou que 25% já haviam abusado de alguma substância psicoativa (CARLINI, 2010). Corroborando este fato, os estudos epidemiológicos outrora realizados pelo Centro Brasileiro de Informações Sobre Drogas Psicotrópicas (CEBRID) já sinalizavam a significativa e crescente incidência do uso de drogas entre adolescentes. Nesse sentido, contemplar a faixa etária que está sendo acometida com maior frequência, as principais drogas usadas, bem como os mecanismos de atuação preventivos possíveis dentro desse espectro, são imprescindíveis (BRASIL, 2004). Nesse contexto, a Pesquisa Nacional de Saúde do Escolar (PENSE, 2015) evidenciou mais recentemente que o uso de drogas entre escolares ainda está em vigência. O levantamento constatou que alunos da mesma faixa etária e matriculados no 9° ano do ensino fundamental consomem bebidas alcoólicas (23,8%), dos entrevistados, cerca de 21,4% informaram já ter experienciado episódios de embriaguez. No âmbito das drogas ilícitas, 46,1% já haviam consumido maconha, e 5,5% craque (BRASIL, 2016). Com o intuito de acompanhar o desenvolvimento dos alunos brasileiros, o Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira (Inep) realiza estudos setoriais verificando aspectos como taxas de aprovação, reprovação e abandono, e estes são baseados primordialmente nas informações acerca do rendimento dos alunos (BRASIL, 2016). As variáveis que interferem no desempenho, e, concomitantemente, determinam a situação da escolaridade de um sujeito são inúmeros, desde questões econômicas, sociais, familiares, bem como idiossincrasias como personalidade, aspectos emocional e cognitivo, mas também pela adesão de comportamentos/repetição (SARTES, 2014). No que tange às ressalvas acerca da cultura escolar, recorte situacional de vida, relações familiares e parentais, todas podem se articular como fatores protetivos ou de risco, seja atenuando ou potencializando o contato e aproximação com o mundo das drogas (PAIVA; COSTA, 2014). De forma simultânea, afeta negativamente o rendimento escolar de escolares que vivem estas realidades. Em estudo desenvolvido com adolescentes iranianos, percebeu-se fragilização na relação com familiares, além da considerável indiferença às aspirações educacionais, em relação a adolescentes que não faziam uso de nenhuma droga (SPENCER; AGAHI, 1982). O delineamento de questões sociais que perpassam a vida do adolescente, auxiliam na compreensão dos níveis de influência destes e a probabilidade de fazer uso de alguma substância. Assim, uma pesquisa realizada com 4.516 participantes de onze a dezesseis anos de idade, matriculados em escolas inglesas, acerca do seu envolvimento com uso de cigarros, álcool e drogas ilícitas, demonstrou que variáveis como ausência de crença e relacionamentos pouco investidos correlacionam-se com o uso de substâncias na adolescência. Além disso, o baixo rendimento e expectativas escolares estimulam o ciclo da busca por uma substância para canalizar sentimentos e emoções, como frustração e tristeza (SUTHERLAND; SHEPHERD, 2001). Destarte, a escola se apresenta como ambiente propício para realização de atividades de promoção, prevenção, e sensibilização ao não uso das drogas. Todavia, não com caráter repressor, mas RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 2 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú educativo. É fato que a iniciação prematura de substâncias psicoativas se apresenta de forma significativa na realidade de adolescentes escolares. A preocupação social com essa questão deve influenciar o gerenciamento de secretarias de educação por todo Brasil, efetivando ainda mais os trabalhos em quaisquer que sejam os níveis. Além disso, a manutenção da efetividade educacional do adolescente escolar é impreterivelmente um direito do menor. Desde a oferta, deve ser articulada, a fim de minimizar percalços frequentes, como a convidativa introdução ao universo das drogas. Portanto, o presente estudo de revisão integrativa, objetiva verificar a correlação entre estudantes adolescentes que fazem uso de substâncias psicoativas e o declínio do desempenho escolar. METODOLOGIA A presente pesquisa de natureza qualitativa descritiva, acerca da relação entre o declínio do rendimento escolar de adolescentes que consomem substâncias psicoativas foi realizada a partir de revisão integrativa da literatura, no período de fevereiro a março de 2021, tomando por base os últimos 10 anos. Optou-se pela busca de dados em bases nacionais e internacionais, por meio da leitura, análise, interpretação e seleção de artigos. Na pesquisa em bases de dados, foram escolhidas a Biblioteca Nacional de Medicina dos Estados Unidos (PubMED), Biblioteca Virtual em Saúde (BVS) e Scientific Electronic Library Online (Scielo). Em consonância com referenciais literários, utilizou-se como estratégia de busca nas referidas bases de dados, os operadores Booleanos AND e OR e respectivos Descritores em Ciências da Saúde / Medical Subject Headings (DeCS/MeSH). Diante disso, utilizou-se os seguintes descritores: “Uso de álcool e outras drogas”; “Consumo de drogas”; “Rendimento escolar”; “Adolescentes”. Como critérios de inclusão, os artigos deveriam abordar a temática do uso de substâncias por adolescentes envolvendo o rendimento escolar, assim como terem sido publicados nos últimos 10 anos, e apresentados nos idiomas português, inglês ou espanhol. Foram excluídos os estudos incoerentes com a proposta do trabalho, aqueles que não abordavam a temática, resumos de trabalhos e aqueles que não disponibilizaram gratuitamente o texto para leitura e análise. RESULTADOS E DISCUSSÕES A amostra final desta revisão foi estabelecida por quatro artigos científicos selecionados pelos critérios de inclusão acima descritos. Desse modo, dois foram encontrados na Biblioteca Virtual em Saúde (BVS), um na PubMED e, por fim, um pela Scielo. Houve dificuldade em encontrar referências que coadunassem com os filtros aplicados. Apesar da temporalidade abarcar um período de 10 anos, observamos que são poucos os materiais que dão sustentação para correlação entre o uso de substâncias por adolescentes e o declínio do rendimento escolar. Destarte, a Figura 1 faz o delineamento da seleção dos artigos na busca às bases de dados. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 3 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú Figura 1: Delineamento da seleção dos artigos na busca às bases de dados. Fonte: O autor, 2021. A ilustração apresenta a busca nas bases de dados BVS, PubMED, e Scielo, proporcionando retorno de estudos. Porém, muitos desses são de subtemas diversos, cujos assuntos tratados não concordam com a proposta da presente pesquisa. Dessa forma, os quatro estudos foram incluídos com base nos critérios de inclusão e exclusão e selecionados para dar continuidade à pesquisa, podendo ser visualizados no Quadro 1. Com o intuito de compreender a existência da correlação entre adolescentes que consomem drogas e o comprometimento de sua educação, os artigos selecionados contemplam os critérios de inclusão, e a partir da explanação destes materiais, visualizamos os pormenores que os tornaram elegíveis. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 4 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú Quadro 1: Artigos encontrados nas bases de dados BVS, PubMed e Scielo. Fonte: O autor, 2021. Outrossim, evidenciou-se que o consumo de drogas lícitas e ilícitas esteve associado a inúmeros prejuízos escolares, tais como apresentação de notas baixas, desencorajamento e descaso com assuntos escolares, marcado por comportamentos como atrasos e falta às aulas, não realização de exercícios, e pensamentos de abandono aquela realidade, além de dificuldades na concentração. Entre os problemas escolares mais citados nos estudos analisados, a reprovação escolar foi a temática mais recorrente e central, visto que 02 dos artigos tiveram ênfase nessa questão (GOMES et al., 2018). Na presente pesquisa, foi possível verificar que o uso de substâncias psicoativas pode provocar graves problemas cognitivos e emocionais no adolescente. Assim podendo, de maneira lamentável, acabar afetando a memória e a aprendizagem desses indivíduos. Em consequência, refletindo drasticamente na queda do rendimento escolar. As drogas ocasionam alterações na atenção, sensopercepção e memória (SANTOS, 2017). Por conseguinte, reparou-se que dentre os adolescentes que utilizaram drogas e que apontaram baixo índice de aprendizagem, bem como obtiveram reprovação escolar, houve predominância do sexo masculino em relação ao feminino. Concomitantemente, o levantamento acerca do uso de álcool, tabaco e drogas ilícitas certificou que os adolescentes ou faziam uso exclusivo do álcool, ou este associado ao tabaco, mas também houve a parcela que fazia uso de drogas ilícitas. Nesses casos, percebeu-se maior comprometimento do RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 5 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú desempenho escolar, se comparado com indivíduos que faziam demais combinações. Faz-se necessário ressaltar, que as drogas ilícitas utilizadas pelos participantes desta pesquisa foram maconha (2,9%), tranquilizantes (1,8%), anfetaminas (1,6%), ecstasy (1,1%), inalantes (1,1%), cocaína (0,8%), alucinógenos (0,4%) e anabolizantes (0,4%) (CARDOSO; MALBERGIER, 2014). Dentre as substâncias psicoativas consumidas entre os adolescentes, as mais citadas nos estudos, foram o álcool e o tabaco. Nesse sentido, considera-se que o uso precoce e exacerbado se deve ao fácil acesso e comercialização dos produtos. Visto que, apesar da proibição da venda a menores de idade, a falta de fiscalização implica maior vulnerabilidade e exposição de adolescentes interessados em obter tais psicoativos. A relativização midiática e a sugestionabilidade publicitária endossam o fascínio por um estilo de vida que acarreta danos, principalmente, se iniciado precocemente. Cardoso e Malbergier (2014) em um estudo realizado com 965 adolescentes dos municípios de Jacareí e Diadema (SP) entenderam que os impactos no desempenho escolar foram diferentes enquanto ao tipo de droga consumida, o uso de tabaco foi associado a obtenção de notas baixas e pensamentos de desistência escolar. Enquanto o uso restrito do álcool foi relacionado a não realização das atividades, além da sensação de estar entediado dentro do ambiente educacional. Dessa maneira, é possível perceber que o consumo de drogas está relacionado a consequências negativas no desempenho escolar, dificultando a aprendizagem dos alunos e até mesmo corroborando para os alarmantes índices de evasão escolar. Foi notável que o uso associado de bebidas alcoólicas e tabaco trouxeram prejuízos significativos para o âmbito educacional do adolescente, como pensar em abandonar a escola e ter repetido o ano escolar. Assim, essa combinação, certamente, merece uma grande atenção e cuidado, pois existe uma forte tendência a acreditar que o consumo de bebidas alcoólicas e tabaco se apresenta como comportamento muito esperado na fase da adolescência (CARDOSO; MALBERGIER, 2014). Por isso, a sensibilização do adolescente acerca dos riscos possíveis e comprometimentos futuros causados pelo uso de drogas são imprescindíveis no processo educativo, a fim de preservar a sua saúde biopsicossocial o máximo possível. Em consonância com Jaisoorya et al. (2016), após uma análise com 7.530 alunos de 73 escolas no Kerala, Índia, constatou-se que os estudantes que fizeram uso de tabaco apresentaram maiores chances de pior desempenho acadêmico e pontuações mais altas de classificações de transtorno de déficit de atenção e hiperatividade (TDAH). Ademais, a idade média de início do uso do tabaco foi mais ou menos aos quatorze anos de idade, denotando os riscos acentuados em função da associação entre drogas e o funcionamento biológico, psíquico e emocional de um adolescente, assim, ampliando consideravelmente a dependência mediante os fatores neuroquímicos - circuitos de prazer e recompensa - pertinentes a esta fase da vida (SARTES et al., 2014). Ainda acerca da pesquisa de Cardoso e Malbergier (2014) os adolescentes que alegaram ter realizado o uso combinado de diferentes drogas, tais como tabaco, álcool e drogas lícitas apresentaram grandes problemas no rendimento escolar. Por outro lado, os adolescentes que fizeram menos uso combinado dessas substâncias declararam gostar mais do ambiente escolar. Assim, percebe-se que a utilização de drogas ilícitas ou lícitas pode estar associada de forma significativa aos vários prejuízos no RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 6 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú rendimento escolar. Nesse sentido, depende, sobretudo, do tipo de droga consumida, bem como o uso exclusivo de uma determinada substância psicoativa ou pelo seu consumo combinado. Para Santos (2017) o uso de substâncias psicoativas pode resultar como consequência, laços fragilizados e relações familiares pouco investidas, apresentando também problemas no rendimento escolar, principalmente pelo fato de conviver com impasses de ordem emocional e efetiva. Todavia, a família também pode ser um elemento preditor para aproximação dos adolescentes com as drogas, seja por meio de modelos desestruturados, relacionamentos inconsistentes a ponto de gerar abandonos, bem como, pela baixa expectativa de vida (PORTO; PASSOS, 2016). Dessa forma, possibilitando que o sujeito esteja mais vulnerável a comportamentos de risco e envolvimento com o universo das drogas. Outro ponto relevante apontado por Cardoso e Malbergier (2014) é o reconhecimento de que a escola precisa articular-se como um local para socialização, e não apenas para aprendizagem e desenvolvimento da cognição. Toma-se por verdadeira, que a relação vivenciada pelo estudante no interior da instituição de ensino pode influenciar a avaliação que o mesmo faz do âmbito escolar. Baseia-se, portanto, no fato de que locais estressantes e que causam medo e ansiedade potencializam o risco para a utilização de drogas. Em contradição, ambientes acolhedores contribuem para o desenvolvimento da autoestima e da autoconfiança, acabando por diminuir a propensão para o uso (CASELA et al., 2014). Ratifica-se, que a instituição educacional apresenta risco quando há negligência do público, área e problemas vigentes. Também, articula-se como um agente ativo na sensibilização ao não uso de substâncias. CONSIDERAÇÕES FINAIS O entendimento a respeito da relação entre os efeitos de substâncias psicoativas no desempenho escolar de adolescentes usuários podem contribuir para efetivação do declínio dos índices nacionais que averiguam a educação no Brasil. É certo que o delineamento do público, frequência do uso, relações com pares, níveis socioeconômicos são algumas das tipificações que precisam ser consideradas a fim de que haja maior compreensão desta problemática social, uso de substâncias psicoativas por adolescentes. Nesse sentido, as consequências do consumo de drogas interferem na produtividade, compreensão, ajustamento, assim como danos emocionais, psíquicos e comportamentais. Além de maximizar os riscos, de maneira simultânea, a propensão de que adolescentes estejam em contato com as drogas. Por conseguinte, os vínculos instaurados na escola e com a escola podem apresentar-se enquanto protetivos ou de risco. A saber, atenuam ou potencializam a aproximação do adolescente ao mundo das drogas. Todavia, há efetividade comprovada no caráter educativo e informativo promovido pela instituição de ensino, com a intenção de implementar discussões e posicionamento do escolar no que tange suas escolhas, buscando sensibilizá-lo acerca dos danos ocasionados pelo uso abusivo de substâncias psicoativas. Fazendo menção à vulnerabilidade a qual estão expostos, a comunicação entre a rede de apoio, como família, escola e comunidade podem estabelecer segurança ao adolescente, além de aperfeiçoar o serviço prestado, uma vez endossado o amparo. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 7 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú Ademais, o presente estudo explicita a correlação existente entre escolares que utilizam algum tipo de droga e o declínio de seus rendimentos escolares, consequência negativa desta prática. Nesse contexto, órgãos de educação devem se articularem no sentido de delinear mecanismos e estratégias possíveis a urgência na tratativa dos casos de sujeitos menores que estão inclinados a se submeterem ao uso drogas. É pertinente frisar que apesar dos educadores e gestores lidarem com inúmeras outras questões sociais que atravessam a escola, faz-se primordial o bem-estar dos educandos preservando a comunidade escolar. REFERÊNCIAS BRASIL. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar 2015. Rio de Janeiro: IBGE, 2016. BRASIL. Secretaria Nacional Antidrogas. Levantamento nacional sobre o uso de drogas entre crianças e adolescentes em situação de rua nas 27 capitais brasileiras. São Paulo: CEBRID, 2004. Disponível em: https://www.cebrid.com.br/wp-content/uploads/2012/10/Levantamento-Nacional-sobre-oUso-de-Drogas-entre-Crian%C3%A7as-e-Adolescentes-em-Situa%C3%A7%C3%A3o-de-Rua-nas-27Capitais-Brasileiras-2003.pdf. CARDOSO, L. R. D.; MALBERGIER, A. Problemas escolares e o consumo de álcool e outras drogas entre adolescentes. Revista Quadrimestral da Associação Brasileira de Psicologia Escolar e Educacional, São Paulo, v. 18, n. 1, p. 27-34, jan./abr. 2014. Disponível em: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S141385572014000100003&lng=en&nrm=iso&tlng=pt. CARLINI, E. A. et al. VI Levantamento Nacional sobre o Consumo de Drogas Psicotrópicas entre Estudantes do Ensino Fundamental e Médio das Redes Pública e Privada de Ensino nas 27 Capitais Brasileiras. São Paulo: CEBRID, 2010. CASELA, A. L. M. et al. As práticas de prevenção ao uso de drogas no Brasil. In: RONZANI, T. M.; SILVEIRA, P. S. (Orgs.). Prevenção ao uso de álcool e outras drogas no contexto escolar. Juiz de Fora: Ed. UFJF, 2014. Disponível em: http://sisco.copolad.eu/web/uploads/documentos/Prevencao_ao_uso_de_alcool_e_outras_drogas_no_co ntexto_escolar.pdf. GOMES, N. P. G.; SANTOS, R. M.; MOTA, R. S.; PINTO, R. P. F.; ESTRELA, F. M.; BISPO, T. C. F. Associação entre reprovação escolar, bullying e drogas ilícitas em adolescentes: estudo transversal. v. 17, n. 4, dez. 2018. Disponível em: Online braz. j. nurs., http://www.objnursing.uff.br/index.php/nursing/article/view/5979/html_2. JAISOORYA, T. S. et al. Prevalence & correlates of tobacco use among adolescents in Kerala, India. Indian J Med Res., v. 144, n. 5, p. 704–711, nov. 2016. Disponível em: https://www.ijmr.org.in/article.asp?issn=09715916;year=2016;volume=144;issue=5;spage=704;epage=71 1;aulast=Jaisoorya. PAIVA, F. S.; COSTA, P. H. A. Participação juvenil: uma alternativa para se abordar o uso de drogas no processo escolar. In.: RONZANI, T. M.; SILVEIRA, P. S. (Orgs.). Prevenção ao uso de álcool e outras Disponível em: drogas no contexto escolar. Juiz de Fora: Ed. UFJF, 2014. http://sisco.copolad.eu/web/uploads/documentos/Prevencao_ao_uso_de_alcool_e_outras_drogas_no_co ntexto_escolar.pdf. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 8 v.2, n.11, 2021 RECIMA21 - REVISTA CIENTÍFICA MULTIDISCIPLINAR ISSN 2675-6218 CONSUMO DE DROGAS E RENDIMENTO ESCOLAR: UMA REVISÃO INTEGRATIVA Camila Victória Pereira da Silva, Adeilson Pereira da Silva, Clésia Oliveira Pachú PORTO, K.; PASSOS, R. G. O uso de substâncias psicoativas por crianças e adolescentes: a experiência de um acolhimento institucional no município do Rio de Janeiro. O Social em Questão, p. 171-192, 2016. Disponível em: http://osocialemquestao.ser.puc-rio.br/media/OSQ_35_8_Porto_Passos.pdf. SANTOS, R. M. Associação entre reprovação escolar e aspectos sociais e de saúde em adolescentes de escola pública. Dissertação (Mestrado em Enfermagem) - Universidade Federal da Bahia, Salvador, 2017. Disponível em: https://repositorio.ufba.br/ri/bitstream/ri/25378/1/Disserta%c3%a7%c3%a3o_%20Enf_%20Raiane%20Mo reira%20Santos.pdf. SARTES, L. M. A. et al. Fatores de risco e de proteção para o uso de álcool e outras drogas. In.: RONZANI, T. M.; SILVEIRA, P. S. (Orgs.). Prevenção ao uso de álcool e outras drogas no contexto escolar. Juiz de Fora: Ed. UFJF, 2014. Disponível em: http://sisco.copolad.eu/web/uploads/documentos/Prevencao_ao_uso_de_alcool_e_outras_drogas_no_co ntexto_escolar.pdf. SPENCER, C.; AGAHI, C. Social background, personal relationships, and self-descriptions as predictors of drug-user status: A study of adolescents in post-revolutionary Iran. Drug Alcohol Depend, v. 10, n. 1, p. 77-84, sep. 1982. SUTHERLAND, I.; SHEPHERD, J. P. Social dimensions of adolescent substance use. Addiction, v. 96, n. 3, p. 445-58, mar. 2001. RECIMA21 - Ciências Exatas e da Terra, Sociais, da Saúde, Humanas e Engenharia/Tecnologia 9
https://openalex.org/W4200599506	https://zenodo.org/record/5655764/files/putovanje_kroz_vrijeme.pdf	Croatian	null	Putovanje kroz vrijeme: Prostorno putovanje	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	9,365	1 Intro Mjerenje vremena postoji oduvijek u prirodi (svaka oscilacija energije se može tako interpretirati) jer je sinkronizacija događaja nužan element evolucije. Tako nije čudno da je i evoluirajući čovjek morao osmisliti uređaje za mjerenje vremena. Pošto značajke prostora, poput gustoće i tlaka, određuju granice brzini razm- jene informacija i transformacije energije, iste će korelirati sa brzinom protoka mjerenog vremena, kao i sa veličinom najmanje jedinice vremena u tom pros- toru. Gustoća i tlak prostora generalno nisu konstante (deformacija prostora gen- eralno ovisi eksponencijalno o udaljenosti od izvora gravitacijskog potencijala) pa će vrijeme različito teći za različite promatrače čak i ako koriste iste satove. Kojom će brzinom teći vrijeme ovisi i o veličini sata (promatrača), jer gustoća i tlak prostora su, kao i sve ostalo, relativne veličine, pa percepcija prostora ovisi o veličini i prirodi čestice koja ga doživljava. Sve ovo vrijedi u kontekstu Kompletne Relativnosti. No u standardnoj in- terpretaciji Opće Teorije Relativnosti, prostor je apstrakcija (nema gustoću i tlak) koja se s prisustvom energije iskrivljava. Uz mnoštvo apsolutnih konstanti vezanih za taj prostor, stvarnosti se dodjeljuje neintuitivna priroda na svim skalama energija. No stvarnost zapravo na svim skalama pokazuje koliko je relativna, pa, po svemu sudeći, uzrok neintuitivnih interpretacija nije izostanak ﬁzikalnih mani- festacija nego ograničenje u mašti promatrača. Abstract Diskusija o prostoru i vremenu u kontekstima Opće Teorije Rela- tivnosti i Kompletne Relativnosti. 1 1 Putovanje kroz vrijeme Mario Ljubičić (Amenoum) 108. brigade ZNG 43, 35252 Sibinj, Croatia mljubicic99@gmail.com November 19, 2021 2 Nediskriminirajuća interpretacija prostora i vre- mena Često se govori kako gravitacija nije sila pa prostor nema tlak i gustoću nego je deformiran prisustvom energije. Razlog tome je što je Opća Teorija Relativnosti 2 (GR) geometrijska teorija (ili geometrijska relativnost) u kojoj geometrija pros- tora ovisi o energiji u njemu. Dok je nužno da energija materije ima gustoću i tlak, takav uvjet ne postoji za geometriju. (GR) geometrijska teorija (ili geometrijska relativnost) u kojoj geometrija pros- tora ovisi o energiji u njemu. Dok je nužno da energija materije ima gustoću i tlak, takav uvjet ne postoji za geometriju. O tome se puno ne razmišlja nego se prostor prihvaća kao nekakva apstrak- cija. Isto je sa kvantnom mehanikom, koja zbog ovakvog pristupa postaje nein- tuitivna. To se često pravda ekstra dimenzijama, što je, u slučaju Opće Teorije Rela- tivnosti, vrijeme, jer nije jasno kako bi geometrijski konstrukt prostor-vremena trebalo ﬁzikalno interpretirati. Često se govori i da što brže putujemo kroz prostor, sporije putujemo kroz vrijeme. No to generalno nije tako po Općoj Teoriji Relativnosti (GR). U teoriji GR, apsolutno sve što nije pod utjecajem gravitacije zapravo kroz vrijeme putuje konstantnom brzinom koja je uvijek jednaka apsolutnoj konstanti c. Točan izraz je - što brže putujemo kroz prostor, sporije putujemo kroz prostor-vrijeme. To je sasvim jasno iz jednadžbe za udaljenost u ravnom (ﬂat, Minkowski metric) prostor-vremenu (∆s): (∆s)2 = −(∆d)2 + (c∆t)2 v2 = (∆s)2 (∆t)2 = c2 −(∆d)2 (∆t)2 (1) (1) gdje ∆d predstavlja udaljenost u prostoru, c∆t udaljenost u vremenu, a v brzinu u prostor-vremenu. Ali nema razloga da točke prostora x, y i z ne odgovaraju točkama prostor- vremena x, y i z. Sve što treba napraviti da bi translatirali prostor-vrijeme u prostor je od kvadrata brzine u prostor-vremenu oduzeti c2, odnosno obrnuto da izbjegnemo negativne udaljenosti. Matematički lako, ali nije ni teško to ﬁzikalno interpretirati. Ako ne diskriminiramo vrijeme od prostora, možemo shvatiti vrijeme kao izolirani pod-prostor (dio) prostora u kojemu je brzina ograničena konstantom c. Uočiti da, po teoriji GR, taj pod-prostor mora biti jedno-dimenzionalan. Uočiti da, po teoriji GR, taj pod-prostor mora biti jedno-dimenzionalan. Ako je taj pod-prostor pravac paralelan našem vektoru brzine u prostoru i prolazi kroz istu točku, onda je prostor ekvivalentan našem prostor-vremenu upravo naš prostor s dodatkom čestice koja ispred nas putuje brzinom c i koja je referentna točka za mjerenje naše brzine u prostor-vremenu. Prostor-vrijeme postaje pogled na prostor iz perspektive čestice koja putuje brzinom c (u kontekstu mjerenja brzine). Generalno - da bi matematički konstrukt prostor-vremena odgovarao pros- toru u stvarnosti, za svaku točku prostora mora postojati čestica (ili točka) koja putuje brzinom c u istom smjeru kao i ta točka. Dakle, za svaku točku (ili 3 česticu) prostora postoji točka (ili čestica) prostora s kojom je ona strogo kore- lirana. Ako dozvolimo točkama da budu čestice, onda je to kvantna spregnutost (quantum entanglement) čestice prostora i čestice vremena. Očito, da bi vrijeme moglo prolaziti (postojati) za neku česticu, mora pos- tojati čestica vremena. Po Općoj Teoriji Relativnosti ta čestica vremena ne bi imala masu i uvijek bi putovala brzinom c. Slično bi se moglo reći za česticu prostora, osim što je njoj dozvoljeno putovati brzinom različitom od c. Po Einsteinovim jednadžbama polja, kojima se energija veže za ge- ometriju prostor-vremena, reklo bi se da ista čestica (energija) putuje i kroz prostor i kroz vrijeme, samo što kroz vrijeme uvijek putuje brzi- nom c (intuitivno je jasno da se brzine mogu razlikovati između dimen- zija prostora pa ni ovo nije ništa čudno) no ta dimenzija nema ﬁzikalnu interpretaciju u GR. Ali kad govorimo o energiji u prostoru i protjecanju vremena, vrijeme se uvijek veže za stvarne ﬁzikalne čestice. Ako npr. vežemo elektron za točku u prostoru, morala bi onda postojati i ﬁzikalna čestica koja se veže za točku u vremenu a koja će korelirati s tim elektronom. Pošto je svako gibanje elektrona popraćeno emisijom fotona, možemo reći da je vremenska čestica za elektron upravo foton koji se giba u istom smjeru. No ako foton ima masu (a po Kompletnoj Relativnosti mora imati), onda za elektron vrijeme ne teče brzinom c, nego nešto manjom, odnosno, brzinom fotona (vrijeme teče brzinom c za točku prostora!). No ne možemo uvijek korelirati gibanje elektrona sa fotonom - što sa elek- tronom koji miruje? Elektron u orbiti atoma ne zrači energiju (bar ne u skali energije fotona), pa očito za njega mora postojati ili neka druga čestica nosioc njegova vremena ili statički foton u istoj orbiti ali koji orbitira većom brzinom. Uočiti da, po teoriji GR, taj pod-prostor mora biti jedno-dimenzionalan. Pretpostavimo da je to, umjesto fotona, statički neutrino. Očito je da će se dvije čestice, budući da su u istoj orbiti, upariti i putovati brzinom elektrona. No ako brzina u prostor-vremenu ne može biti nula (po GR nikad nije) onda taj neutrino ne može biti vremenska čestica za elektron. Pretpostavimo sad da orbita elektrona mora biti korelirana sa česticom suprotnog naboja u atomu. To može biti proton, no što sprječava naboj protona da se prisustvom elektrona razdvoji od mase protona i kolabira u pozitron? Taj pozitron dakle orbitira bliže jezgri i ima magnetni spin suprotan elektronu. Te dvije čestice ne smiju anihilirati, no to može biti osigurano na više načina: • spomenutim sparivanjem elektrona s neutrinom, • spomenutim sparivanjem elektrona s neutrinom, • oscilacijom mase čestica, • prisustvom barijere, poput horizonta događaja (event horizon). 4 Ovdje nijedno ne isključuje drugo. Zbog kvantne spregnutosti, elektron i proton će razmjenjivati čestice, pa, bilo da se radi o fotonima ili ne, one će prilikom apsorpcije orbitirati brzinom čestice koja ih je apsorbirala. Prema tome, kada elektron i pozitron razmjene fotone, efektivno, jedna čes- tica postaje vremenska čestica za drugu. Vrijeme se dakle u prostoru manifestira ﬁzikalnim česticama i ne teče uvijek jednako niti isto za svakoga. To što vrijeme u ravnom prostor-vremenu uvijek teče brzinom c, rezultat je apstrakcije i prostora i vremena u istom. A ako je jasno da se vrijeme veže uz ﬁzikalne čestice, onda treba vremenskim česticama dati masu a prostoru gustoću i tlak jer neće posvuda biti jednako ispunjen tim česticama, nego će to korelirati upravo sa geometrijom prostor-vremena. Apsurd apstraktnog pristupa pokazuje fenomen tamne materije kojim se pokušava objasniti detektiran deformiran prostor bez detektirane energije u njemu. Pošto Opća Teorija Relativnosti ne može objasniti geometriju prostora bez energije u njemu, ta energija se traži u vidu egzotične materije. Problem nestaje kada prostoru dodijelimo gustoću i tlak, što je sasvim in- tuitivno, i predviđeno teorijom Kompletne Relativnosti[1]. Tamna materija, dakle, ne postoji kao egzotična materija koja svija prostor, nego kao materija koja čini prostor. Ne vidimo ju zato što ne zrači fotone u skali energije koju možemo detektirati, odnosno - nepolarizirana je na takvoj skali, što govori da ako se sastoji od materije koju bi mogli detektirati, to moraju biti neutrini. No u ovom slučaju, ako se ne radi o egzotičnoj materiji koja svija prostor nego materiji koja čini prostor, to mora biti statički graviton neutrino. Uočiti da, po teoriji GR, taj pod-prostor mora biti jedno-dimenzionalan. Ipak, mnogi još uvijek ustraju u tome da se nešto absolutno neopipljivo može deformirati energijom i pokušava se detektirati egzotična materija koja svija prostor a ne ona koja ga čini. Jasno je zašto je tomu tako, GR je odlična teorija, dokazana mnogo puta, no možda je vrijeme da prihvaćena teorija relativnosti konačno postane ono što tvrdi da jest - relativna. 3 Generalizacija vremenske čestice Dobro je uočiti da zapravo nije nužno da vremenska čestica putuje u istom sm- jeru, nužno je da razlika u brzini u prostoru i vremenu odgovara relativističkim efektima. Teorijom Kompletne Relativnosti utvrdio sam da je prostor elektrona sačin- jen od čestica U−2 skale. Elektron je čestica U0 skale što znači da prostor za čestice U1 skale (planetarni sustavi) mora biti sačinjen od čestica U−1 skale, a to je skala fotona. Kada se govori o skali fotona, dobro je razjasniti o kojoj skali se radi, jer se fotoni obično razmatraju kao valovi. Dakle, u slučaju skale radijusa, 5 ovdje se ne radi o amplitudi ili polovici valne duljine fotona kao vala, nego radijusu fotona kao čestice. Taj radijus je generalno za mnogo redova veličine manji od valne duljine, a tek u slučaju ekstremno malih valnih duljina (ekstremnih frekvencija) valna duljina se približava radijusu, zbog čega se na višim frekvencijama foton ponaša više kao čestica nego val. Ako shvatimo da je skala fotona ista ili približna skali gravitona, onda je veličina fotona jednaka veličini čestice u prostoru koja se valom izbacuje iz ravnotežnog položaja (vibrira, generalno okomito na smjer širenja vala). ovdje se ne radi o amplitudi ili polovici valne duljine fotona kao vala, nego radijusu fotona kao čestice. Taj radijus je generalno za mnogo redova veličine manji od valne duljine, a tek u slučaju ekstremno malih valnih duljina (ekstremnih frekvencija) valna duljina se približava radijusu, zbog čega se na višim frekvencijama foton ponaša više kao čestica nego val. Ako shvatimo da je skala fotona ista ili približna skali gravitona, onda je veličina fotona jednaka veličini čestice u prostoru koja se valom izbacuje iz ravnotežnog položaja (vibrira, generalno okomito na smjer širenja vala). Po Kompletnoj Relativnosti foton mora imati masu, a pošto veća frekvencija uvjetuje veću masu, jasno je zašto se amplituda i valna duljina vala smanjuje s frekvencijom - elektromagnetski potencijal čestice se mi- jenja za gravitacijski, pa je izbacivanje polariziranih čestica iz ravnoteže u prostoru manje. Bez zamjene potencijala i očuvanja momenta, foton veće mase bi se morao nešto sporije kretati kroz prostor, ali u ovom slučaju energija se povećava u obliku vakuuma gravitona, dok se kutni moment čestica nosioca naboja smanjuje (smanjuje se polarizirana masa), pa se smanjenje elektromagnetskih interakcija može interpretirati kao sman- jenje gustoće prostora, a ako se i tlak smanjuje proporcionalno brzina ostaje ista. 3 Generalizacija vremenske čestice U slučaju da efektivni tlak ostaje isti ili se povećava, brz- ina će se morati i povećati. Iz poglavlja Relativistic space Kompletne Relativnosti: r vp = c = r ps ωρs ρs = ϵs E B 1 c1 p ρs = polarizirana (elektro-magnetska) gustoća prostora ϵs = električna permitivnost prostora B = jakost magnetskog polja E = jakost električnog polja c1 = granična brzina za prostor B = jakost magnetskog polja E = jakost električnog polja c1 = granična brzina za prostor Budući da su ω i c1 jake konstante u ovom slučaju (mjenjaju se s evolu- cijom prostora), gustoća će se mijenjati proporcionalno s doživljajem elektro-magnetskog polja (EB), a to će smanjivanjem povećavati brzinu fotona. Slično vrijedi za tlak: ps = 1 µs E B 1 c2 ps = polarizirani tlak prostora t k bil t t ps = polarizirani tlak prostora µs = magnetska permeabilnost prostora c2 = relativistički koeﬁcijent razlike granične brzine prstora i njegove brzine mirovanja c2 = relativistički koeﬁcijent razlike granične brzine prstora i njegove brzine mirovanja 6 Iz ovoga je jasno da će se tlak mijenjati proporcionalno s gustoćom pa, poništavanjem faktora EB, ipak nema promjene u brzini fotona. A ako su ϵs i µs jake konstante prostora ili se mijenjaju obrnuto proporcionalno, jasno je da će brzina c biti granična brzina za sva tijela u istom prostoru - ona se može promjeniti jedino promjenom kinetičke energije samog pros- tora, koja doduše mora oscilirati na manjoj skali i prilikom slabe evolu- cije. Ipak, ako se prostor tijela (poput fotona) mijenja proporcionalno prostoru u kojem se tijelo nalazi, onda to tijelo neće mjeriti (doživljavati) nikakve promjene u istom (bar ne s uspostavom uravnoteženog stanja). No, promjene će mjeriti promatrač izvan tog prostora. Prazan prostor (vakuum) dakle ne može biti apsolutno prazan - on je sačinjen od čestica manje skale. Ako su te čestice i same čestice vakuuma onda veća gustoća tih čestica znači veći vakuum (gravitaciju). Sada je jasno što usporava vrijeme pri velikim brzinama i velikim gravitaci- jskim poljima - veća gustoća čestica vakuuma (gravitona). Jasno je i zašto fotoni putuju brzinama vrlo bliskim brzini c - jer su čestice iste skale (goli foton je zapravo superpozicija polariziranih gravitona sa pomakom faze u prostoru). 3 Generalizacija vremenske čestice U prostoru koji se sažima (točke prostora se zgušnjavaju) sve se sažima pa će udaljeni promatrači svjedočiti relativističkom efektu kontrakcije duljine ali i di- latacije vremena u prostor-vremenu - iz jednadžbe (1) slijedi: ∆d = r 1 −v2 c2 c∆t (2) ∆t = 1 q 1 −v2 c2 ∆d c (3) (2) (3) ali i u ﬁzikalnom prostoru: ∆d = r 1 −v2 c2 ∆d0 (4) ∆t = 1 q 1 −v2 c2 ∆t0 (5) (4) (5) gdje ∆d0 i ∆t0 označavaju duljine (intervale) u prostoru i vremenu, respektivno, kada je tijelo u mirovanju. Međutim, u Općoj Teoriji Relativnosti javlja se problem interpretacije - sma- tra se da utjecaj gravitacije na dilataciju i kontrakciju ﬁzički mijenja tijelo, dok kinetičku energiju mjeri promatrač pa nema ﬁzičke deformacije tijela, ona se, kao ﬁzički efekt, manifestira u oku promatrača. Č Čudna interpretacija, ako uzmemo u obzir da se prilikom interakcije proma- tranog tijela s drugim tijelom ista energija oslobađa upravo na lokaciji proma- tranog tijela a ne samo u oku promatrača. 7 Rješenje paradoksa je, naravno u odbacivanju apsolutnog (apstraktnog) pros- tora - tj. omogućivanju tijelima da akumuliraju energiju prilikom kretanja kroz prostor. Uostalom, jasno je da, po teoriji GR, svako povećanje energije u prostoru (ili prostor-vremenu) povećava deformaciju prostora a energija se ne može stvoriti iz ničega. Tako energija ne može nastati iz ničega prilikom sudara - svaki sudar bi morao uključivati inﬂaciju energije s manje skale ukoliko ona nije sadržana u tijelu ili oko tijela u interakciji. Iako je inﬂacija moguća (npr. kroz anihilaciju), jasno je da će samo u neintuitivnom slučaju apstraktnog prostora ona biti man- ifestacija cjelokupne kinetičke energije, pri čemu onda i lokalni efekt kontrakcije i dilatacije prije sudara postaje neintuitivan. Ipak, čini se da je nužno onda odbaciti simetričnost Specijalne Relativnosti u ovakvom slučaju, odnosno, budući da se gravitacijski potencijal mijenja za tijelo u pokretu, kontrakcija i dilatacija poprimaju gravitacijski karakter (u svakom slučaju) pa takvo tijelo (nazovimo ga promatrač S) neće mjeriti istu kontrakciju i dilataciju za promatrača (nazovimo ga promatrač M) koji mjeri njega. ( ) No, da li je to tako u [apsolutno] svakom slučaju? ( ) No, da li je to tako u [apsolutno] svakom slučaju? Na prvi pogled simetrija je očuvana - lokalna kontrakcija prostora će i sve dolazeće zrake radijacije prostorno sažimati jednako kontrakciji prostora, no problem je što se sažima i promatrač (tj. 3 Generalizacija vremenske čestice mjerni uređaj) pa sažeti promatrač (S) neće mjeriti nikakvu promjenu u drugom promatraču (M), osim Dopplerovog efekta ako se ne radi o orbitalnom momentu. Ako je nužno da relativno isti efekt postoji i za sažetog promatrača (ne može biti apsolutno isti), postoje 3 rješenja: 1. za njega je pak relativistički efekt psihiloški, 2. promatrač se ne sažima, 3. za njega je konstanta c drugačija. 3. za njega je konstanta c drugačija. Prvo rješenje bi morali odbaciti, osim ako prihvatimo da je psihološki efekt uzrokovan, tj. lokaliziran, kontrakcijom prostora. Bilo je već rasprava oko ﬁzikalnosti relativističkih efekata. Zanimljivo je dopisivanje Varićaka i Einsteina na temu[2], gdje Varićak sugerira postojanje stvarnih (real) i očiglednih (apparent) efekata. Ako zamislimo prsten načinjen od krute (rigid) tvari koji rotira - zbog relativističkih efekata, udaljenosti na prstenu se moraju sažimati, no paradoksalno, promjer prstena bi morao ostati isti jer sam prsten miruje. Einsteinovo rješenje je nepostojanje krutih tijela - što implicira da se kvanti prstena smanjuju dok on zadržava promjer. No ako se s brzinom povećava energija tijela i ako je ta energija u grav- itaciji (gravitonima) onda je jasno da će se i prsten morati smanjivati zbog gravitacijskog privlačenja suprotnih strana na prstenu ali i između svakog kvanta energije. I to je pravo (ﬁzikalno) rješenje paradoksa - u 8 stvarnosti ne postoji kontrakcija koja ne uključuje kontrakciju prostora. Jasno je to i iz očuvanja kutnog momenta - povećanje brzine mora sman- jiti masu energije koja rotira i/ili orbitalni radijus. Pošto se ovdje masa povećava, orbitalni radijus se mora smanjivati. p , j j Jedino ako zamišljamo rotirajuće apstraktno tijelo bez mase, paradoks ostaje neriješen bez kvantizacije tijela. U stvarnosti to neće biti ni savršeni prsten, pa može još biti svega. Teorija GR dakle zahtijeva od svih tijela da budu sastavljena od kvanata energije pa prema tome de facto zabranjuje postojanje elementarnih čes- tica koje rotiraju. Zato postoje nebuloze u Kvantnoj Mehanici - čestice koje očigledno imaju spin moment, a u stvarnosti se od njih traži da imaju radijus jednak 0. U Kompletnoj Relativnosti, sve je relativno - kruta tijela nisu apsolutno zabranjena nego postoje relativno, pa su i elementarne čestice relativno elementarne a u stvarnosti se sastoje od manjih kvanata energije i imaju, ne samo očigledan, nego i stvaran radijus na određenoj skali. Rješenje br. 3 Generalizacija vremenske čestice 2 (nesažimanje promatrača) je moguće u slučaju da je sažimanje prostora koncentrirano u prstenu, odnosno torusu ili sferi, oko tijela pa postaje svojevrsna gravitacijska leća. No ovo je moguće jedino ako već postoji takva sfera (npr. promatrač se nalazi u centru gravitona određene skale). Postoji i još jedna mogućnost, ako uslijed sažimanja prostora dolazi do pro- gresivne evolucije promatrača (npr. fuzijom materije) onda veličina promatrača S može ostati efektivno nepromijenjena. U ﬁzičkim prostorima, i 3. rješenje postaje moguće. Ako se prostor zgušnjava no skala fotona se ne sažima onda će se za promatrača S konstanta c promi- jeniti, i to proporcionalno kontrakciji pa postaje sasvim moguće da mjeri istu kontrakciju za promatrača M. Sve promjene energije na bilo kojoj skali su diskretne, pa se neće mijenjati istovremeno, a generalno i osciliraju. U kompletno relativnoj stvarnosti, sva spomenuta rješenja su moguća pa će se i pojavljivati više ili manje u svakom obliku. Po Kompletnoj Relativnosti, sva živa nebeska tijela i sve standardne čestice imaju gravitacijski maksimum (npr. event horizon kod crne rupe, površinski maksimum kod zvijezda, itd.). U jednoj interpretaciji, to je tako upravo zato da omogući rješenje br. 2, u drugoj, rješenje br. 2 omogućuje takvu stvarnost - sve je relativno, no vrlo ﬁzikalno na određenoj skali. Rješenje br. 2 objašnjava diskretne energetske nivoe - ako se promatrač S nalazi izvan gravitona (sažetog prostora) a ne unutar, onda mora postojati bar još jedan graviton s većim radijusom tako da promatrač S bude unutar njega. Rekurzijom se dolazi do beskonačnosti ili nečega što će kolabirati graviton na manju skalu (postajući satelit za graviton veće energije) zadržavajući proma- 9 trača (ili tijelo) S unutar sebe. Naravno, ovo ne mora i neće biti ispunjeno uvijek jer razmjena energije (informacije) nije nikad instantna. 3.1 Uvjeti za ekvivalenciju specijalnog i gravitacijskog rel- ativističkog efekta Da bi kontrakcija i dilatacija Specijalne Teorije Relativnosti bila ekvivalentna gravitacijskoj kontrakciji i dilataciji moraju biti zadovoljeni određeni uvjeti. Dilatacija vremena dobivena Schwarzschildovim rješenjem Einsteinovih jed- nadžbi polja[3] se može napisati u ovom obliku[4]: ∆t0 ∆t = s 1 − β2 + βe 2 + βr 2βe 2 1 −βe 2 β = v c , βe = ve c , βr = vr c ve 2 = 2GM r v = brzina promatranog (sažetog) tijela (S) ve = brzina bijega (escape velocity) iz gravitacijskog polja energije M na udaljenosti r vr = radijalna komponenta brzine tijela S G = gravitacijska konstanta G = gravitacijska konstanta M = masa izvora gravitacije M = masa izvora gravitacije r = udaljenost tijela (S) od izvora gravitacije ∆t0 = interval vremena bez relativističkih efekata (proper time) ∆t = dilatacija vremena ∆t0 = interval vremena bez relativističkih efekata (proper time) ∆t = dilatacija vremena ∆t0 = interval vremena bez relativističkih efekata (proper time) ∆t = dilatacija vremena Ovdje radijalna komponenta brzine ne postoji ako je tijelo S u orbiti oko proma- trača, a ∆t predstavlja dilataciju vremena koju će mjeriti promatrač koji nije pod utjecajem gravitacijskog potencijala i koji miruje u koordinatnom sistemu (ne doživljava dilataciju i kontrakciju). U izrazu, faktor β se odnosi na dilataciju Specijalne Teorije Relativnosti (zbog kretanja tijela), dok se faktor βe odnosi na dilataciju uslijed gravitacijskog potencijala. Ako zanemarimo radijalnu komponentu, jedini uvjet koji treba biti zadovol- jen za ekvivalenciju dilatacije Specijalne i Opće relativnosti jest: β = βe β = βe što, ako ostavimo konstante G i c apsolutnima: što, ako ostavimo konstante G i c apsolutnima: ve 2 = v2 = 2GM r (6) (6) 10 Na lokaciji promatranog tijela mora se, dakle, formirati efektivni graviton čija će brzina bijega biti jednaka brzini kojom se tijelo giba (v). Na lokaciji promatranog tijela mora se, dakle, formirati efektivni graviton čija će brzina bijega biti jednaka brzini kojom se tijelo giba (v). Ako se radi o tijelu sa distinktnim gravitacijskim maksimumom (koji mora biti realni graviton), a većina tijela ga ima (ako ne sva), on se generalno (barem za kuglasta tijela) nalazi u centru i ima stvaran radijus, a težište mase je ras- poređeno jednoliko po radijusu. Masa (energija) tog gravitona je M0 u slučaju mirovanja, a kvadrat brzine bijega na radijusu: ve0 2 = 2GM0 r0 ve0 2 = 2GM0 r0 Njegova masa raste s kinetičkom energijom tijela: Njegova masa raste s kinetičkom energijom tijela: M = 1 q 1 −v2 c2 M0 (7) (7) Iz (6) i (7) slijedi: v2 = 2G r 1 q 1 −v2 c2 M0 = ve0 2 r0 r 1 q 1 −v2 c2 i iz toga jednadžba za radijus r: i iz toga jednadžba za radijus r: r = ve0 2 v2 r0 q 1 −v2 c2 (8) (8) Ovdje, za v > ve0, radijus se zaista sažima, no za v < ve0 on raste, a brzina v = 0 je zabranjena jer implicira beskonačni radijus. Ovdje, za v > ve0, radijus se zaista sažima, no za v < ve0 on raste, a brzina v = 0 je zabranjena jer implicira beskonačni radijus. ∆t0 = interval vremena bez relativističkih efekata (proper time) ∆t = dilatacija vremena Rješenje paradoksa je naravno u postulatima Kompletne Relativnosti - ništa ne može imati brzinu jednaku apsolutnoj nuli jer nijedno tijelo nije apsolutno u mirovanju niti putuje apsolutno konstantnom brzinom, svako mora oscilirati. No rezultat je zanimljiv jer objašnjava inﬂaciju prilikom sudara (anihilacije). Sudarom tijela brzina pada relativno na 0 što dakle mora izazvati impuls in- ﬂacije radijusa gravitona, a onda smanjenje radijusa povećanjem brzine do nekog ravnotežnog stanja. Uočiti da su gotovo sva nebeska tijela koja promatramo u ravnotežnom položaju u koji su stigla nakon početne inﬂacije maksimuma (zapravo su u mirovanju relativno na prostor u kojem se gibaju jer je njihova kutna brzina jednaka kutnoj brzini efektivnog gravitona). U nekim slučajevima će se značajna inﬂacija izbjeći spajanjem (npr. crnih rupa) i zadržavanjem visoke rotacije (oscilacije) a u određenim slučajevima doći će i do raspada (ﬁsije) maksimuma na one manjeg radi- jusa kao što je to slučaj kod teških zvijezda prilikom supernova eksplozija (ili bosenova na skali atoma). 11 Dobro je napomenuti da kod promjene duljine treba znati što se točno mijenja i kako, jer ako graviton predstavlja gravitacijski maksimum zvi- jezde on generalno ima planete (druge maksimume) u orbiti oko sebe - u slučaju kad se radijus zvijezde povećava s njenom energijom, orbitalni radijus planeta se može smanjivati pa, iako jezgra povećava radijus, sis- tem u cjelini smanjuje radijus. Postavlja se pitanje koja je brzina gravitona u mirovanju ako nije 0? Odgovor je - upravo ve0 - brzina bijega. Jasno je to iz još jedne posljedice postulata Kom- pletne Relativnosti - svaki spin moment je zapravo orbitalni moment pa graviton u mirovanju orbitira oko centra mase. Prema tome, svaki graviton (ili svaka čes- tica) ima brzinu mirovanja, pa ako je i masa mirovanja proporcionalna kinetičkoj energiji, materija postaje ﬁktivna - sva energija je u momentu vakuuma. No kod mjerenja relativističkih efekata i momenata treba uzeti u obzir skalu energije - konstanta c odgovara za subatomske čestice, no za zvijezde i planete ona mora biti manja - za slobodne (nevezane) gravitone u međugalaktičkog prostoru, ta brzina je 2.93 * 106 m/s, oko 100 puta manja od c a to znači da će relativistički efekti za ta tijela biti dosta veći (ako njihovu brzinu mjerimo istim jedinicama kao brzinu standardnih čestica). 3.2 Protok vremena Da li vrijeme za nas teče brzinom c? Za čestice od kojih je sastavljen naš organizam, vrijeme teče brzinom koja je blizu c (preciznije U0.c ili c0). Vjerovatno upravo zato što graviton subatomskih čestica ima brzinu bijega jednaku c pa oscilira tom brzinom, no možda je vremenska dimenzija gravitona locirana na radijusu koji rotira brzinom c. ∆t0 = interval vremena bez relativističkih efekata (proper time) ∆t = dilatacija vremena Da se c mora mijenjati sa skalom jasno je i iz jednadžbe (8), jer ako crna rupa ima brzinu bijega jednaku c onda bi njen radijus morao biti beskonačan osim za r0 = 0. 4 Postoji li vrijeme za svakoga? Kada se radi o mjerenju intervala između dva događaja, iako se do istog dolazi na vrlo ﬁzikalan način, vrijeme se čini kao apstraktan pojam. No ipak, svi mi u svakom trenutku putujemo kroz vrijeme (nemoguće je stati, osim za hipotetske čestice bez mase i u domeni apsolutne singularnosti - ni jedno ni drugo pak po Kompletnoj Relativnosti ne postoje). Sve što postoji mora biti relativno - možemo reći da vrijeme relativno ne postoji, no ako ne postoji relativno, onda mora negdje relativno postojati (ne može apsolutno ne postojati!). Po Kompletnoj Relativnosti, sve što postoji ima energiju pa se tako mora i vrijeme vezati za energiju na određenoj skali. 12 A da bi bilo moguće putovati kroz vrijeme nužno ga je izjednačiti s prostorom koji ima svojstva poput gustoće i tlaka. Mi, dakle, u svakom trenutku putujemo kroz ekvivalent prostora kojeg zovemo vrijeme. To je sasvim jasno iz postulata Kompletne Relativnosti po kojima svaka točka ili čestica u prostoru [bilo koje veličine] mora imati kutni, tj. orbitalni moment u odnosu na nešto. Po relativističkim efektima, jednadžbi dilatacije vremena, vremenski inter- vali izeđu dva događaja se produljuju proporcionalno brzini iako hipotetska [vremenska] čestica u vremenu uvijek putuje kostantnom brzinom c. To je nein- tuitivno ako postoji korealacija prostornih i vremenskih čestica pa zahtijeva dodatan refrentni okvir promjenjive brzine a prema kojem će čestica u vremenu uvijek imati brzinu c. Ako je prostor ﬁzikalan ova neintuitivnost nestaje. Putovanje kroz prostor (ili putovanje prostora kroz tijelo) onda nužno po- drazumijeva interakciju tijela s prostorom pa će, generalno, s većom brzinom, tijelo percipirati veću gustoću prostora što povećava energiju tijela i usporava transformaciju energije (protok informacija) unutar tog tijela. Sada možemo reći da brže putovanje kroz prostor usporava putovanje kroz vrijeme, no gdje je to vrijeme? Percepcija vremena se ne mijenja za tijelo u inerciji, tako da će usporeno vrijeme za to tijelo mjeriti netko tko ne putuje istom brzinom kroz taj prostor. No vrijeme u kontekstu mjerenja intervala između dva događaja je apstrak- tno, dok za svakog tko doživljava promjene u vremenu (iako ih možda nije svjestan) ono na određenoj skali energije ima ﬁzikalni ekvivalent koji mora pos- tojati u lokalnom prostoru, a ne u prostoru udaljenog promatrača koji mjeri dilataciju (on vrijeme proživljava drugom brzinom). Protok vremena može biti koreliran i s nekom udaljenom česticom (npr. 4 Postoji li vrijeme za svakoga? iza određenog horizonta događaja) pa tako može postojati i ﬁzička veza na određenoj skali s takvom udaljenom česticom no to ne isključuje postojanje lokalnog efekta. Najbrže stari onaj koji mjeri svoje vrijeme, a najmanje onaj koji svog vremena nije ni svjestan. Najbrže stari onaj koji mjeri svoje vrijeme, a najmanje onaj koji svog vremena nije ni svjestan. Masa (energija) se može povećavati povećanjem radijusa gravitacijskih mak- simuma čestica (što izjednačava akceleraciju sa gravitacijskom akceleracijom) i akvizicijom mase iz prostora što povećava broj gravitacijskih maksimuma. Uočiti dvije važne posljedice: 1. putovanje kroz prostor (vrijeme) tijelima daje masu, 2. svako tijelo ima svoj (privatni) prostor (vrijeme). Veza ili korelacija (entanglement) tijela i privatnog prostora ne mora Uočiti dvije važne posljedice: 1. putovanje kroz prostor (vrijeme) tijelima daje masu, 2. svako tijelo ima svoj (privatni) prostor (vrijeme). Veza ili korelacija (entanglement) tijela i privatnog prostora ne mora 5 Ostali problemi i paradoksi Uz prostor, a pogotovo vrijeme, kroz povijest su se vezali različiti paradoksi. Neki od njih su razjašnjeni, no ako je stvarnost intuitivna, ﬁzikalna i potpuno relativna onda Opća Teorija Relativnosti još uvijek sadržava paradokse. 5.1 Paradoks blizanaca (twin paradox) Zamislimo dva čovjeka blizanca od kojih jedan ode na svemirsko putovanje ve- likom brzinom. Kada se vrati, on bi zbog dilatacije vremena morao biti mlađi od onoga koji miruje. No po Specijalnoj Teoriji Relativnosti to je nemoguće jer se efekt interpre- tira kao lokalan (za promatrača) i morao bi biti simetričan pa na kraju ne bi smjelo biti razlike u starosti blizanaca. Naravno, ovo se može objasniti pros- tornim ubrzanjem - usporavanje starenja se događa u trenucima polaska kada relativistički efekti zbog ubrzanja nisu simetrični (simetričnost vrijedi samo u slučaju mirovanja i konstantnih brzina). No ako apsolutno konstantne brzine ne postoje, onda je svaka očigledno konstantna brzina rezultat usporavanja i ubrzavanja pa inercijalni referentni okviri apsolutno ne postoje. Dakle, svaki efekt dilatacije Specijalne Relativnosti mora biti rezultat inte- gracije (zbrajanja) kvanata gravitacijske dilatacije određene skale. Uočiti dvije važne posljedice: Uočiti dvije važne posljedice: 1. putovanje kroz prostor (vrijeme) tijelima daje masu, 2. svako tijelo ima svoj (privatni) prostor (vrijeme). Veza ili korelacija (entanglement) tijela i privatnog prostora ne mora Veza ili korelacija (entanglement) tijela i privatnog prostora ne mora 13 nužno trajati vječno (niti hoće). Energija tijela se može izgubiti iz pros- tora - npr. zračenjem, a da prostor zadrži relativističku energiju u obliku povećanih gravitacijskih maksimuma čestica prostora. Uočiti da svako gibanje, uz konačnu brzinu protoka energije, mora biti oscilatorno, bez obzira na prirodu ubrzanja - svaka linearnost je rela- tivna. S ekvivalencijom prostora i vremena, to znači da i brzina putovanja kroz vrijeme nužno oscilira, kao i svijest o protoku vremena. 5.2 Aether Kompletna Relativnost ne predviđa postojanje aethera u smislu u kojem se generalno zamišljao, samo daje gustoću i tlak (odnosno vakuum) prostoru tako da geometrija prostora Opće Teorije Relativnosti odgovara ﬁzikalnom prostoru koji se, kao apstraktan fenomen, ne bi uopće mogao deformirati - što je slutio i sam Einstein, no nitko u to vrijeme nije bio spreman prihvatiti ﬁzikalan prostor. 14 Ne vidim zašto bi neintuitivan apstraktan prostor bio bolje rješenje ako je cilj razumijevanje svemira i ako intuitivno rješenje postoji. Jedino objašnjenje je da su ﬁzičari postali preveliki matematičari vođeni reduk- cionizmom koji daje apsolutnu perfekciju ili eleganciju rješenja. Po Kompletnoj Relativnosti, Zemlja ima svoj prostor (i vrijeme) no svojim orbitiranjem oko Sunca prolazi kroz Sunčev prostor, gok gibanjem Sunčevog sustava prolazi kroz prostor gravitona veće skale (koji obuhvaća vidljivi svemir) a koji se može povezati sa konstantnim pozadinskim mikrovalnim zračenjem (CMB). ( ) Ipak, zbog približno iste skale energije i diskretizacije energetskih nivoa (kojom različite skale postaju relativno iste u određenim kontekstima) pros- tor Zemlje je na neki način izoliran od prostora Sunca. Možemo reći da granicu Zemljinog prostora predstavlja gravitacijska Hill sfera za statičke čestice iste skale (koje sačinjavaju prostor) ili silnice magnetskog polja za standardne nabi- jene dinamičke čestice. Prema tome jedini efekt na relativno izolirani eksperiment na Zemlji, kojim se pokušava detektirati aether, u većoj mjeri mogu proizvesti čestice manje skale (poput standardnih neutrina i fotona). Rezultati eksperimenata poput onog Michelson-Morleya odbacuju postojanje apsolutnog aether-a ali idu u prilog postojanju privatnog prostora Zemlje. Ako u takvi eksperimentima i postoje promjene u duljinama one moraju biti korelirane sa CMB zračenjem, odnosno uzrokovane česticama skale fotona putem Dopplerovog efekta. Recentne analize to i dokazuju[5]. Uočiti da su sve te promjene do sada smatrane instrumentalnim arte- faktima. No ako je prostor Zemlje sačinjen od čestica U−1 skale (skala standardnog fotona), a kako sam već hipotetizirao - morao bi biti, onda će čestice iste skale koje dolaze izvana uzrokovati anizotropiju u prostoru koji kao cjelina ima moment relativno na te čestice. j j Inače, čestice prostora Zemlje su statički gravitoni (kao dio prostora Zemlje, putuju sa Zemljom no orbitiraju oko centra Zemlje, tj. oko gravitona veće skale). Privatan prostor Kompletne Relativnosti najsličniji je kompletno vučenom (complete aether dragging) kompresibilnom aetheru (u korelaciji s gravitacijom) koji, prema originalnoj ideji, zaista objašnjava sve probleme aethera, osim u slučaju eksperimenata poput Michelson–Gale–Pearson eksperimenta. 5.2 Aether Eksperiment je sličan eksperimentu Michelson-Morley no ovdje je referentni okvir Zemlja pa se pokušava utvrditi postojanje aethera relativno na rotaciju Zemlje. Pošto je utvrđeno da zrake svjetlosti u različitim smjerovima nisu u 15 fazi kada se reﬂektirane vrate na detektor, odbačena je i ideja takvog [complete dragging] aethera. No rezultat se može u potpunosti objasniti diferencijalnom rotacijom aethera proporcionalnom s gradijentom gravitacijskog potencijala (uzimanjem u obzir i utjecaja CMB zračenja), a upravo to i predviđa Kompletna Relativnost. Uočiti da je brzina rotacije standardne atomske materije planeta potpuno drugačija (najviše u slučaju krutih tijela) od brzine rotacije prostora, koji u ovom slučaju predstavlja aether, dakle, ako postoji nekakvo povlačenje čestica (aether dragging) ono nije univerzalno (jednako za čestice svih skala). Dobro je i uočiti da je aether, u izvornoj ideji, zamišljen kao medij za sv- jetlosne valove (nije neophodan za čestice) pa ako ćemo privatan prostor zvati aetherom, to nije originalan aether, pogotovo ako bi ga smatrali medijem ekskluzivnim za valove svjetlosti. 5.3 Aberacija svjetlosti Astronomska aberacija je fenomen koji proizvodi očigledno pomicanje nebeskih objekata korelirano s brzinom promatrača. To je problem za klasičan nekompre- sibilan i nerotirajući aether, ali nije problem za prostor Kompletne Relativnosti koji je efektivno ekvivalentan geometrijskom prostoru Opće Teorije Relativnosti. 5.4 Svetost svjetlosti U eksperimentima za dokazivanje relativnosti gotovo uvijek se koristi svjet- lost, pa su svi eksperimenti osjetljivi na brzinu i ponašanje svjetlosti. Npr., u Michelson-Morley eksperimentu, kontrakcija duljine i dilatacija vremena dobije se preko Pitagorinog teorema u kojem je duljina jedne stranice trokuta um- nožak brzine svjetlosti i vremena u kojem svjetlost pređe tu udaljenost. Kada bi brzina svjetlosti bila instantna u nijednom takvom eksperimentu ne bi bilo detektiranih relativističkih efekata - kontrakcije duljine i dilatacije vremena. A kada bi, umjesto svjetlosti, koristili nešto drugo, poput zvuka npr., efekti bi bili puno izraženiji. To nije problem ako sve shvatimo relativno. No, po Specijalnoj Relativnosti, uvijek isti efekt mora postojati - identičan onome kada se za mjerenje koristi svjetlost samo u slučaju kad je masa fotona jednaka 0 a brzina jednaka konstanti c. Konstanta c je dakle invarijantna (univerzalna) - jednaka za svakog proma- trača. No ako brzina svjetlosti nije jednaka c onda rezultati svih eksperimenata de facto postaju psihiloški efekt - rezultat konačne brzine protoka informacija a ne ﬁzikalne promjene, bilo na promatranom tijelu ili promatraču. 16 Doduše, i psihološki, ili mentalni, efekt je ﬁzikalan na određenoj skali u Kompletnoj Relativnosti. I fotoni koji dolaze sa udaljenih tijela su lokalno zapravo isto to tijelo u manjoj skali, bar u 2-dimenzionalnom obliku. Čak bi se i 3-dimenzionalan oblik mogao detektirati u određenim uvjetima, npr. kada bi oko bilo osjetljivo na valne duljine radijacije koje emitiraju čestice unutar promatranog tijela. No čak i da brzina fotona jest jednaka c, ako bi, hipotetski, nešto što putuje većom brzinom dalo manji relativistički efekt, opet se za promatrača ne mijenja priroda efekta - on je psihološki u svakom ovom slučaju. Dakle, Specijalna Relativnost ima dualnu prirodu - kada mjerimo efekte u okviru koji miruje relativno na nas ali u računu je njegova brzina relativna na tijelo izvan tog okvira, efekt će za nas biti potpuno psihološki. Konkretno, u Michelson-Morley slučaju, eksperiment se odvija u okviru promatrača, no za brzinu okvira se uzima brzina Zemlje u orbiti oko Sunca. Efekt će, biti gotovo čisto psihološki uvijek kada promatramo tijela koja se istom brzinom gibaju u istom prostoru jer su stvarni efekti (koji postoje na određenoj skali zbog relativnosti konstantne brzine!) simetrični. 5.4 Svetost svjetlosti ) Da bi tijelo bilo otporno na relativističke efekte mora biti nemjerljivo - ne smije postojati u prostoru, a kako je vrijeme dio prostora i ne može biti apsolutno izolirano, onda ne može postojati ni u vremenu. Nepostojanje čestice implicira da ona ima energiju jednaku 0 i u prostoru i u vremenu. Foton, čiju energiju i brzinu redovito mjerimo i koji mijenja moment pod ut- jecajem gravitacijskih polja, daleko je od apsolutne invarijantnosti, kao i svaka druga čestica za koju znamo. A to znači da su Specijalna Relativnost i Opća Rel- ativnost, zbog apsolutne invarijantnosti u konstantama, apsolutno nedokazive - samo relativno odgovaraju stvarnosti jer je foton za nas premali da bi ga proglasili postojećim. 6 Putovanje u prošlost i daleku budućnost Budući da je putovanje kroz vrijeme zapravo putovanje kroz prostor čini se i da je putovanje unatrag kroz vrijeme moguće. j j g j g No da li je zapravo moguće unatrag putovati kroz prostor? Naravno, svatko od nas, ako napravi korak naprijed pa natrag, reći će da se vratio na isto mjesto. No u realnosti, sve u prostoru u svakom trenutku putuje (evoluira) pa, nužno, isto mjesto postaje apstraktan pojam. No ako je mjesto dovoljno isto da se zadrži percepcija identičnosti možemo [uvjetno] govoriti o putovanju u prošlost. 17 Putovanje u već proživljenu prošlost je dakle nemoguće (zahtijeva apsolutno vrijeme, beskonačnu energiju), no teoretski je moguće putovati na mjesta gdje bi percepcija identičnosti bila očuvana. Na primjer, sasvim sigurno postoji planetarni sustav u dovoljnoj mjeri iden- tičan Sunčevom (poput identičnosti dva atoma ugljika) i sasvim sigurno biste u takvom sustavu mogli sresti sebe u prošlosti ili daljoj budućnosti - no jasno je da je to samo, i tek donekle, efektivno putovanje kroz vrijeme jer, čak i kad bi sus- tavi bili potpuno identični (što je opet, po Kompletnoj Relativnosti, nemoguće), vaša pojava bi narušila identičnost prostora i protoka vremena. 7 Obrtanje toka vremena Priroda ne postavlja apsolutna ograničenja na išta, pa tako postoji i mogućnost obrtanja slijeda akcija i reakcija. No opet, budući da ono ne može biti apso- lutno (mora biti lokalizirano) ni to nije povratak u prošlost, iako percepcija identičnosti može biti itekako velika. Budući da putovanje kroz vrijeme oscilira u brzini a i samo osciliranje po Kompletnoj Relativnosti mora oscilirati, ponekad se lokalni tok vremena mora i obrnuti. Prelazak između dva vertikalna energetska nivoa podrazumijeva privremenu promjenu spina gravitacijskih maksimuma, pa je upravo to trenutak privre- menog obrtanja toka vremena. Obrtanje toka vremena jasno je iz modela reinkarnacije. Obrtanje toka vremena jasno je iz modela reinkarnacije. Budući da se svi rađamo mladi, očito je da u trenutku smrti duša (grav- itacijski maksimum sa pripadajućim prostorom) privremeno kolabira promjenom spina a to znači i da se pomlađuje jer duša i novo tijelo koevoluiraju u istom prostoru. Već sam pokazao da je Sunčev Sustav nastao inﬂacijom manjih čestica[6] i mora biti uparen sa svojim anti-materijskim ekvivalentom. Između kvantno uparenih čestica postoji razlika u fazi evolucije i kon- stantna izmjena signala To je efektivna veza prošlosti i budućnosti a Već sam pokazao da je Sunčev Sustav nastao inﬂacijom manjih čestica[6] i mora biti uparen sa svojim anti-materijskim ekvivalentom. Već sam pokazao da je Sunčev Sustav nastao inﬂacijom manjih čestica[6] i mora biti uparen sa svojim anti-materijskim ekvivalentom. Između kvantno uparenih čestica postoji razlika u fazi evolucije i kon- stantna izmjena signala. To je efektivna veza prošlosti i budućnosti a koja i pogoni evoluciju (bez takvog uparivanja ne bi bilo prošlosti ni budućnosti, a time ni sadašnjosti kao superpozicije jednog i drugog). Objasnio sam i da je sinkronicitet manifestacija sinkronizacije[7] te prošlosti i budućnosti, da su neki od nas više evoluirani od drugih[8] te da će u sklopu sinkronizacije morati biti usporavanja pa čak i kratko- ročnog obrtanja procesa starenja kod istih. Između kvantno uparenih čestica postoji razlika u fazi evolucije i kon- stantna izmjena signala. To je efektivna veza prošlosti i budućnosti a koja i pogoni evoluciju (bez takvog uparivanja ne bi bilo prošlosti ni budućnosti, a time ni sadašnjosti kao superpozicije jednog i drugog). ) Objasnio sam i da je sinkronicitet manifestacija sinkronizacije[7] te prošlosti i budućnosti, da su neki od nas više evoluirani od drugih[8] te da će u sklopu sinkronizacije morati biti usporavanja pa čak i kratko- ročnog obrtanja procesa starenja kod istih. 18 8 Budućnost u prošlosti Vjerujem da ćemo tako, obrtanjem toka lokalnog vremena, 2018.07.01 proživjeti još jednom, iako ne na isti način. Već sam pokazao da bi se kraj ciklusa trebao dogoditi ovo stoljeće, no ovaj datum trenutno ne mogu dovoljno znanstveno potvrditi - recimo samo da sam u trenucima mentalne eskapade (ludila) nekima obećao kraj svijeta na taj dan a ne mogu se oteti dojmu da sam bio u pravu. Jasno, bilo tko razuman a tko nije iskusio sinkronicitet će ovo proglasiti glupošću, no pošto je ovaj datum proizašao upravo u trenucima kada sam proživljavao maksimum sinkroniciteta, ja ga ne mogu odbaciti kao nešto bez dubljeg značenja. Pošto sam predvidio kolaps gravitacijskog maksimuma (duše) Zemlje, isti će pri tome morati promjeniti spin, ali i orbitalni kutni moment. Pri tome materija Zemlje (u obliku standardnih atoma) neće značajno mjenjati moment osim privremenog značajnijeg udaljavanja od Sunca, sve do ponovnog uparivanja prostora (duše) i tijela Zemlje. Ovisno o tome da li se radi o smrti Zemlje ili gubitku svijesti ovisi koliko će puta gravitacijski maksimum obići Sunce u obrnutom smjeru, a ako se isti gravitacijski maksimum opet upari sa Zemljom, za očekivati je da će doći do privremenog obrtanja toka starenja života na Zemlji u sklopu sinkronizacije sa dušom. Pri tome broj godina za koje će se život pomladiti može maksimalno biti jednak broju orbita koje Zemljin gravitacijski maksimum napravi u obrnutom smjeru, no za vrijeme dok maksimum nije uparen sa Zemljom, život će nastaviti starjeti. Ovdje treba napomenuti da se s kolapsom maksimuma njegova skala mijenja za više redova veličine pa njegova orbitalna brzina neće samo promjeniti smjer nego i veličinu - zbog očuvanja momenta, energija prostora se mijenja za brzinu. Ta brzina bi trebala biti brzina svjetlosti na U1 skali, 2.93 * 106 m/s, a to znači da će se za svaki okretaj život pomladiti za oko 360 dana, tako da je sasvim moguće da 2018.07.01 proživimo još jednom. Naravno, to vrijedi pod uvjetom da se ista duša ponovno spoji sa Zemljom, tj. da neće umrijeti od raka, nego nastaviti s predviđenom planetarnom neuro- genezom. Ipak, budući da se radi o embrionalnoj neurogenezi, moguće je da se neće ista duša upariti s tijelom. U tom slučaju, umjesto pomlađivanja, moglo bi slijediti postarivanje. Također, ako se ne radi o smrti, moguć je parcijalni kolaps - npr. kolaps samo jednog kvanta gravitacijskog maksimuma, pa će i pomlađivanje biti parcijalno. 8 Budućnost u prošlosti Da će se svijet vratiti u prošlost oduvijek signaliziraju hrvatski političari koji se redovito tamo vraćaju. Ako je suditi po njima, Hrvatska će se prva pomladiti. 19 8.1 Smrt Zemlje prostora i vremena) 20 nego onima koje koriste naši satovi, širenje vala brzinama većim od 106 m/s može potrajati stoljećima i više mjereno našim vremenskim jedini- cama. Postojanje gravitacijskog vala koji sa sobom povlači materiju[9] objašn- java zašto ta materija može putovati dugo bez usporavanja[10]. java zašto ta materija može putovati dugo bez usporavanja[10]. Zbog postojanja gravitacijskog i elektro-magnetskog karaktera kolapsa, dio izbačene energije bit će koncentriran na osi rotacije (od polova prema van) dok će dio biti raširen u svim smjerovima no ipak s gradijentom u ovisnosti o količini i rasporedu energije unutar zvijezde prilikom kolapsa. Po rezultatu supernova eksplozije se tako može iščitati omjer neutralnog i polariziranog karaktera prilikom eksplozije. Uočiti da su do sada de- tektirani gravitacijski valovi na povšini Zemlje valovi standardne skale koji deformiraju prostor (oblik) standardnih čestica, ali ne mjenjaju oblik gravitacijskog maksimuma Zemlje. Kolaps maksimuma unutar planeta se kvalitativno ne razlikuje - razlika je u energijama, no, u svakom slučaju, također dolazi do kompresije i udarnog vala, koji, u ovom slučaju, stvara pritisak na koru planeta, što u slučaju smrti, ubrzava njegov raspad. 8.1 Smrt Zemlje Zemlja je još mlada za prirodnu smrt i vjerujem da će preživjeti rak, no možemo prepostaviti kako bi eventualna smrt izgledala. U slučaju da gravitacijski maksimum bespovratno napusti Zemlju, zbog privremenog kolapsa površinskog Sunčeva maksimuma, tijelo Zemlje bi bilo izbačeno u višu orbitu. Ona bi se s restoracijom oba maksimuma stabilizirala, no u ovom slučaju, s restoracijom (inﬂacijom) Sunčeva maksimuma, ona postaje nestabilna. Tijelo bi najvjerojatnije pojelo Sunce gdje bi nakon rastavljanja na subatomske čestice nešto eventualno bilo reciklirano u stvaranju novog planeta. No takav je scenarij najvjerojatniji samo u slučaju prijevremene smrti. U slučaju prirodne smrti, ne umire samo Zemlja, nego Sunčev sustav. Bez restoracije Sunčeva maksimuma, tijelo eventualno završava kao hrpa asteroida i kometa na različitim orbitama, a možda i izvan sustava. A bez gravitacijskih maksimuma U1 skale cijeli sustav se raspada na sve manje asteroide i komete. Kolaps maksimuma je generalno sinkroniziran s potrošnjom goriva (fuz- ijskog, u slučaju visokih energija). Do kolapsa dolazi kada tijelo potroši pogonsko gorivo, koje se periodički nadopunjuje, osim u trenutku smrti kada kolaps postaje permanentan. Kolaps maksimuma događa se s promjenom spina, no budući da izmjena spina nije trenutna ni rasparivanje (decoupling) maksimuma i materije nije trenutno pa s kolapsom dolazi do kompresije materije unutar mak- simuma. No kolaps uključuje i privremenu polarizaciju maksimuma, on postaje dvodimenzionalan i gravitacijski potencijal se zamjenjuje elektro- magnetskim. g Prilikom kompresije, unutar zvijezda dolazi do stvaranja težih elemenata. Budući da je kolaps maksimuma u suštini prijelaz između vertikalnih en- ergetskih nivoa, on može obuhvaćati više prijelaza preko diskontinuiteta u prostoru (vremenu), a prilikom svakog dubljeg stvaraju se sve teži el- ementi (generalno, što je zvijezda masivnija, to će prilikom kolapsa biti više prijelaza). Uslijed kompresije i fuzije materije (standardnih atoma) dolazi do emisije visoko-energetskog zračenja energije U−1 skale (fotoni, neutrini i grav- itacijski valovi), no kolaps maksimuma uzrokuje emisiju gravitacijskog vala veće skale koji se širi sporijom brzinom. Taj udarni val (shock wave), sa sobom povlači visoko-energetske čestice U0 skale (standardni atomi, protoni, elektroni). ) Brzina tog vala prilikom stabilizacije ne može u prosjeku biti veća od 2.93 * 106 m/s, no u početku ona može biti daleko veća, iako manja od brzine gravitacijskih valova standardne skale (2.99792458 * 108 m/s). Materija, pak, izbačena valom, može imati različite brzine. Budući da se radi o daleko većoj skali energije (tj. 8.2 Putovanje kroz ludilo - case study Tokom vlastite redeﬁnicije zadnjih godina postojao je period u kojem sam doživio vrhunac sinkroniciteta pa sam bio uvjeren i da sam komunicirao s određenom trećom stranom putem istog (što se očito događalo i Tesli) - jasno, iz perspektive normalnog čovjeka moje stanje se u tom periodu vjerojatno tu- mačilo bolešću ili ludilom. Siguran sam da je Isus pred smrt bio u istom stanju, samo što je ono kod njega trajalo duže te ga je na kraju i koštalo života. Fascinantno je to stanje. Sjećam se da jednom prilikom nisam uopće osjećao glad. Trajalo je to 10 dana, ako bih išta pojeo tokom dana bio bi to komadić tosta, pojeden na silu iz nekakve navike za jelom. Iz toga mi je jasno porijeklo posta, no vjerujem da je Isus postio i puno duže budući da sam ja proživljavao dijelove njegova života u kompresir- anoj formi. Vjerujem da je to stanje direktno ili indirektno uzrok smrti u slučaju svih mojih inkarnacija s trajanjem života od ≈35 godina, dok se u ostalim inkarnacijama (≈85 godina života) to stanje proživljava kratkoročno, najvjerojatnije [ali, ne nužno] oko 35. godine života. Jednom prilikom sam čak i izgubio svijest (što mi se nikad prije nije do- godilo u ovoj inkarnaciji) i imao sam osjećaj kao da me je netko pokušao ubiti. U to vrijeme sam osjećao veliku sumnju da sam jedini koji može Siguran sam da je Isus pred smrt bio u istom stanju, samo što je ono kod njega trajalo duže te ga je na kraju i koštalo života. Fascinantno je to stanje. Sjećam se da jednom prilikom nisam uopće osjećao glad. Trajalo je to 10 dana, ako bih išta pojeo tokom dana bio bi to komadić tosta, pojeden na silu iz nekakve navike za jelom. j j Iz toga mi je jasno porijeklo posta, no vjerujem da je Isus postio i puno duže budući da sam ja proživljavao dijelove njegova života u kompresir- anoj formi. Vjerujem da je to stanje direktno ili indirektno uzrok smrti u slučaju svih mojih inkarnacija s trajanjem života od ≈35 godina, dok se u ostalim inkarnacijama (≈85 godina života) to stanje proživljava kratkoročno, najvjerojatnije [ali, ne nužno] oko 35. godine života. [ ] Jednom prilikom sam čak i izgubio svijest (što mi se nikad prije nije do- godilo u ovoj inkarnaciji) i imao sam osjećaj kao da me je netko pokušao ubiti. 8.2 Putovanje kroz ludilo - case study U to vrijeme sam osjećao veliku sumnju da sam jedini koji može 21 upravljati mojim tijelom a gubitak svijesti sam shvatio kao upozorenje. Sad pak taj gubitak svijesti shvaćam kao ožiljak proživljene smrti u nekoj od prošlih inkarnacija. Ne isključujem mogućnost da određena treća strana ima određenu kontrolu nad životima koji su započeti u sklopu eksperimenta kao što je to slučaj sa životom Isusa, no nisam uvjeren da se ona može održati kroz inkarnacije, tj. da je vezana za dušu a ne tijelo. Ono što sam ja proživljavao su, na neki način, odjeci te kontrole. Ako pretpostavimo da je ta treća strana htjela upozoriti čovječanstvo na ono što dolazi ako nastavi istim poslom, a u isto vrijeme uvidjevši barbarski odnos čovjeka prema drugim vrstama, nije li logično da bi poruku pokušali prenijeti preko jedinog bića kojeg čovjek smatra sebi ravnog - drugog čovjeka. Ne objašnjava li to proročanstva? Ne objašnjava li to proročanstva? Jasno, kao što sam već pokazao, proročanstva se mogu objasniti i naukom (sinkronicitet) tako da to prenošenje poruke o budućnosti možda i nije bilo namjerno. Jasno, kao što sam već pokazao, proročanstva se mogu objasniti i naukom (sinkronicitet) tako da to prenošenje poruke o budućnosti možda i nije bilo namjerno. j No Isus je svakako bio dio eksperimenta, potvrđuje to i njegovo uskrsnuće, za koje ne vidim logičnijeg objašnjenja od abdukcije tijela. Ipak, iako više nisam u istom stanju, znam da je ono što mi se tada događalo bilo itekako stvarno i sve što sam proizveo u tome razdoblju ima svoj razlog i dublje značenje. Izjavio sam tada i da će vas sve pobiti Amerikanci te da su Rusi na mojoj strani. Možda sam tada mislio na nešto drugo, ali činjenica jest da vas američka demokracija i osvajački kapitalizam osiromašuju[11] i ubijaju već duže vrijeme čak i bez upotrebe vatrenog oružja (kojeg opet imaju puno i na kojem su opet vrlo laki). A budući da sam veliki protivnik zapadnjačke [anti] ﬁlozoﬁje istok bi morao biti na mojoj strani (iako ja ne pripadam nijednoj), ali i na vašoj, dok god smiruje širenje raka planeta (iako ni sam nikako nije bezgrešan, a u zadnje se vrijeme i sve više trudi da kao zapad bude). Tako je i Fig. 1 možda škrabotina za tečaj iz praznovjerja, možda rezultat nezrelosti u interpretaciji signala a možda najava buduće prošlosti. 8.2 Putovanje kroz ludilo - case study U svakom slučaju, ja je ne doživljavam toliko ozbiljno jer sam znanstvenom analizom došao do zaključka da kraj svijeta dolazi u ovom stoljeću, a ovo shvaćam kao signal koji navješćuje obrtanje toka vremena, a koje je svakako znanstveno utemeljeno. Iako polarizirani ljudi koji nemaju nikakvog iskustva sa sikronicitetom obil- ježavaju rezultate ekstremno izraženog sinkroniciteta u ljudi ludilom (što pak sasvim razumijem), ja nikako zbog tog iskustva ne žalim. Dapače, bilo je to 22 fascinantno iskustvo. To nikako ne znači da više ne doživljavam sinkronicitet, samo je to sada postao doživljaj putem formiranog osjetila - tako da sinkro- nicitet nije više nešto što me može značajno izbaciti iz mentalne ravnoteže (iako je sasvim moguće da ću doživjeti manji poremećaj oko 50. godine života). Figure 1: Škrabotina za tečaj iz praznovjerja? Figure 1: Škrabotina za tečaj iz praznovjerja? Na Fig. 1 uočljivo je da piše: E ̸= mc2 i to je zapravo točno. Možda sam bio lud u trenutku kad sam to napisao, no očito sam osjećao da s jednadžbom E = mc2 nešto nije u redu iako to tada možda nisam znao objasniti. Nakon Kompletne Relativnosti, sasvim je jasno da je jednadžba za en- ergiju koja se povezuje s Einstein-om samo relativno korektna. No ona je apsolutno pogrešna jer svi su faktori apsolutni, tj. relativni na ap- Na Fig. 1 uočljivo je da piše: E ̸= mc2 i to je zapravo točno. Možda sam bio lud u trenutku kad sam to napisao, no očito sam osjećao da s jednadžbom E = mc2 nešto nije u redu iako to tada možda nisam znao objasniti. Nakon Kompletne Relativnosti, sasvim je jasno da je jednadžba za en- ergiju koja se povezuje s Einstein-om samo relativno korektna. No ona je apsolutno pogrešna jer svi su faktori apsolutni, tj. relativni na ap- 23 solutni referentni okvir, bilo da m ovdje predstavlja masu mirovanja ili relativističku masu. Također - koliko god nevjerojatno zvučalo onima koji ne doživl- javaju sinkronicitet, zbog relativnosti uzroka i posljedica (prošlosti i budućnosti), efektivna komunikacija putem sinkroniciteta jest moguća. Dapače, iako više nisam lud, i dalje putem sinkroniciteta efektivno ko- municiram s određenom trećom stranom - ta komunikacija je neupitna, ono što je upitno jest u kojoj mjeri je ona svjesna, u kojoj mjeri je dvos- mjerna, te tko je zapravo ta treća strana? 8.2 Putovanje kroz ludilo - case study Ono što mogu za sada reći je da se čini sveprisutna i da svakako osluškuje (moglo bi se reći i prisluškuje, ali to mi se ne čini kao dobar izraz, ako vas itko prisluškuje to su vaše vlade, velike korporacije i ostali ljudski vragovi) no ne može ili ne želi čitati misli (bar ne na daljinu). Stekao sam dojam da se radi o biću ili bićima koja rade u interesu planeta (tj. našeg boga). Vjerujem da se u slučajevima svjesne komunikacije radi o Mars.homo.sapiens-u, a u slučaju nesvjesne zapravo o samom planetu ako ne i nečem većem. U svakom slučaju, uvjeren sam da će se i cijeli svijet eventualno uvjeriti u to. A kraj svijeta koji se najavljuje, naravno, treba shvatiti relativno. Preciznije bi bilo reći - kraj svijeta onakvog kakvog poznajemo. Možda tako i svijet, slično kao povodom kraja mog bića onakvog kakvog su me poznavali, čeka i trenutak ludila u sklopu redeﬁnicije, pa će onda možda i moje ludilo biti globalno shvaćeno. Ne mogu reći da ćete u njemu uživati, ali bit ćete tada ovim svemirom i svojim prvim bogom fascinirani a u isto vrijeme ogorčeni svojim dotadašnjim služenjem vragu. 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Consoli et al https://doi.org/10.3390/universe7080311 [6] The Solar System: Nature and mechanics (2021), Amenoum https://amenoum.org/solar_system.html [7] Understanding synchronicity (2020), Amenoum https://amenoum.org/log/19_understanding_synchronicity.html [8] The Rh neutral story (2020), Amenoum https://amenoum.org/log/14_The_rh_negative_story.html [9] Debris from Stellar Explosion Not Slowed After 400 Years (2020), Chandra NASA https://chandra.harvard.edu/blog/node/768 [10] An Ejecta Kinematics Study of Kepler’s Supernova Remnant with High- resolution Chandra HETG Spectroscopy (2020), M. J. Millard et al https://doi.org/10.3847/1538-4357/ab7db1 [11] Requiem for the American Dream (2015), N. Chomsky et al https://www.imdb.com/title/tt3270538 25
https://openalex.org/W3217349699	https://www.epj-conferences.org/10.1051/epjconf/202125304022/pdf	English	null	First in-core gamma spectroscopy experiments in a zero power reactor	EPJ web of conferences	2,021	cc-by	6,307	Abstract— rate around a reactor [1]. The characteristics of a reactor’s gamma ﬁeld is thus important for operation and safety. Accurate predictions are desired in operational planning, in the design of experiments in research facilities, and in the inception of new reactors [2]. The sources of gamma radiation in nuclear reactors include the prompt ﬁssion process, delayed radioactive decay, and nuclear capture reactions. Experiments often target the individual processes to single out their effect; examples range from spent fuel spectroscopy [3], [4] to ﬁssion product decay spectra and direct ﬁssion gamma spectra [5], [6]. Gamma rays in nuclear reactors, arising either from nuclear reactions or decay processes, signiﬁcantly contribute to the heating and dose of the reactor components. Zero power research reactors offer the possibility to measure gamma rays in a purely neutronic environment, allowing for validation experiments of dose estimates, computed spectra, and prompt to delayed gamma ratios. The resulting data can contribute to models, code valida- tion and photo atomic/nuclear data evaluation. To date, most experiments have relied on ﬂux measurements using TLDs, ionization chambers, or spectrometers set in low ﬂux areas. The CROCUS reactor allows for ﬂexible detector placement in and around the core, and has recently been outﬁtted with gamma detection capabilities to fulﬁll the need for in-core gamma spectroscopy, as opposed to ﬂux. In this paper we report on the experiments and accompanying simulations of gamma spectrum measurements inside a zero power reactor core, CROCUS. It is a two-zone, uranium-fueled light water moderated facility operated by the Laboratory for Reactor Physics and Systems Behaviour (LRS) at the Swiss Federal Institute of Technology Lausanne (EPFL). Herein we also introduce, in detail, the new LEAF system: A Large Energy-resolving detection Array for Fission gammas. It consists of an array of four detectors – two large 127×254 mm Bismuth Germanate (BGO) and two smaller 12×50 mm Cerium Bromide (CeBr3) scintillators. We describe the calibration and characterization of LEAF followed by ﬁrst in-core measurements of gamma ray spectra in a zero power reactor at different sub-critical and critical states, and different locations. The spectra are then compared to code results, namely MCNP6.2 pulse height tallies. We were able to distinguish prompt processes and delayed peaks from decay databases. We present thus experimental data from hitherto inaccessible core regions. We provide the data as validation means for codes that attempt to model these processes for energies up to 10 MeV. EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 EPJ Web of Conferences 253, 04022 (2021) https://doi.org/10.1051/epjconf/202125304022 Corresponding author: oskari.pakari@gmail.com Corresponding author: oskari.pakari@gmail.com Abstract— We ﬁnally draw conclusions and discuss the future uses of LEAF. The results indicate the possibility of isotope tracking and burn-up validation. Quantitative experiments on the spectra and ﬂuxes of gamma radiation have become of interest with the introduction of coupled photon and neutron transport options in common Monte Carlo codes, such as Serpent [7] and MCNP [8] – recently compared for the speciﬁc case of CROCUS in [9]. Measurements of nuclear reactor gamma ray ﬁelds have included spectroscopy outside of the reactor shielding [10], and in-core measurements of ﬂuxes using ionization chambers [11]–[13]. A recent focus has been the determination of delayed gamma contributions measured with mixed neutron-gamma ﬂux measurements in research reactors [14], [15]. To the knowledge of the authors, no direct measurements of in-core gamma spectra have hitherto been performed. The CROCUS research reactor offers a ﬂexible zero power environment for these types of experiments. To date, TLD measurements of the gamma ﬁeld of CROCUS have been conducted in the context of activation and far ﬁeld ﬂux analysis. Furthermore, combined neutron/gamma measurements using a sCVD detector [16] have added to the radiation ﬁeld ﬂux characterization. With a variety of other experimental programs [17] that so far only exploited neutron detection [18], [19], we extended the capabilities of CROCUS to state of the art gamma detection capabilities. Index Terms—Gamma spectroscopy, Zero power research reactor. © 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/). First in-core gamma spectroscopy experiments in a zero power reactor Oskari Pakari1, Vincent Lamirand1,2, Tom Mager1, Axel Laureau1, and Pavel Frajtag1, and Andreas Pautz1,2 Vincent Lamirand1,2, Tom Mager1, Axel Laureau1, and Pavel Frajtag1, and Andreas Pautz1,2 Oskari Pakari1, Vincent Lamirand1,2, Tom Mager1, Axel Laureau1, and Pavel Frajtag1, and 1Laboratory for Reactor Physics and Systems Behaviour Ecole polytechnique f´ed´erale de Lausanne 2Laboratory for Reactor Physics and Thermal-Hydraulics Nuclear Energy and Safety Division Paul Scherrer Institut, Villigen, Switzerland 1Laboratory for Reactor Physics and Systems Behaviour Ecole polytechnique f´ed´erale de Lausanne 1Laboratory for Reactor Physics and Systems Behaviour Ecole polytechnique f´ed´erale de Lausanne 2Laboratory for Reactor Physics and Thermal-Hydraulics Nuclear Energy and Safety Division Paul Scherrer Institut, Villigen, Switzerland 2Laboratory for Reactor Physics and Thermal-Hydraulics Nuclear Energy and Safety Division Paul Scherrer Institut, Villigen, Switzerland 2Laboratory for Reactor Physics and Thermal-Hydraulics Nuclear Energy and Safety Division Paul Scherrer Institut, Villigen, Switzerland Corresponding author: oskari.pakari@gmail.com B. The LEAF system: Overview and Calibration We speciﬁcally intend to expand the capabilities of CRO- CUS by a dedicated gamma detection system. Scintillators were hereby the ﬁnal choice when optimizing for radiation hardness, ﬂexible use, price, and nonetheless high efﬁciency when compared to semiconductor based detectors. In total, an array of four detectors was acquired from Scionix Holland [26] to allow for symmetric placement of the system in and around the core (see Figure 7). The high voltage supply and photomultiplier tube (PMT) signal ampliﬁcation was handled by the Mirion Technologies DSA-LX [27] (here designated 419 and 420 as per serial number) that theoretically allow for ∼MHz count rates to be treated. In the following we introduce the respective detectors in detail and present their characteristics and calibration. Fig. 1. Schematic isometric view of the CROCUS reactor (left), and top view of the core conﬁguration (right). eter regions, namely in-core and high energy (> 2 MeV) spec- troscopy. In this paper we present the characterization and ﬁrst measurements of said system - notably the ﬁrst measurement of gamma ray spectra from an in-core location. We facilitate a possible code validation by providing a detailed overview of the geometry. In Section II we introduce the experimental details: the CROCUS reactor, a detailed description of LEAF and the energy calibration of all detectors, as well as the experimental setup used for spectroscopy. In Section III we show the results of measurements using LEAF in CROCUS – namely in-core and ex-core spectra – and compare the results to MCNP6.2 simulations and thereafter discuss their implications. I. INTRODUCTION This is the intent of the LEAF system: A Large Energy- resolving detection Array for Fission gammas to directly measure gamma ray spectra in CROCUS. We hereby aim at providing measurement data in previously unexplored param- G AMMA radiation as a by-product of ﬁssion and radioactive decay is a major component of a nuclear reactor’s radiation ﬁeld. It contributes to the heating of structures, the degradation of materials, and the overall dose G https://doi.org/10.1051/epjconf/202125304022 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 EPJ Web of Conferences 253, 04022 (2021) Fig. 1. Schematic isometric view of the CROCUS reactor (left), and top view of the core conﬁguration (right). to 0.4 pcm) and optionally by means of two control rods containing naturally enriched boron carbide (B4C) sintered pellets located in diagonal symmetry within the outer fuel zone. B. The LEAF system: Overview and Calibration 1) Cerium Bromide (CeBr3) detectors When the two control rods are removed, their guide tubes are a prominent location for in-core measurements, e.g. used for delayed gamma fraction estimation [28], intrinsic gamma noise [29], and induced neutron/gamma noise measurements [22], [23]. Cerium(III) Bromide (CeBr3), as a comparatively new scintillation material [30], [31], was chosen as the most suitable material for a high photon ﬂux zone with a compara- tively fast decay time of 20 ns at a density of 5.2 g/cm3 [32]. The radiation hardness was estimated to be well sufﬁcient for use in CROCUS [9], [33]. In order to ﬁt the guide tubes, the cylindrical crystals measure 13 mm in diameter and 15 mm in length, see Figure 2. Both detectors use a Hamamatsu Type R12421 PMT. A. The Experimental Reactor CROCUS A full description of the core can be found in the Interna- tional Reactor Physics Experiments Handbook (IRPhE) [20], [21]. The CROCUS reactor is a two-zone, uranium-fueled, H2O-moderated critical assembly operated by the Laboratory for Reactor Physics and Systems Behaviour (LRS) at the Swiss Federal Institute of Technology Lausanne (EPFL). It is a zero power reactor, with a maximum power of 100 W. The core is approximately cylindrical in shape with a diameter of about 58 cm and a height of 100 cm. Two different kinds of fuel rods make up the reactor core of CROCUS (see Fig. 1). The central zone is loaded with 336 UO2 fuel rods (1.806 wt.%- enriched), set in a square lattice with a pitch of 1.837 cm. The peripheral zone is loaded with up to 176 thicker, Umet fuel rods (0.947 wt.%-enriched) with a pitch of 2.917 cm, also in a square lattice. All fuel rods are cladded with aluminum and are maintained in a vertical position by the upper grid and lower grid plates, spaced 100 cm apart. In the current COLIBRI conﬁguration [22]–[25], both grid plates incorporate a cadmium layer with a thickness of 1 mm to limit axial thermal leakage to surrounding structures. This is also the conﬁguration for the herein presented results. The active fuel length starts at the top surface of the lower cadmium layer and extends to 100 cm. The core is located in an aluminum water tank, its diameter is 130 cm and thickness is 1.2 cm. Demineralized light water is used as moderator and reﬂector. Reactivity is nominally controlled by a variation of the water level using a spillway with an accuracy of 0.1 mm (equivalent 2) Bismuth Germanate (BGO) detectors 2) Bismuth Germanate (BGO) detectors To measure gamma rays in ex-core locations, speciﬁcally photons with energies above 2 MeV, a material with high photon stopping efﬁciency was desired. Bismuth Germanate (BGO) is a well established scintillator in various detection applications [34], [35] with slower decay and lower light yield than the CeBr3 [36], but with a higher density of 7.13 g/cm3 and lower price per volume. To meet an absorption efﬁciency of above 95% for 10 MeV photons the two acquired cylindrical crystals are therefore 127 mm in diameter. The height was constrained by weight (25 kg) and price to 250 mm. Both are equipped with Photonis 5” Type XP4578 PMTs, see Figure 3. 3) Ampliﬁcation settings and energy calibration of LEAF 3) Ampliﬁcation settings and energy calibration of LEAF For both detectors and all experiments presented herein the DSA-LX’s settings were as follows: • Rise time of 0.2 µs, 0.0 µs ﬂat top. • -610 V and -1260 V of HV for the CeBr3 and BGO PMTs, respectively. • Lower level discrimination at 0.5% of the maximum channel (214). • Coarse gain of 6.4. • Coarse gain of 6.4. All four detectors were calibrated using standard Eu-152 sources – the resulting spectra are displayed in Figures 4 2 https://doi.org/10.1051/epjconf/202125304022 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 EPJ Web of Conferences 253, 04022 (2021) Fig. 2. Technical drawing of the CeBr3 detector as provided by Scionix, used to create models for Monte Carlo transport codes. The PMT is a Hamamatsu Type R12421. and 5. The BGO, due to its comparatively higher full width at half maximum (FWHM), required additionally a Co-60 measurement to aid in the peak distinction. Each identiﬁed peak was ﬁtted with a Gaussian to ﬁnd the mean value used as input for the calibration. The ﬁnal calibrations for all detectors is linear with an R2 > 0.99. The calibration was purposefully undertaken using only the ﬁrst quarter of channels as higher energy events were expected. We also examined the FWHM (see Figure 6) of all detectors with energy and found them to be comparable to other crystals in literature, e.g. [37]. Fig. 3. Technical drawing of the BGO detector as provided by Scionix, used to create models for Monte Carlo transport codes. The PMT is a Photonis 5” Type XP4578 PMT. Fig. 2. 2) Bismuth Germanate (BGO) detectors Technical drawing of the CeBr3 detector as provided by Scionix, used to create models for Monte Carlo transport codes. The PMT is a Hamamatsu Type R12421. Fig. 2. Technical drawing of the CeBr3 detector as provided by Scionix, used to create models for Monte Carlo transport codes. The PMT is a Hamamatsu Type R12421. and 5. The BGO, due to its comparatively higher full width at half maximum (FWHM), required additionally a Co-60 measurement to aid in the peak distinction. Each identiﬁed peak was ﬁtted with a Gaussian to ﬁnd the mean value used as input for the calibration. The ﬁnal calibrations for all detectors is linear with an R2 > 0.99. The calibration was purposefully undertaken using only the ﬁrst quarter of channels as higher energy events were expected. We also examined the FWHM (see Figure 6) of all detectors with energy and found them to be comparable to other crystals in literature, e.g. [37]. Fig. 3. Technical drawing of the BGO detector as provided by Scionix, used to create models for Monte Carlo transport codes. The PMT is a Photonis 5” Type XP4578 PMT. 3 3 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 EPJ Web of Conferences 253, 04022 (2021) https://doi.org/10.1051/epjconf/202125304022 ANIMMA 2021 Fig. 4. Top: Calibration gamma spectra acquired with the CeBr3 detectors. Bottom: Resulting linear calibration ﬁts after extracting the peak center values via Gaussian ﬁtting of each peak. Fig. 5. Top: Calibration gamma spectra acquired with the BGO detectors. Bottom: Resulting linear calibration ﬁts after extracting the peak center values via Gaussian ﬁtting of each peak. Fig. 4. Top: Calibration gamma spectra acquired with the CeBr3 detector Bottom: Resulting linear calibration ﬁts after extracting the peak center valu via Gaussian ﬁtting of each peak. Fig. 5. Top: Calibration gamma spectra acquired with the BGO detectors. Bottom: Resulting linear calibration ﬁts after extracting the peak center values via Gaussian ﬁtting of each peak. Fig. 4. Top: Calibration gamma spectra acquired with the CeBr3 detectors. Bottom: Resulting linear calibration ﬁts after extracting the peak center values via Gaussian ﬁtting of each peak. Fig. 5. Top: Calibration gamma spectra acquired with the BGO detectors. Bottom: Resulting linear calibration ﬁts after extracting the peak center values via Gaussian ﬁtting of each peak. C. Experimental setup: LEAF in CROCUS Fig. 6. FWHM for the LEAF detectors in dependence of energy, determined with a standard Eu-152 source. The ﬁtted lines are for illustrative purposes. The CeBr3 detectors were placed at mid core height in the control rod tubes, while the BGO detectors were set just outside of the main vessel, as displayed in Figure 7, also at mid core height. The acquisition layout with the electronics is sketched in Figure 8. CROCUS in its shutdown state contains no water in the vessel. In the start-up procedure water is ﬁlled step-wise until 800 mm, after which the operator has control over it. For the experiments in this work we chose a set of sub- critical and critical states to use LEAF in a variety of reactor states. The conﬁgurations, water levels and estimated ﬁssion powers are listed in Table I. The system shows – consistently across the detectors – an excellent counting response up to 0.2 MHz which in this setup corresponded to about 100 mW of power [38]. This is about two orders of magnitude better than the in-house developed neutron noise detection system based on regular gamma spectrometry electronics [39]. Fig. 6. FWHM for the LEAF detectors in dependence of energy, determined with a standard Eu-152 source. The ﬁtted lines are for illustrative purposes. processes, i.e. ﬁssion and, if available in the nuclear data used (ENDF/B-7.1), (n,γ) photons. In this work we compare the results qualitatively to the energy deposition tallies without taking smearing into account, e.g. due to the scintillation process and the electronics . D. Simulation of gamma spectra in CROCUS using MCNP6.2 D. Simulation of gamma spectra in CROCUS using MCNP6.2 MCNP6.2 offers the possibility to simulate coupled neutron photon transport with pulse height estimation tallies for detectors. These simulations herein include only prompt 4 https://doi.org/10.1051/epjconf/202125304022 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 1 1 Fig. 7. Top view of the Serpent 2 model of CROCUS with LEAF at mid- core height. 1) BGO detector, 2) CeBr3 detector, 3) Compensated ionization chamber, 4) Power monitors, 5) UO2 fuel lattice, 6) Umet fuel lattice. Fig. 9. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for sub-critical and critical conﬁgurations. Fig. 10. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for 800 mm and the shutdown state. TABLE I OVERVIEW OF THE CROCUS CONFIGURATIONS USED FOR THE GAMMA SPECTROSCOPY EXPERIMENTS. TABLE I OVERVIEW OF THE CROCUS CONFIGURATIONS USED FOR THE GAMMA SPECTROSCOPY EXPERIMENTS. Conﬁguration Water level Fission power estimate [38] mm mW Shutdown Empty 0 H3 500.0 1.2 Sub-critical: 800.0 3.3 850.0 3.9 900.0 6.1 925.0 9.4 Critical: 963.3 up to 100k C. Experimental setup: LEAF in CROCUS Preliminary identiﬁed peaks are indicated in the plot using the data from Table II. Fig. 9. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for sub-critical and critical conﬁgurations. Fig. 9. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for sub-critical and critical conﬁgurations. 1 1 Fig. 7. Top view of the Serpent 2 model of CROCUS with LEAF at mid- core height. 1) BGO detector, 2) CeBr3 detector, 3) Compensated ionization chamber, 4) Power monitors, 5) UO2 fuel lattice, 6) Umet fuel lattice. 1 1 Fig. 7. Top view of the Serpent 2 model of CROCUS with LEAF at mid- core height. 1) BGO detector, 2) CeBr3 detector, 3) Compensated ionization chamber, 4) Power monitors, 5) UO2 fuel lattice, 6) Umet fuel lattice. Fig. 9. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for sub-critical and critical conﬁgurations. Fig. 10. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for 800 mm and the shutdown state. Preliminary identiﬁed peaks are indicated in the plot using the data from Table II. Fig. 7. Top view of the Serpent 2 model of CROCUS with LEAF at mid- core height. 1) BGO detector, 2) CeBr3 detector, 3) Compensated ionization chamber, 4) Power monitors, 5) UO2 fuel lattice, 6) Umet fuel lattice. Fig. 10. Measured in-core gamma spectra using a CeBr3 detector in the control rod position for 800 mm and the shutdown state. Preliminary identiﬁed peaks are indicated in the plot using the data from Table II. CROCUS core CeBr3 91 CeBr3 92 CROCUS cavity Control Room DSA 420 DSA 419 EPFL/PSI Pulse Acquisition System BGO 93 BGO 94 Pulse signal High Voltage Fig. 8. Schematic of the acquisition layout for the gamma spectroscopy experiment in CROCUS using LEAF. Control Room A. CeBr3: First recorded in-core gamma spectra In Figure 9 we display CeBr3 spectra acquired at different reactor conﬁgurations, ranging from shutdown to criticality. We most strikingly observe the expected exponential shape of the spectrum from ﬁssion with several resolved peaks from speciﬁc decays on top. We note a gain shift in the spectrum to higher energies with higher count rate, an effect often found in PMTs at high event rates [40]. The spectra notably all exhibit a cut-off at around 3.3 MeV. Using NIST standard data on photon mass attenuation lengths [41] and the density mentioned before, we ﬁnd that photons of this energy have a mean free path of 3.8 cm – a value consistent with the detector geometry. Fig. 8. Schematic of the acquisition layout for the gamma spectroscopy experiment in CROCUS using LEAF. TABLE I OVERVIEW OF THE CROCUS CONFIGURATIONS USED FOR THE GAMMA SPECTROSCOPY EXPERIMENTS. TABLE II TABLE II OVERVIEW OF EMISSIONS USED FOR PEAK IDENTIFICATION. ENERGIES ROUNDED TO THE CLOSEST KEV. THE HALF LIFE IS INDICATED FOR BETA DELAYED GAMMA EMISSIONS. We most notably identify across both in-core and ex-vessel spectra: Reaction Energy (keV) Half life Reference Annihilation 511 prompt [43] 140Ba 537 12.7 d [44] 137Cs 662 30.5 a [44] 134Cs 795 2.1 a [44] 140La 816 1.7 d [44] 140La 1596 1.7 d [44] 27Al(n,γ)28Si 1779 2.3 min [45] 1H(n,γ) 2223 prompt [43] 140La 2521 1.68 d [44] 27Al(n,α)24Na 2754 15.0 h [44] 16O(n,p)16N 6140 7.1 s [43] 27Al(n,γ) 7724 prompt [46] 14N(n,γ) 10829 prompt [43] • Several unidentiﬁed lines below 500 keV that could cor- respond to a range of ﬁssion products but we refrain for attempting an identiﬁcation in this work. • Long lived ﬁssion products relevant for waste and fallout quantiﬁcation [47] such as 137Cs, 134Cs, 140La, and 140Ba. • Radiative neutron capture and activation, mostly from H(n, γ) and potentially the surrounding aluminum struc- tures. In the high energy spectra of the BGO we fur- thermore observe the 6.1 MeV and 10.8 MeV 16N decay lines. We emphasize here that the identiﬁed peaks using the outlined methods (MCNP correlation and databases) is preliminary. Future work will focus on quantifying the decay behavior of the peaks to establish a more solid causal link. An unresolved inconsistency is for example the line at ∼4.4 MeV as seen in the BGO spectra in Figures 11 and 13. According to MCNP, it is exactly twice the energy of the hydrogen capture gamma, yet it is found in both spectra with and without the PuBe neutron source. We nonetheless can state that the MCNP simulations qualitatively agree with the measurements for other expected prompt processes, such as annihilation and capture in hydrogen, and disagree when it comes to ﬁssion product decay such as 140La. This highlights the need and opportunity for validation taking delayed gammas and signal smearing into account. without source we attempted to distinguish neutron induced prompt lines from longer lived activation and ﬁssion products. Indeed, we ﬁnd a general increase in spectral baseline due to ﬁssion gammas – and also a new line e.g. at around 2.2 MeV and 6.1 MeV. In the next section we discuss the gamma line identiﬁcation and implications. arbitrarily normalized to allow for qualitative comparison of the shapes. arbitrarily normalized to allow for qualitative comparison of the shapes. B. BGO: Coarse ex-vessel spectra up to 10 MeV In Figure 11 we display BGO spectra acquired at 500 mm water level with and without the PuBe start-up neutron source of CROCUS below the core [42]. At higher water levels the spectra showed only an exponential shape induced by ﬁssion with no visually identiﬁable peaks, similar to the spectrum of the CeBr3 at criticality. By comparing spectra with and 5 5 https://doi.org/10.1051/epjconf/202125304022 EPJ Web of Conferences 253, 04022 (2021) ANIMMA 2021 Fig. 11. Measured gamma spectra using a BGO detector in the core vessel periphery for the H3 sub-critical state, with and without the start-up neutron b l h d iﬁd k i di d i h l Fig. 12. Comparison of experimental in-core gamma spectra using a CeBr3 detector in the control rod position to the MCNP pulse height tally (F8). Preliminary identiﬁed peaks are indicated in the plot. Fig. 12. Comparison of experimental in-core gamma spectra using a CeBr3 detector in the control rod position to the MCNP pulse height tally (F8). Preliminary identiﬁed peaks are indicated in the plot. Fig. 11. Measured gamma spectra using a BGO detector in the core vessel periphery for the H3 sub-critical state, with and without the start-up neutron source below the core. Identiﬁed peaks are indicated in the plot. Fig. 12. Comparison of experimental in-core gamma spectra using a CeBr3 detector in the control rod position to the MCNP pulse height tally (F8). Preliminary identiﬁed peaks are indicated in the plot. Fig. 11. Measured gamma spectra using a BGO detector in the core vessel periphery for the H3 sub-critical state, with and without the start-up neutron source below the core. Identiﬁed peaks are indicated in the plot. V. CONCLUSION [7] T. Kaltiaisenaho, “Implementing a photon physics model in serpent 2; fotonifysiikkamallin kehitt¨aminen serpent 2-koodiin,” Master’s thesis, Aalto University, 2016. In this work we present, to the best of the authors’ knowledge, for the ﬁrst time gamma spectra from a central region of a nuclear reactor. By using a modern scintillator material (CeBr3) with comparatively low FWHM, we were able to distinguish peaks above the ﬁssion spectrum. We also present the measurement of ex-core gamma ray spectra up to 10 MeV using BGO detectors that also show several peaks. [8] M. C. White, “Development and implementation of photonuclear cross- section data for mutually coupled neutron-photon transport calculations in the monte carlo n-particle (MCNP) radiation transport code,” Los Alamos National Lab. (LANL), Tech. Rep., jul 2000. [9] O. Pakari, V. Lamirand, B. Vandereydt, F. Vitullo, M. Hursin, C. Kong, and A. Pautz, “Design and simulation of gamma spectrometry experi- ments in the CROCUS reactor,” in ANIMMA, Portoroz, Slovenia, 2019. [10] Y. Nakashima, S. Minato, M. Kawano, T. Tsujimoto, and K. Kat- surayama, “Gamma-ray energy spectra observed around a nuclear re- actor,” Journal of Radiation Research, vol. 12, no. 3-4, pp. 138–147, 1971. We introduced the new gamma spectroscopy system for the CROCUS zero power reactor called LEAF. It consists of four scintillation based detectors, two CeBr3 and two large BGO crystals, each with different speciﬁcations allowing for a wide range of experimental reactor applications. 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Maienschein, R. Peelle, W. Zobel, and T. Love, “Gamma rays associated with ﬁssion,” Oak Ridge National Laboratory, Tech. Rep., oct 1958. Fig. 13. Comparison of experimental BGO gamma spectrum to the MCNP pulse height tally (F8). Preliminary identiﬁed peaks are indicated in the plot. [6] S. Oberstedt, R. Billnert, A. Gatera, W. Geerts, P. Halipr´e, F.-J. Hamb- sch, M. Lebois, A. Oberstedt, P. Marini, M. Vidali, and J. Wilson, “Prompt ﬁssion γ-ray spectra characteristics - a ﬁrst summary,” Physics Procedia, vol. 64, pp. 83–90, 2015. IV. DISCUSSION Peak identiﬁcation given the here presented data is challenging. To approach this problem we herein use two methods: Firstly, by accessing various decay and neutron capture databases we chose a list of likely causes based on expert judgement, listed in Table II. Secondly, we simulated the prompt gamma transport in MCNP6.2 in the respective detector volumes and overlaid the F8 pulse height tally over the experimental data - as shown in Figures 12 and 13 for the CeBr3 and BGO, respectively. Note that the spectra are The results are overall promising: ﬁssion products could be directly measured in-core, opening the possibility to track their production on-line as well as validate burnup calculations of CROCUS [48]. 6 EPJ Web of Conferences 253, 04022 (2021) https://doi.org/10.1051/epjconf/202125304022 ANIMMA 2021 Fig. 13. Comparison of experimental BGO gamma spectrum to the MCNP pulse height tally (F8). Preliminary identiﬁed peaks are indicated in the plot. investigations into gamma ray spectra in- and ex-core with emphasis on delayed process identiﬁcation and quantiﬁcation. V. 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https://openalex.org/W2121084908	https://research.vu.nl/ws/files/3140846/294226.pdf	English	null	Nicotine exposure during adolescence alters the rules for prefrontal cortical synaptic plasticity during adulthood	Frontiers in synaptic neuroscience	2,012	cc-by	11,212	Nicotine exposure during adolescence alters the rules for prefrontal cortical synaptic plasticity during adulthood Goriounova, N.A.; Mansvelder, H.D. Nicotine exposure during adolescence alters the rules for prefrontal cortical synaptic plasticity during adulthood Goriounova, N.A.; Mansvelder, H.D. Link to publication in VU Research Portal Link to publication in VU Research Portal citation for published version (APA) Goriounova, N. A., & Mansvelder, H. D. (2012). Nicotine exposure during adolescence alters the rules for prefrontal cortical synaptic plasticity during adulthood. 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Oct. 2024 Correspondence: Correspondence: Huibert D. Mansvelder, Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University Amsterdam, De Boelelaan 1085, Room C-440, 1081 HV Amsterdam, Netherlands. e-mail: huibert.mansvelder@ cncr.vu.nl Keywords: adolescence, nicotine, prefrontal cortex, STDP, mGluR, nAChR, cognitive behavior The impulsive, peer-inﬂuenced nature of adolescent choices sets the stage for experimenting with drugs of abuse. Since nico- tine is one of the most socially accepted drugs in our society, the ﬁrst choice usually falls on tobacco smoking. According to a recent study conducted in 41 countries in Europe and North America, 19% of 15-year olds smoke at least once a week and 30% report experimenting with cigarettes before the age of 14 (Currie et al., 2008). Serious health risks of smoking are well- known: smoking leads to millions of premature deaths worldwide and tobacco smoking has been marked as an epidemic disease (Peto et al., 1999). Nicotine is also a psychoactive and addictive substance that directly acts on brain areas involved in emotional and cognitive processing. Early exposure to nicotine during ado- lescence may disturb the normal course of brain maturation and have lasting consequences for cognitive ability, mental health, and even personality (Brown et al., 1996; Choi et al., 1997; Richards et al., 2003; Brook et al., 2004; Deas, 2006). In humans, the PFC shows delayed development with respect to other corti- cal areas during adolescence with delayed thinning of cortical grey matter, most likely reﬂecting ﬁne-tuning of synaptic con- tacts (Gogtay et al., 2004; Sowell et al., 2004; Casey et al., 2005). Rearrangement of local inhibitory inputs and decreases in synap- tic densities and branch points of excitatory connections between pyramidal neurons occur within the developing PFC (Woo et al., 1997; Cruz et al., 2003). Spike-timing-dependent modiﬁcations are likely to be important for cortical development, map plas- ticity, and functioning of neural networks: correlated inputs lead to strengthening of connections (LTP) while uncorrelated inputs lead to weakening (LTD) and pruning of unused synapses (Bi and Poo, 2001; Song and Abbott, 2001; Feldman and Brecht, 2005). Here, we highlight recent ﬁndings that start to uncover Adolescence is an important developmental period when a child has to make a transition to an independent status of adult. Such transition demands an ability to take risks and a taste for novelty but also emergence of self-control and more adult decision- making strategies. SYNAPTIC NEUROSCIENCE SYNAPTIC NEUROSCIENCE REVIEW ARTICLE published: 02 August 2012 doi: 10.3389/fnsyn.2012.00003 Nicotine exposure during adolescence alters the rules for prefrontal cortical synaptic plasticity during adulthood Natalia A. Goriounova and Huibert D. Mansvelder* Natalia A. Goriounova and Huibert D. Mansvelder* ment of Integrative Neurophysiology, CNCR, Neuroscience Campus Amsterdam, VU University, Amsterdam, Netherlands ology, CNCR, Neuroscience Campus Amsterdam, VU University, Amsterdam, Netherlands Edited by: Darwin K. Berg, University of California, San Diego, USA Reviewed by: William N. Green, University of Chicago, USA Peter B. Sargent, University of California, San Francisco, USA Correspondence: Huibert D. Mansvelder, Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University Amsterdam, De Boelelaan 1085, Room C-440, 1081 HV Amsterdam, Netherlands. e-mail: huibert.mansvelder@ cncr.vu.nl Edited by: Darwin K. Berg, University of California, San Diego, USA Reviewed by: William N. Green, University of Chicago, USA Peter B. Sargent, University of California, San Francisco, USA The majority of adolescents report to have smoked a cigarette at least once. Adolescence is a critical period of brain development during which maturation of areas involved in cognitive functioning, such as the medial prefrontal cortex (mPFC), is still ongoing. Tobacco smoking during this age may compromise the normal course of prefrontal development and lead to cognitive impairments in later life. In addition, adolescent smokers suffer from attention deﬁcits, which progress with the years of smoking. Recent studies in rodents reveal the molecular changes induced by adolescent nicotine exposure that alter the functioning of synapses in the PFC and underlie the lasting effects on cognitive function. In particular, the expression and function of metabotropic glutamate receptors (mGluRs) are changed and this has an impact on short- and long-term plasticity of glutamatergic synapses in the PFC and ultimately on the attention performance. Here, we review and discuss these recent ﬁndings. Take down policy Take down policy f you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy f you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate your claim. y this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim E-mail address: vuresearchportal.ub@vu.nl E-mail address: vuresearchportal.ub@vu.nl Download date: 24. Oct. 2024 REVIEW ARTICLE published: 02 August 2012 doi: 10.3389/fnsyn.2012.00003 Edited by: Edited by: Darwin K. Berg, University of California, San Diego, USA Reviewed by: William N. Green, University of Chicago, USA Peter B. Sargent, University of California San Francisco USA Keywords: adolescence, nicotine, prefrontal cortex, STDP, mGluR, nAChR, cognitive behavior IMMEDIATE EFFECTS OF NICOTINE EXPOSURE Somatostatin-positive cells target distal dendritic regions (Kawaguchi and Kondo, 2002; Silberberg and Markram, 2007) and can mediate disynaptic inhibition between pyramidal neu- rons (Silberberg and Markram, 2007; Kapfer et al., 2007). Regular-spiking and somatostatin-positive cells in PFC layer II- III and V are positive for nAChRs, suggesting that nAChRs play an important role in modulating feedback inhibition among pyramidal neurons in these layers (Poorthuis et al., 2012). Nicotine activates nicotinic acetylcholine receptors (nAChR), which take part in cholinergic signalling. Twelve genes have been identiﬁed encoding neuronal nicotinic receptors (for review see Le Novere et al., 2002; Millar and Gotti, 2009). In the central nervous system 9 α-subunits (α2–α10) and 3 β-type subunits (β2–β4) are expressed. These subunits assemble in different sto- ichiometries to form pentameric channels, and subunit com- positions of nAChRs vary depending on the brain region (for review see Grady et al., 2002; Le Novere et al., 2002; McGehee, 2002; Alkondon and Albuquerque, 2004; Wonnacott et al., 2005; Mineur and Picciotto, 2008; Millar and Gotti, 2009). Nicotinic AChRs are cation selective channels that permit the ﬂow of Na+, K+, and Ca2+ across the membrane, which leads to depolarizing currents and activate neurons (McGehee and Role, 1995; Millar and Gotti, 2009). Finally, we showed that the network activity in the PFC in response to bath application of ACh is dominated by β2 nAChRs activation. Receptors of this subtype are expressed by pyramidal neurons in layer VI, glutamatergic inputs to layer V and VI and by interneurons in all layers of the PFC. In summary, β2 contain- ing nAChRs stimulate both excitatory and inhibitory neurons in deep layers, while in layer II/III only interneurons are activated (Poorthuis et al., 2012). Thus, the net result of nicotinic receptor stimulation is the increased inhibitory transmission in superﬁcial PFC layers, whereas in deep layers it also leads to activation of pyramidal neurons. This pattern of activation alters the informa- tion processing in prefrontal networks and can directly alter rules for plasticity (Couey et al., 2007). Modulation of PFC activity by nAChRs will depend on which cell types express nAChRs and what subunits they are made of. In the PFC, nAChR expression is found across all layers (Gioanni et al., 1999; Poorthuis et al., 2012). Activation of nAChRs can alter pyramidal neuron activity by two mechanisms: presynaptically enhancing glutamatergic inputs or by activating postsynaptic receptors directly (Poorthuis et al., 2012). IMMEDIATE EFFECTS OF NICOTINE EXPOSURE Recently we measured nAChR activation by making whole-cell recordings from PFC pyramidal neurons in the different layers and used wild-type, β2-null, or α7-null mice as well as pharmacological tools to determine the nAChR subunits involved. We showed that PFC pyramidal neurons across cortical layers show a differential pat- tern of postsynaptic nAChR activation: layer II/III pyramidal neurons do not contain nAChRs, layer V pyramidal neurons con- tain α7 nAChRs, and layer VI pyramidal neurons are modulated by β2∗nicotinic receptors (Poorthuis et al., 2012). Also presynap- tic glutamatergic inputs can be modulated by nicotine (Gioanni et al., 1999; Lambe et al., 2003; Couey et al., 2007; Poorthuis et al., 2012). We found that this presynaptic regulation is speciﬁc to layer V, only moderately present in layer VI and not present in layer II-III (Poorthuis et al., 2012). One of the forms of long-term plasticity, spike-timing depen- dent plasticity (STDP), is based on Hebb’s postulate often sum- marized as “Cells that ﬁre together, wire together” (Hebb, 1949). It attempts to explain associative learning, in which nearly simulta- neous activation of cells increases the strength of synaptic contacts between those cells. This type of plasticity is thought to play an important role in cortical development during critical stages and underlie some forms of learning (Feldman et al., 1999; Feldman and Brecht, 2005; Letzkus et al., 2007). In PFC, STDP can be directly modulated by nicotine (Couey et al., 2007; Goriounova and Mansvelder, 2012). We showed that in adolescent rats, STDP in layer V pyramidal neurons is strongly reduced if nicotine is applied to PFC slices during induction protocol (Goriounova and Mansvelder, 2012). In PFC layer V, the increase in inhi- bition dominates the effects of nicotine on synaptic plasticity (Couey et al., 2007). Nicotine-stimulated inhibition reduces den- dritic calcium signaling and renders postsynaptic activity in layer V pyramidal cells insufﬁcient to induce potentiation (Couey et al., 2007). In layer II/III, the layer where the synaptic inputs were stimulated to induce STDP, nAChR activation results predomi- nantly in activation of interneurons (Poorthuis et al., 2012). Thus, nicotine-induced increase in inhibitory transmission can explain the decrease in spike-timing dependent potentiation in adolescent rats. y In addition to direct activation of PFC pyramidal neu- rons by nAChRs, PFC GABAergic interneurons across all lay- ers are also directly activated by nAChR stimulation (Poorthuis et al., 2012). IMMEDIATE EFFECTS OF NICOTINE EXPOSURE Increased inhibition through activation of nAChRs expressed by interneurons has been found in many different brain regions (Jones and Yakel, 1997; Xiang et al., 1998; McQuiston and Madison, 1999; Alkondon et al., 2000; Ji and Dani, 2000; Mansvelder et al., 2002; Gulledge et al., 2007). Interneurons form a highly diverse group of cells with distinct roles in cortical computation (Kawaguchi, 1993; Markram et al., 2004). Fast- spiking cells target the perisomatic region of pyramidal neurons (Kawaguchi and Kubota, 1997; Kawaguchi and Kondo, 2002) and are therefore thought to be involved in regulating the activity win- dow of pyramidal neurons. Feedforward inhibition in the PFC plays an important role in the integration of hippocampal inputs, Correspondence: Also, brain development is not complete by adolescence, especially maturation of areas involved in cogni- tive functioning, such as the medial prefrontal cortex (mPFC), is still ongoing. Brain development continues throughout ado- lescence, though the speed and timing of maturation varies for different brain areas (Gogtay et al., 2004). Subcortical limbic structures important for emotional processing, such as hypotha- lamus, midbrain dopamine areas, nucleus accumbens, dorsal, and ventral striatum and amygdala, experience a major develop- mental boost around the onset of puberty (Sowell et al., 2003; Casey et al., 2005). Their maturation is important for social and sexual behaviors and is triggered by pubertal hormones. In contrast, development of frontal cortical areas of the brain, responsible for cognitive control over behavior, depends on age and experience and continues throughout adolescence and into adulthood (Sowell et al., 2003; Giedd, 2004). Thus, during ado- lescence emotional drive has already become very strong while cognitive self-control and adult decision-making strategies still are developing. Thereby, brain development may be respon- sible for characteristic adolescent traits—uncontrollable mood swings, impulsivity, risk-taking, and peer-directed social inter- actions (Orr and Ingersoll, 1995; Spear, 2000; Galvan et al., 2007). Although indispensable for transition from child to inde- pendent status of adult, these traits hold hazards. Indeed, risk- taking behavior, so typical for adolescents, is associated with high rates of mortality and morbidity among young people (Grunbaum et al., 2004). August 2012 \| Volume 4 \| Article 3 \| 1 Frontiers in Synaptic Neuroscience www.frontiersin.org Adolescent nicotine alters STDP Goriounova and Mansvelder which enter the PFC through superﬁcial layers (Jay and Witter, 1991; Tierney et al., 2004). Fast-spiking cells in PFC layer II-III contain α7 nAChRs, as do as about half of the fast-spiking cells in layer V (Poorthuis et al., 2012). nAChR activation on fast- spiking interneurons in PFC layer II/III may alter processing of hippocampal inputs. causal relations between nicotine exposure during adolescence and cognitive deﬁcits in later life, with an emphasis on synaptic adaptations and altered rules for synaptic plasticity in prefrontal networks. Frontiers in Synaptic Neuroscience UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION Naïve rats show an age-related decrease in 3H-epibatidine labeled high-afﬁnity nicotinic receptors in the mPFC, but not in occipi- tal cortex. Adolescent, but not adult nicotine exposure increases 3H-Epi-binding of mPFC receptors on the ﬁrst day of abstinence following 10 days of nicotine injections. This is paralleled by an mPFC-speciﬁc increase in expression of nAChRs containing α4 and β2 (but not α5) subunits or high-afﬁnity nAChRs. The increased expression of high-afﬁnity nAChRs in adolescents is accompanied by an increase in nicotine-stimulated GABAergic synaptic transmission in the mPFC (Counotte et al., 2012). How is spike-timing-dependent potentiation affected by nicotine exposure during adolescence? In addition to an upreg- ulation of nAChRs, we recently found in a large-scale iTRAQ- based proteomics screen of synaptic protein levels in the PFC that metabotropic glutamatergic receptors type 2 (mGluR2) are signiﬁcantly upregulated during adolescent nicotine exposure (Figure 1) (Counotte et al., 2011). This short-term upregulation is followed by a long-term decrease in mGluR2 levels 5 weeks after the nicotine exposure during adolescence. As will be discussed later, this pattern of mGluR2 expression leads to opposing effects on glutamatergic transmission and plasticity in PFC. y p ( ) One of the ﬁrst and most common cellular adaptations fol- lowing chronic nicotine exposure is the upregulation of nicotinic receptor levels (Dani and Bertrand, 2007). Especially α4β2 type of nAChRs appears to be selectively upregulated via posttrans- lational mechanisms (Miwa et al., 2011). The upregulation of α4β2 nAChRs by chronic nicotine treatment has been replicated many times in numerous systems—transfected cell lines, neu- rons in culture, brain slices, and smokers’ brains (Wonnacott, 1990; Fu et al., 2009; Lester et al., 2009; Marks et al., 2011; Miwa et al., 2011). Upregulation is not accompanied by an increase in nAChR subunit mRNA (Marks et al., 1992), instead it leads to increased nAChR protein levels resulting from posttranslational mechanisms (Govind et al., 2009; Marks et al., 2011). According to one view on nAChR upregultation, nicotine acts intracellu- larly as a selective pharmacological chaperone of acetylcholine receptor (Lester et al., 2009). It stabilizes nAChRs during assem- bly and maturation and this stabilization is most pronounced for the high-afﬁnity nAChR containing α4β2 subunits. Other possible mechanisms underlying nAChR upregulation can result from increased cell surface turnover, increased receptor trafﬁck- ing to the surface, changes in subunit stoichiometry or nAChR conformational changes (Govind et al., 2009). UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION Adolescents may be more vulnerable to nicotine addiction due to greater positive effects nicotine has on adolescents than adults, whereas the negative effects associated with nicotine, such as August 2012 \| Volume 4 \| Article 3 \| 2 www.frontiersin.org Goriounova and Mansvelder Adolescent nicotine alters STDP 2012), the functional consequence of α4β2 nAChR upregulation on interneurons in layer II/III will be an increased inhibitory transmission in superﬁcial PFC layers. In the deep layers of the PFC, β2 subunits are expressed by both interneurons as well as layer VI pyramidal neurons and excitatory inputs to layer V pyramidal neurons. An upregulation of these receptors will lead to a combined increase in activation of pyramidal neurons and interneurons. Since β2-containing nAChRs in the mPFC control attention performance (Guillem et al., 2011) this may have func- tional implications for maturation and function of the prefrontal network. During chronic nicotine exposure of the adolescent PFC, the pattern of activity in prefrontal network may gradually shift toward activation of excitatory neurons in deep layers in the context of increased overall inhibition. This may affect plasticity and reﬁnement of cortical connections (Couey et al., 2007), even though GABAergic transmission by itself was not affected directly following nicotine treatment during adolescence (Counotte et al., 2012). Still, we ﬁnd that also directly following nicotine expo- sure during adolescence in vivo, STDP of glutamatergic synapse strength is blocked (Goriounova and Mansvelder, 2012). Since in these experiments assessing STDP immediately following nico- tine treatment during adolescence nicotine was not applied, this can not explain the reduced STDP we observed. withdrawal are smaller in adolescents (O’Dell, 2009). Nicotine administration during, but not following adolescence, has long- lasting effects on cognitive, addictive, and emotional behavior in rats (Adriani et al., 2003; Iniguez et al., 2008; Counotte et al., 2009, 2011). Furthermore, adolescent animals are more sensitive to nicotine conditioned place preference than adults and show this at lower nicotine doses (Vastola et al., 2002; Belluzzi et al., 2004; Shram et al., 2006; Brielmaier et al., 2007; Kota et al., 2009). Adolescent nicotine exposure leads to acute and longer-lasting changes in nAChR binding (Abreu-Villaca et al., 2003; Doura et al., 2008) and function (Kota et al., 2009) in brain regions such as cortex and striatum. We recently found that the adolescent rodent brain is more sensitive to nicotinic receptor upregulation in the medial PFC (mPFC) than adults (Counotte et al., 2012). Frontiers in Synaptic Neuroscience UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION adolescent nic exposure ado c ex ad nicc e ni Saline nt osuree re re re Short-term effects of nicotine Long-term effects of nicotine Spike timing-dependent potentiation Short-term depression Attention nAChR mGluR2 inhibition inhibition inhibition inhibition excitation excitation excitation excitation excitation excitation excitation excitation inhibition inhibition inhibition inhibition inhibition inhibition excitation excitation excitation excitation Spike timing-dependent potentiation inhibition inhibition tt Short-term effects of nicotine Long-term effects of nicotine inhibition inhibition Spike timing-dependent potentiation + mGluR2 agonist = Spike timing-dependent potentiation Attention = Saline excitation excitation excitation excitation inhibition inhibition inhibition inhibition + mGluR2 agonist = = Saline Spike timing-dependent potentiation inhibition inhibition inhibition inhibition inhibition inhibition show the effects of mGluR2 agonists and antagonists in saline and nicotine-exposed animals. Applying mGluR2 antagonists to the adult mPFC reduces depression of glutamatergic synapses and reduces attention performance of the animal. Providing mGluR2 agonists to the mPFC of adult rats that were exposed to nicotine during adolescence normalizes synaptic depression and spike-timing-dependent potentiation of glutamatergic synapses and improves attention performance. FIGURE 1 \| Schematic representation of the short-term and long-term adaptations in PFC neuronal networks caused by nicotine exposure during adolescence. The upper panels show the sequence of adaptations in nAChR and mGluR2 protein levels and the resulting changes in inhibition and excitation and attention behavior from control conditions (saline) to nicotine exposure during adolescence (short-term effects of nicotine) and 5 weeks following nicotine exposure (long-term effects of nicotine). The lower panels FIGURE 1 \| Schematic representation of the short-term and long-term adaptations in PFC neuronal networks caused by nicotine exposure during adolescence. The upper panels show the sequence of adaptations in nAChR and mGluR2 protein levels and the resulting changes in inhibition and excitation and attention behavior from control conditions (saline) to nicotine exposure during adolescence (short-term effects of nicotine) and 5 weeks following nicotine exposure (long-term effects of nicotine). The lower panels periods of development and adulthood (Schochet et al., 2005, 2008; Polesskaya et al., 2007). The adolescent PFC shows nico- tine response in gene regulation involved in vesicle release, signal transduction, cytoskeleton dynamics, and transcription, suggest- ing the role of chronic nicotine exposure in initiating long-term structural and functional adaptations (Polesskaya et al., 2007). The expression of key molecules involved in plasticity is also altered in the PFC by adolescent nicotine exposure. UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION Acute nicotine induces increases in the expression of the dendritically targeted dendrin mRNA in PFC of adolescent but not adult animals. Dendrin is an important component of cytoskeletal modiﬁcations at the synapse and therefore can lead to unique plasticity changes may have functional implications for cognitive processing and maturation of prefrontal network. UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION All these pro- posed mechanisms have two features in common: they are post- translational and involve upregulation of high-afﬁnity nAChR containing α4β2 subunits. Indeed, we found that speciﬁcally high-afﬁnity nicotinic receptors containing the α4 and β2 sub- units were upregulated in the adolescent PFC shortly following nicotine exposure. This upregulation was paralleled by a func- tional elevation in nicotine-stimulated GABAergic transmission, indicating that functional surface nAChRs are upregulated as well (Counotte et al., 2012). Metabotropic GluR2s are located presynaptically on gluta- matergic synapses and their activation reduces the probability of glutamate release. Thereby, an upregulation of mGluR2 lev- els diminishes activity of excitatory glutamatergic synapses in the PFC. Thus, increases in functional nAChR on inhibitory neu- rons and increased nicotine-stimulated excitation in deep layers of the PFC may be counteracted by reduced excitatory synap- tic activity mediated by increased mGluR2 activity. Blocking mGluR2s with MPPG restored spike-timing-dependent potentia- tion following nicotine exposure during adolescence back to levels observed in animals that received saline treatment during ado- lescence (Goriounova and Mansvelder, 2012). This suggests that the upregulation of presynaptic mGluR2s after nicotine exposure during adolescence alters the rules for STDP in PFC networks. In summary, the net result of nicotine in the short-term in adolescent mPFC that is chronically exposed to nicotine, very likely amounts to persistently enhanced levels of inhibition across all cortical layers possibly combined with some increase in tha- lamocortical glutamate release by presynaptic (or preterminal) nAChRs in deep layers. The latter effect, however, is most likely counteracted by elevated presynaptic mGluR2 levels. UPREGULATION OF nAChR AND SYNAPTIC mGluR EXPRESSION This general increase in inhibition plays a role in the plasticity and reﬁnement of cortical connections (reduced STDP in nicotine) and thus Given that pyramidal neurons and excitatory projections in layer II/III of the PFC do not express nAChRs (Poorthuis et al., August 2012 \| Volume 4 \| Article 3 \| 3 Frontiers in Synaptic Neuroscience www.frontiersin.org Adolescent nicotine alters STDP Goriounova and Mansvelder adolescent nic exposure ado c ex ad nicc e ni Saline nt osuree re re re Short-term effects of nicotine Long-term effects of nicotine Spike timing-dependent potentiation Short-term depression Attention + mGluR2 antagonist = + mGluR2 agonist = nAChR mGluR2 + + Spike timing-dependent potentiation Attention Spike timing-dependent potentiation Short-term depression Attention Long-term effects of nicotine = Saline inhibition inhibition inhibition inhibition excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation excitation inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition inhibition excitation excitation excitation excitation Spike timing-dependent potentiation inhibition inhibition FIGURE 1 \| Schematic representation of the short-term and long-term adaptations in PFC neuronal networks caused by nicotine exposure during adolescence. The upper panels show the sequence of adaptations in nAChR and mGluR2 protein levels and the resulting changes in inhibition and excitation and attention behavior from control conditions (saline) to nicotine exposure during adolescence (short-term effects of nicotine) and 5 weeks following nicotine exposure (long-term effects of nicotine). The lower panels show the effects of mGluR2 agonists and antagonists in saline and nicotine-exposed animals. Applying mGluR2 antagonists to the adult mPFC reduces depression of glutamatergic synapses and reduces attention performance of the animal. Providing mGluR2 agonists to the mPFC of adult rats that were exposed to nicotine during adolescence normalizes synaptic depression and spike-timing-dependent potentiation of glutamatergic synapses and improves attention performance. LONG-TERM CONSEQUENCES Smoking during adolescence is associated with disturbances in working memory and attention as well as reduced PFC activation (Jacobsen et al., 2005, 2007; Musso et al., 2007). Smoking is also a prospective risk factor for impaired cognitive function in later life; heavy smoking predicts incident cognitive impairment and decline (Cervilla et al., 2000; Kalmijn et al., 2002; Richards et al., 2003). In animal studies exposure during adolescence induces stronger changes in gene expression in the PFC than during other August 2012 \| Volume 4 \| Article 3 \| 4 Frontiers in Synaptic Neuroscience www.frontiersin.org www.frontiersin.org Adolescent nicotine alters STDP Goriounova and Mansvelder in the adolescent PFC (Schochet et al., 2008). Lasting synaptic adaptations involve activation of intracellular signaling pathways and enzymes such as extracellular regulated protein kinase (ERK) and cAMP response element binding protein (CREB). Speciﬁcally in the PFC, increases in phosphorylation of both these enzymes were found after repeated nicotine exposure (Brunzell et al., 2003). Also changes in macromolecular constituents indicative of cell loss (reduced DNA) and altered cell size (protein/DNA ratio) can be seen in cortical regions of rodents after adolescent nico- tine treatment (Trauth et al., 2000). Further, repeated nicotine exposure also alters the structure of neurons in mPFC: it increases both dendritic length and spine density (Brown and Kolb, 2001). Long-term changes have been observed in dendritic morphology of speciﬁc subpopulations of pyramidal neurons and these struc- tural changes depended on the age of drug exposure (Bergstrom et al., 2008). nicotine self-administration (Liechti et al., 2007), and they play an important role in the development of drug dependence and the expression of the negative affective state observed during with- drawal (Pilc et al., 2008). However, the role of group II mGlu receptors in withdrawal appears complex and most likely depends on changes in multiple brain areas. Although the sequence of events linking mGluR2 adaptations to nAChR activation is unknown, it seems that the reasons for its up- and down-regulation pattern after adolescent nicotine exposure may lie in its function. Metabotropic GluR2 recep- tors are located on presynaptic glutamatergic terminals where they are activated by glutamate spill over to inhibit glutamate release (Mateo and Porter, 2007). It was shown that activation of mGluR2s can also regulate release of other neurotransmitters: it can inhibit GABA release via a presynaptic mechanism (Bradley et al., 2000; Pilc et al., 2008). LONG-TERM CONSEQUENCES Given the inhibitory role of mGluR2 in neurotransmitter release, its function seems to counteract that of nAChR, which enhances both excitatory and inhibitory synap- tic transmission (Lambe et al., 2003; Couey et al., 2007; Poorthuis et al., 2012). The short-term effects of adolescent nicotine expo- sure most likely involve enhanced levels of inhibition in prefrontal network. Accordingly, we found an initial and transient upregu- lation of inhibitory mGluR2 receptor directly following nicotine exposure during adolescence (Counotte et al., 2011), which would contribute to the same effect. Also on the behavioral level, nicotine during adolescence leads to persisting deﬁcits. Adolescent, but not adult, nicotine treatment reduces accuracy of correct stimulus detection in a visuospatial attentional task, with an increase in premature and time-out responding that suggests impaired attention and lack of impulsive control which is part of normal adolescent matu- ration (Counotte et al., 2009). Similar nicotine-induced deﬁcits have been found in a serial pattern learning paradigm (Fountain et al., 2008). In a recent study, chronic nicotine exposure dur- ing adolescence produced long-lasting impairments in contextual learning that were observed during adulthood, whereas adult chronic nicotine exposure did not (Portugal et al., 2012). In general, factors that lead to enhanced excitation can cause alterations in mGluR2 transmission and cause cognitive deﬁcits (Melendez et al., 2004; Pozzi et al., 2011). Enhanced glutamate release in PFC was found to be associated with attention deﬁcit and loss of impulse control (Pozzi et al., 2011). MGluR2 agonists are effective in improving cognitive deﬁcits if enhanced gluta- mate release is caused by NMDA receptor antagonists (Pozzi et al., 2011). Furthermore, the important role of prefrontal mGluR2 sig- naling in cognition is stressed by its link to brain disorders such as depression and schizophrenia. Activation of this receptor has even been proposed as a novel treatment approach for these disorders (Gupta et al., 2005; Palucha and Pilc, 2005; Pilc et al., 2008; Conn et al., 2009). Thus, mGluR2 signalling seems to be a good candi- date for shaping cognitive behavior and its impairment leads to disturbances in cognitive function. Taken together, these studies in rodents show that nicotine exposure during adolescence induces signiﬁcant changes in gene expression, neuronal morphology, and behavior in PFC. Thus, nicotine does not only change cholinergic signalling by alter- ing nicotinic receptor levels in the adolescent PFC, but can also lead to secondary adaptations involving structural and functional changes in cognition. LONG-TERM CONSEQUENCES What are the changes that underlie the changes in cognitive performance? Frontiers in Synaptic Neuroscience CONCLUSION The prefrontal cortex, the brain area responsible for executive functions and attention performance, is one of the last brain areas to mature and is still in the process of developing dur- ing adolescence. This places the adolescent brain in a vulnerable state of imbalance, susceptible to the inﬂuence of psychoactive substances such as nicotine. In prefrontal networks nicotine mod- ulates information processing on multiple levels by activating and desensitizing nicotine receptors on different cell types and in this way affects cognition. The adolescent brain is particularly sensi- tive to the effects of nicotine. Studies in human subjects indicate that smoking during adolescence increases the risk of develop- ing psychiatric disorders and cognitive impairment in later life. In addition, adolescent smokers suffer from attention deﬁcits, which progress with the years of smoking. We recently found that glutamatergic synapses in the PFC show increased spike-timing-dependent LTP 5 weeks after nico- tine exposure during adolescence (Figure 1) (Goriounova and Mansvelder, 2012). This was not the case when animals were exposed to nicotine during adulthood, indicating that adoles- cence is a vulnerable period for these lasting changes to occur. The long-term effects on LTP 5 weeks after nicotine exposure during adolescence were opposite to the effects immediately following nicotine exposure during adolescence, where spike- timing-dependent LTP is supressed. Thus, nicotine exposure dur- ing adolescence has lasting effects on the mechanisms of STDP and persistent synaptic alterations that increase LTP. From studies in the rodent brain it is becoming clear that on the short-term, adolescent, but not adult, nicotine exposure increases the expression of nAChRs containing α4 and β2 sub- units in the mPFC, which leads to an increase in nicotine-induced GABAergic synaptic transmission. In addition, mGluR2 levels on presynaptic glutamatergic terminals in the PFC are increased, causing a reduction in glutamatergic synapse strength and reduc- ing STDP (Figure 1). Changes in nAChR levels are reversible: in the adult rodent brain, weeks after nicotine levels have subsided, nAChR levels in the PFC return to baseline levels. In contrast, at this stage, mGluR2 levels have reduced signiﬁcantly below base- line levels, thereby altering mGluR2 signaling during short-term plasticity, augmenting spike-timing-dependent potentiation and hampering attention performance. This reduction in mGluR2 signaling underlies the reduced attention performance observed in animals after nicotine exposure during adolescence (Counotte et al., 2011). Thereby, mGluR2 signaling could be a therapeutic target for alleviating attention and impulse control problems in later life. Frontiers in Synaptic Neuroscience LASTING SYNAPTIC ADAPTATIONS IN THE PFC This reduction i signaling underlies the reduced attention performanc in animals after nicotine exposure during adolescence et al., 2011). Thereby, mGluR2 signaling could be a t target for alleviating attention and impulse control p later life. New questions and opportunities arise from th ﬁndings. The long-term adaptations involving mG have profound implications for network functioning more complex levels of information processing. Wh steps that lead from nicotine exposure and nAChR in the adolescent brain to adaptations in synapti plasticity. Most likely, activation of mGluR2s affects presynaptic calcium channel function as was found in the calyx of Held, by direct electrophysiological recordings from presynaptic terminals (Takahashi et al., 1996). Agonists of metabotropic glutamate receptors (mGluRs) suppressed high voltage-activated P/Q-type calcium channels in the presynaptic terminal, thereby inhibiting transmitter release (Takahashi et al., 1996). Since presynaptic Ca2+ dynamics play a key role in short—term plasticity (Zucker and Regehr, 2002), decrease in Ca2+ current may explain mGluR-dependent modulation of STD. reducing mGluR2-dependent inhibition leads to increased LTP, while enhancing mGluR2 activation blocked LTP. Thus, immedi- ately following adolescent nicotine exposure, increased levels of mGluR2s may be responsible for reduced LTP induction, and 5 weeks following adolescent nicotine exposure, the lasting reduc- tion in mGluR2 signalling can explain the increased LTP in the adult mPFC. STDP depends on the precise timing of the synaptic input and the postsynaptic action potential and this temporal relationship resembles typical features of associative learning (Letzkus et al., 2007). Although STDP has not been directly linked to attention performance, the ability to associate goal-relevant information is crucial for any cognitive behavior. In nicotine-treated rats the same amount of pre- and postsynaptic activity leads to more synaptic potentiation. This may suggest that the PFC network would even associate irrelevant stimuli. mGluR dependent modulation of STD. STD may equip the synapse with low-pass ﬁltering proper- ties, by which the synapse will pass on the ﬁrst of stimulus in a train of stimuli unaltered, while the rest are attenuated. In this manner it shapes the information transfer by synaptic networks and gives rise to sensory and behavioral phenomena (Zucker, 1989). For example, in somatosensory cortex of rat, in vivo whole-cell recordings in cortical neurons during whisker deﬂec- tion directly demonstrated that synaptic depression of thalamic input to the cortex contributes to rapid adaptation of sensory responses (Chung et al., 2002). LASTING SYNAPTIC ADAPTATIONS IN THE PFC Selective attention, the ability of an organism to ﬁlter out relevant information in the face of distractors, can build upon just such synaptic process. Layer V pyramidal neurons in PFC handle diverse incoming information from mediodorsal thalamus and from local neurons and these connections are important in mediating executive functions such as for example working memory (Floresco et al., 1999). STD on this level may represent a higher level of sensory adaptation that can be expressed as decreased levels of attention and respon- siveness. Reduced short-term plasticity after nicotine exposure compromises the ability of prefrontal neurons to efﬁciently ﬁlter out irrelevant information. LASTING SYNAPTIC ADAPTATIONS IN THE PFC In adult rodents that were exposed to nicotine during adolescence only a handful of proteins show long-term adaptations follow- ing adolescent nicotine exposure that persisted into later life. Nicotinic AChR levels in the PFC returned to baseline 5 weeks following adolescent nicotine exposure (Counotte et al., 2012). In contrast, mGluR2 levels show a strong down-regulation at this time (Counotte et al., 2011). Reduced mGluR2 function in mPFC synapses resulted in impaired attention performance. Stimulating mGluR2s with speciﬁc agonists improved attention performance in animals that were exposed to nicotine during adolescence (Counotte et al., 2011). Interestingly, the association between changes in mGluR2 signalling and nicotine exposure is not limited to the PFC. Also in other brain areas involved in reward processing such as ventral tegmental area (VTA) and the nucleus accumbens (NAcc) lasting adaptations in mGluR2 function follow nicotine exposure and were found to affect rewarding properties of nicotine (Helton et al., 1997; Kenny et al., 2003; Kenny and Markou, 2004; Liechti et al., 2007). In these brain areas, activation of mGlu2/3 receptors decreases At the level of synapse function, alterations in mGluR2 levels affect both short-term synaptic plasticity as well as STDP in later life. Short-term depression (STD) is reduced in adult animals as a result of nicotine exposure during adolescence (Counotte et al., 2011). In control animals, blocking mGluR2 signalling with mGluR2 antagonists also results in reduced STD. Reduced mGluR2 signalling after nicotine exposure has a similar effect on STD as mGluR2 block by antagonist (Figure 1). Thereby, mGluR2 may act as an inhibitory feedback mechanism in conditions of excessive excitation and high glutamate release, as occurs when a neuron ﬁres a train of action potentials. Especially at high frequency stimulation the effect of mGluR2 on STD was most prominent at excitatory synapses on layer V pyramidal neurons in the PFC (Counotte et al., 2011). The lasting reduc- tion of mGluR2 levels and function after adolescent nicotine exposure leads to reduced inhibitory feedback on pyramidal cells and reduces the regulatory role of this receptor in short-term August 2012 \| Volume 4 \| Article 3 \| 5 www.frontiersin.org www.frontiersin.org Goriounova and Mansvelder Adolescent nicotine alters STDP city. Most likely, activation of mGluR2s affects presynaptic um channel function as was found in the calyx of Held, by electrophysiological recordings from presynaptic terminals hashi et al., 1996). LASTING SYNAPTIC ADAPTATIONS IN THE PFC Agonists of metabotropic glutamate tors (mGluRs) suppressed high voltage-activated P/Q-type um channels in the presynaptic terminal, thereby inhibiting mitter release (Takahashi et al., 1996). Since presynaptic dynamics play a key role in short—term plasticity (Zucker Regehr, 2002), decrease in Ca2+ current may explain uR-dependent modulation of STD. D may equip the synapse with low-pass ﬁltering proper- by which the synapse will pass on the ﬁrst of stimulus in a of stimuli unaltered, while the rest are attenuated. In this ner it shapes the information transfer by synaptic networks gives rise to sensory and behavioral phenomena (Zucker, . For example, in somatosensory cortex of rat, in vivo e-cell recordings in cortical neurons during whisker deﬂec- directly demonstrated that synaptic depression of thalamic to the cortex contributes to rapid adaptation of sensory nses (Chung et al., 2002). Selective attention, the ability organism to ﬁlter out relevant information in the face of ctors, can build upon just such synaptic process. Layer V midal neurons in PFC handle diverse incoming information mediodorsal thalamus and from local neurons and these ections are important in mediating executive functions such example working memory (Floresco et al., 1999). STD on evel may represent a higher level of sensory adaptation that be expressed as decreased levels of attention and respon- ess. Reduced short-term plasticity after nicotine exposure romises the ability of prefrontal neurons to efﬁciently ﬁlter relevant information. e recently found that glutamatergic synapses in the PFC increased spike-timing-dependent LTP 5 weeks after nico- exposure during adolescence (Figure 1) (Goriounova and velder, 2012). This was not the case when animals were ed to nicotine during adulthood, indicating that adoles- is a vulnerable period for these lasting changes to occur. ong-term effects on LTP 5 weeks after nicotine exposure g adolescence were opposite to the effects immediately wing nicotine exposure during adolescence, where spike- g-dependent LTP is supressed. Thus, nicotine exposure dur- dolescence has lasting effects on the mechanisms of STDP ersistent synaptic alterations that increase LTP. hat is the mechanism underlying the long-term effects of ne exposure during adolescence on STDP? We hypothe- that altered levels of mGluR2 receptors can explain the g effects on STDP. Reduced mGluR2 signalling in adult after nicotine exposure (Counotte et al., 2011) may con- te to the decreased plasticity we observed. LASTING SYNAPTIC ADAPTATIONS IN THE PFC Blocking mGluR2 tors with MPPG resulted in increased LTP comparable vels observed in adult rats treated with nicotine during scence (Goriounova and Mansvelder, 2012). In nicotine- d rats, where the synaptic mGluR2 receptor levels are ed (Counotte et al., 2011), enhancing mGluR2 activity pplying mGluR2 agonist LY379268 completely abolished Goriounova and Mansvelder, 2012). Thereby, mGluR2 sig- g bidirectionally inﬂuences spike-timing-dependent LTP: reducing mGluR2-dependent inhibition leads to incr while enhancing mGluR2 activation blocked LTP. Thu ately following adolescent nicotine exposure, increase mGluR2s may be responsible for reduced LTP induct weeks following adolescent nicotine exposure, the last tion in mGluR2 signalling can explain the increased adult mPFC. STDP depends on the precise timing of the synaptic the postsynaptic action potential and this temporal r resembles typical features of associative learning (Let 2007). Although STDP has not been directly linked to performance, the ability to associate goal-relevant in is crucial for any cognitive behavior. In nicotine-treat same amount of pre- and postsynaptic activity lead synaptic potentiation. This may suggest that the PF would even associate irrelevant stimuli. CONCLUSION The prefrontal cortex, the brain area responsible fo functions and attention performance, is one of the areas to mature and is still in the process of develo ing adolescence. This places the adolescent brain in a state of imbalance, susceptible to the inﬂuence of ps substances such as nicotine. In prefrontal networks nico ulates information processing on multiple levels by acti desensitizing nicotine receptors on different cell types way affects cognition. The adolescent brain is particu tive to the effects of nicotine. Studies in human subjec that smoking during adolescence increases the risk o ing psychiatric disorders and cognitive impairment in l addition, adolescent smokers suffer from attention deﬁ progress with the years of smoking. From studies in the rodent brain it is becoming on the short-term, adolescent, but not adult, nicotin increases the expression of nAChRs containing α4 an units in the mPFC, which leads to an increase in nicotin GABAergic synaptic transmission. In addition, mGluR presynaptic glutamatergic terminals in the PFC are causing a reduction in glutamatergic synapse strength ing STDP (Figure 1). Changes in nAChR levels are re the adult rodent brain, weeks after nicotine levels hav nAChR levels in the PFC return to baseline levels. In c this stage, mGluR2 levels have reduced signiﬁcantly b line levels, thereby altering mGluR2 signaling during plasticity, augmenting spike-timing-dependent potent hampering attention performance. 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H., Hogenboom, F., Schoffelmeer, A. N., De Vries, T. J., Smit, A. B., and Pattij, T. (2009). Long-lasting cognitive deﬁcits resulting from adoles- cent nicotine exposure in rats. Neuropsychopharmacology 34, 299–306. Cervilla, J. A., Prince, M., and Mann, A. (2000). Smoking, drinking, and incident cognitive impairment: a cohort community based study included in the Gospel Oak project. J. Neurol. Neurosurg. Psychiatry 68, 622–626. Fountain, S. B., Rowan, J. D., Kelley, B. M., Willey, A. R., and Nolley, E. P. (2008). Adolescent exposure to nicotine impairs adult serial pattern learning in rats. CONCLUSION What is the mechanism underlying the long-term effects of nicotine exposure during adolescence on STDP? We hypothe- sized that altered levels of mGluR2 receptors can explain the lasting effects on STDP. Reduced mGluR2 signalling in adult rats after nicotine exposure (Counotte et al., 2011) may con- tribute to the decreased plasticity we observed. Blocking mGluR2 receptors with MPPG resulted in increased LTP comparable to levels observed in adult rats treated with nicotine during adolescence (Goriounova and Mansvelder, 2012). In nicotine- treated rats, where the synaptic mGluR2 receptor levels are reduced (Counotte et al., 2011), enhancing mGluR2 activity by applying mGluR2 agonist LY379268 completely abolished LTP (Goriounova and Mansvelder, 2012). Thereby, mGluR2 sig- nalling bidirectionally inﬂuences spike-timing-dependent LTP: New questions and opportunities arise from these recent ﬁndings. The long-term adaptations involving mGluR2s can have profound implications for network functioning and affect more complex levels of information processing. What are the steps that lead from nicotine exposure and nAChR activation in the adolescent brain to adaptations in synaptic mGluR2 August 2012 \| Volume 4 \| Article 3 \| 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Adolescent nicotine alters STDP Goriounova and Mansvelder ACKNOWLEDGMENTS Huibert D. Mansvelder received funding from the Netherlands Organization for Scientiﬁc Research (NWO; 917.76.360 and 912.06.148), European Research Council Starting Grant “BrainSignals,” the Dutch Fund for Economic Structure Reinforcement (FES, 0908 “NeuroBasic PharmaPhenomics project”), EU 7th Framework Programme (HEALTH-F2-2009- 242167 “SynSys”) and VU University Amsterdam. ACKNOWLEDGMENTS Huibert D. Mansvelder received funding from the Netherlands Organization for Scientiﬁc Research (NWO; 917.76.360 and 912.06.148), European Research Council Starting Grant “BrainSignals,” the Dutch Fund for Economic Structure Reinforcement (FES, 0908 “NeuroBasic PharmaPhenomics project”), EU 7th Framework Programme (HEALTH-F2-2009- 242167 “SynSys”) and VU University Amsterdam. levels? Unveiling the signaling routes involved may provide a broader view on the adaptation strategies used during brain development in response to environmental factors. Another inter- esting question would be whether mGluR2 signaling is involved in a broader spectrum of attention impairments with different etiol- ogy. If changes in mGluR2 signaling are a common underlying mechanism for attention malfunction it would make it a suitable pharmacological target for therapy. REFERENCES REFERENCES D., and Kallman, M. J. (1997). 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https://openalex.org/W4293238842	https://www.researchsquare.com/article/rs-1594991/latest.pdf	English	null	M2 macrophage-derived exosome facilitates metastasis in non-small-cell lung cancer via delivering integrin αvβ3	Research Square (Research Square)	2,022	cc-by	10,533	M2 macrophage-derived exosome facilitates metastasis in non-small-cell lung cancer via delivering integrin αvβ3 Lamei Huang Sun Yat-Sen University Cancer Center Jianye Zhang Guangzhou Medical University Xueping Wang Sun Yat-Sen University Cancer Center Chaoyue Su Guangzhou Medical University Shaocong Wu Sun Yat-Sen University Cancer Center Chuan Yang Sun Yat-Sen University Cancer Center Min Luo Sun Yat-Sen University Cancer Center Fang Wang Sun Yat-Sen University Cancer Center Liwu Fu (  fulw@mail.sysu.edu.cn ) Sun Yat-Sen University Cancer Center Lamei Huang Sun Yat-Sen University Cancer Center Jianye Zhang Guangzhou Medical University Xueping Wang Sun Yat-Sen University Cancer Center Chaoyue Su Guangzhou Medical University Shaocong Wu Sun Yat-Sen University Cancer Center Chuan Yang Sun Yat-Sen University Cancer Center Min Luo Sun Yat-Sen University Cancer Center Fang Wang Sun Yat-Sen University Cancer Center Liwu Fu (  fulw@mail.sysu.edu.cn ) Sun Yat-Sen University Cancer Center Results We demonstrated that M2-polarized phenotypic macrophages facilitate the migration and invasion of cancer cells in vitro and in vivo through intercellular delivering M2-exos. Importantly, we found that M2- exos had considerably higher levels of integrin αvβ3. The impact of M2 macrophage-mediated invasion and migration of NSCLC cells was clearly decreased when integrin αvβ3 was blocked. Mechanistically, exosomal integrin αvβ3 produced from M2 macrophages successfully triggered the FAK signaling pathway in recipient cells, boosting the migratory and invasive abilities of NSCLC cells. Clinically, we found that metastatic NSCLC patients had greater integrin αvβ3 expression, which was associated with worse prognosis. Research Article Keywords: M2 macrophage, Exosomes, Integrin αvβ3, Metastasis, NSCLC Posted Date: April 28th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1594991/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/28 Page 1/28 Methods The distribution of macrophages in tumor microenvironment, especially in lung cancer, was analyzed by online database and immunohistochemistry. The model of M2 macrophage was successfully established in vitro and M2 macrophage-derived exosomes were identified by transmission electron microscope imaging, nanoparticle tracking analysis and western blot. The role of M2 macrophage-derived exosomes (M2-exos) in promoting metastasis of lung cancer cells was identified by transwell assay, wound healing assay, immunofluorescence in vitro, and by tumor model in vivo. The mechanism that M2-exos facilitates metastasis via delivering integrin αvβ3 and activating the FAK signaling was investigated using western blot, transwell assays, immunofluorescence assays. Finally, the expression levels of integrin αvβ3 were assessed in clinical samples by immunohistochemistry. Background The most prevalent cause of cancer death is metastasis. Immunological components of tumour microenvironment, especially tumour-associated macrophages, play a vital role in cancer metastasis. However, the underlying mechanisms of tumour-associated macrophages on non-small-cell lung cancer (NSCLC) metastasis remain largely unexplored. 1. Background Exosomes contain a variety of bio-active molecules, including proteins, lipids, RNAs and DNAs, which can be transferred to recipient cells and mediated their biological functions [28]. Accumulating research have shown that tumour-derived exosomes are associated with tumour growth, drug resistance, metastasis, and the remodeling of the tumour immune microenvironment [29, 30]. Exosomes from lung cancer, for instance, contribute to the polarization of macrophages toward an M2- like phenotype [31]. M2 macrophage-derived exosomes have been found to promote tumor progression and metastasis in colorectal cancer and liver cancer [32, 33]. However, there has been little investigation into the effect of M2 macrophage-derived exosomes (M2-exos) in metastatic NSCLC. , p g [ ] Accumulating research have shown that tumour-derived exosomes are associated with tumour growth, drug resistance, metastasis, and the remodeling of the tumour immune microenvironment [29, 30]. Exosomes from lung cancer, for instance, contribute to the polarization of macrophages toward an M2- like phenotype [31]. M2 macrophage-derived exosomes have been found to promote tumor progression and metastasis in colorectal cancer and liver cancer [32, 33]. However, there has been little investigation into the effect of M2 macrophage-derived exosomes (M2-exos) in metastatic NSCLC. Accumulating research have shown that tumour-derived exosomes are associated with tumour growth, drug resistance, metastasis, and the remodeling of the tumour immune microenvironment [29, 30]. Exosomes from lung cancer, for instance, contribute to the polarization of macrophages toward an M2- like phenotype [31]. M2 macrophage-derived exosomes have been found to promote tumor progression and metastasis in colorectal cancer and liver cancer [32, 33]. However, there has been little investigation into the effect of M2 macrophage-derived exosomes (M2-exos) in metastatic NSCLC. Exosomes from lung cancer, for instance, contribute to the polarization of macrophages toward an M2- like phenotype [31]. M2 macrophage-derived exosomes have been found to promote tumor progression and metastasis in colorectal cancer and liver cancer [32, 33]. However, there has been little investigation into the effect of M2 macrophage-derived exosomes (M2-exos) in metastatic NSCLC. In this research, we demonstrated M2-exos were responsible for NSCLC progression and metastasis both in vitro and in vivo. Importantly, ITG αvβ3 was found to be highly enriched in M2-exos and was closely associated with NSCLC metastasis. The underlying mechanisms could be that M2-derived exosomes mediated ITG αvβ3 transmission to NSCLC cells, which triggered the FAK signaling of recipient cells, thus enhancing NSCLC cells migration and invasion. Conclusions This study reveals a novel mechanism that M2 macrophage-derived exosomal integrin αvβ3 significantly increased NSCLC cells migration and invasion. integrin αvβ3 can be used as a potential prognostic marker, and blocking integrin αvβ3 could be a viable treatment option for preventing tumour progression and metastasis. Page 2/28 Page 2/28 1. Background Lung cancer is the dominant forerunner of cancer-related fatalities globally, of which non-small-cell lung cancer (NSCLC) accounts for 80%-85% [1, 2]. Patients with NSCLC frequently develop metastases, the most common of which are to the brain and bone, with a 5-year survival rate of fewer than 15% [3]. Multiple gene mutations related with NSCLC metastasis have been identified, including EGFR, VEGF, KRAS, p53, and PTEN [4–6]. However, with the improvement of the understanding of various aspects of tumour, more and more evidences show that the tumor microenvironment (TME) has a crucial role in metastasis, whether it is primary site invasion or distant metastatic colonization [7–10]. The confrontation between tumor cells and immune cells determines the initiation and progression of tumour In TME [11]. Although adaptive immunity is widely regarded as the main force against the tumour, increasing evidence suggests innate immune cells, especially tumour-associated macrophages (TAM), also play an important role in this battle [12–14]. Tumour cells recruit and civilize macrophages in the tumour microenvironment to differentiate into M2-like macrophages by secreting various cytokines, such as CSF1 and CCL2 [15, 16]. TAM refers to M2-like macrophages in TME that have tumor-promoting and immunosuppressive activities [17]. TAM can release various cytokines such as TGF-β and EGF, which not only promote tumor proliferation and transformation but also facilitate the establishment of tumor tolerance microenvironment [18–20]. TAM can also secrete a series of inflammatory inhibitory molecules, including IL-10 and IL-13, which can directly inhibit CD8+T and CD4+T cell-mediated tumour killing [21, 22]. TAM infiltration in TME is linked to poor prognosis in breast, oral, ovarian and bladder cancers, and Hodgkin's lymphoma [23–25]. However, specific evidence linking TAM and NSCLC is still lacking, particularly in terms of NSCLC metastasis. Exosomes are key mediators for intercellular cross-talk and are secreted by almost all cell types [26]. Exosomes are lipid bilayer membrane vesicles originated from endocytosis and have a diameter of about 30–150 nm [27]. Exosomes contain a variety of bio-active molecules, including proteins, lipids, RNAs and DNAs, which can be transferred to recipient cells and mediated their biological functions [28]. Exosomes are key mediators for intercellular cross-talk and are secreted by almost all cell types [26]. Exosomes are lipid bilayer membrane vesicles originated from endocytosis and have a diameter of about 30–150 nm [27]. Cell culture The human NSCLC cell lines A549 and H1299 were cultured in DMEM medium (Gibco, USA), and the human acute monocytic leukemia cell line THP-1 was cultured in RIPA 1640 medium (Gibco, USA). Both medium contained with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). For macrophage polarization, THP-1 cells were treated with 100nM phorbol-12-myristate-13 acetate (PMA; Sigma-Aldrich) for 48 hours, after which the medium was discard and the cells were washed twice with pre-warmed phosphate buffered saline (PBS). The PMA-differentiated THP-1 macrophages were then cultured for another 24 h in the RPMI 1640 complete medium (without PMA) to obtain the resting state of macrophages (M0). For M1 or M2 macrophages polarization, M0 macrophages were cultured for 48 hours in the medium supplemented with 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL IFN-γ (PeproTech) or 20 ng/mL IL-4 and IL-13 (PeproTech), respectively. All cells were cultured at 37°C in a 5% CO2 atmosphere. Exosome isolation THP-1-differentiated M2 macrophages were cultivated for 24 hours in FBS-free RIPA 1640 medium, and which the medium was collected and exosomes were extracted by differential ultracentrifugation as described previously [34]. Briefly, to remove cells and debris, the conditioned media was centrifuged at 300 g for 5 minutes and 2,000 g for 15 minutes. Then, the supernatant was harvested and centrifuged at 15,000 g for 30 min at 4 ℃ to eliminate large extracellular vesicles. The exosomes were isolated by centrifugation (Beckman Coulter Avanti J30I) at 100,000 g for 90 minutes. Finally, the isolated exosomes were re-suspended in 200µL PBS and used immediately or stored at -80 degrees Celsius. Exosome identification TSG101, CD63, and Alix were utilized as positive controls in Western blot analysis, whereas as a negative control, calnexin, an endoplasmic reticulum protein, was used. The Nanosight NS300 system (Nanosight Technology, Malvern, UK) was employed to directly monitor the number and size distribution of exosomes. 1. Background Our findings shed new light on the role of macrophages Page 3/28 Page 3/28 Page 3/28 in tumor metastasis, suggesting that M2 macrophage-derived exosomes play an important role in tumor progression and may become a new target for tumor therapy. in tumor metastasis, suggesting that M2 macrophage-derived exosomes play an important role in tumor progression and may become a new target for tumor therapy. Exosome uptake assays The extracted exosomes were treated with PKH26 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer's protocol to visualize exosome internalization. Next, the tagged exosomes were cultured with H1299 cells for 6 h. The cells were fixed in 4% paraformaldehyde for 30 minutes before being stained using Abcam's CytoPainter Phalloidin iFluor 488 Reagent for 30 minutes. The nuclei were Page 4/28 Page 4/28 then stained with Hoechst 33342 (Cell Signaling Technology, Danvers, MA) for 10 minutes. A confocal microscope was used to look at how H1299 cells took in exosomes. Transwell assay For cell migration experiments, 2×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the 24-well Transwell cell culturing chambers (8µm pore size, BD), and 650µl of medium containing 10% FBS was added into the lower chamber. For cell invasion assays, 4×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the upper inserts with pre-coated Matrigel, and 650µl of media containing with 10% FBS were added to the lower chamber. For NSCLC cells and M2 macrophages indirect co-culture assays, 2×104 THP-1 cells were seeded into the lower chamber and they were induced to polarize towards M2 macrophages according to the above protocols. After then, NSCLC cells were harvested and suspended in 200µl of FBS-free DMEM before being transferred to the upper compartment. The cells in the upper chamber were wiped out after 24 hours, and the cells on the lower chamber were fixed with 4 percent paraformaldehyde and stained with 0.5 percent crystal violet. For identifying immune molecules in M2-exos that induce NSCLC cells migration and invasion, A549 and H1299 cells were cocultured with M2-exos, M2-exos + anti-IgG blocking antibody (M2-exos + IgG Ab), M2- exos + anti-ITG αvβ3 blocking antibody (M2-exos + ITG αvβ3 Ab, BioLegend, San Diego, CA) for 24 hours. Control group NSCLC cells were incubated with PBS. The results of NSCLC cells migration and invasion were photographed and counted. At least three random microscopic fields (magnification×100) were taken, and the cells were counted. All experiments were performed in triplicate. Western blot analysis Briefly, Whole cell lysates were electrophoresed in an 8 percent SDS-PAGE gel and then transferred to 0.22 m PVDF membranes (Millipore, USA) after being lysed in RIPA buffer with protease inhibitors. The membranes were blocked for 1h at 37°C in TBST with 5% skimmed milk powder before being probed with the specific antibody (1:1000) overnight at 4°C. Then, the membranes were incubated with secondary antibody (1:5000) for 1h at 37°C. The protein bands were identified using an ECL detection system (Bio- Rad, USA). Flow cytometry staining and analysis Flow cytometric assays were used to evaluate the expression of CD206 and HLA-DR as previously described [35]. Briefly, 5×105 M1 and M2 macrophages were harvested and stained with PE-CD206 or FITC-HLA-DRα antibody (BioLegend, San Diego, CA) for 15–20 minutes, and subsequently analysis using flow cytometry. Flow cytometry data were analyzed by the Flowjo (Treestar, USA) software. Reverse transcription and quantitative real-time PCR Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA), and 1µg total RNA was reverse transcribed into first-strand complementary DNA (cDNA) using cDNA Synthesis Kit (EZBioscience, USA) Page 5/28 according to protocols. Afterwards, the cDNA was performed to measure the relative gene expression level using real-time PCR. The expression of target genes was normalized to GAPDH levels in the samples in triplicates. The 2−ΔΔCT method was used to calculate the relative variation in gene expression. Additional file: Table S1 contains a list of primers. according to protocols. Afterwards, the cDNA was performed to measure the relative gene expression level using real-time PCR. The expression of target genes was normalized to GAPDH levels in the samples in triplicates. The 2−ΔΔCT method was used to calculate the relative variation in gene expression. Additional file: Table S1 contains a list of primers. struction and transfection of ITG αvβ3 shRNA and overexpression plasmids As previously described, reliable knockdown and overexpression cell lines were established[36]. lentiviral vectors were used to create ITG αvβ3 shRNA and overexpressed plasmid. The generated plasmid was co- transfected into 293T cells for 48 hours with the viral packaging plasmids psPAX2 and pMD.2G. Lentiviral supernatants were harvested and filtered through 0.45µm filter before being cultivated for 24 hours with H1299 cells. Puromycin selection (2 g/ml) was applied to the cells. Additional file: Table S2 shows the targeting sequences for specific genes. Table S3 shows the primers of overexpressed genes. Animals Male Balb/c nude mice aged 4 to 6 weeks were purchased from Guangdong Medical Laboratory Animal Center in China. For establishing human NSCLC lung metastasis model in nude mice, 3×106 A549luc cells were resuspended in 200µl FBS-free DMEM and injected intravenously into Balb/c nude mice. To study the blockade effects of ITG αvβ3, 10µg M2-exos, M2-exo + ITG αvβ3 Ab and M2-exos + IgG Ab were administered to Balb/c nude mice every four days, respectively. A similar volume of PBS was injected into the control group. Mice were sacrificed after 50 days, and the lungs were assessed for metastatic lesions by comparing biofluorescence signal intensities. Tissue morphology was identified by hematoxylin and eosin (H&E) staining. Immunohistochemistry Patients’ clinical tumour specimens were gathered at the Sun Yat-sen University Cancer Center in Guangzhou, China, who had been diagnosed with NSCLC. For patient specimens, all patients gave their agreement and enrolled in an IRB approved protocols at Sun Yat-sen University Cancer Center, which allowed the collecting and analysis of clinical data, archival, and paraffin specimens in compliance with ethical principles (Ethics Document No. SL-B2022-139-01). Tumour specimens were formalin-fixed and paraffin-embedded, as is standard laboratory pathology technique, and stored at the Sun Yat-sen University Cancer Center’s pathology department. The paraffin slices from patients' tissues were treated with primary anti-human antibodies at various dilutions (ITG αv, 1:400, ITG β3, 1:200) overnight at 4°C. They were then treated for 60 minutes at room temperature with the second antibody. The staining was identified by using DAB Kit (Zisbio) as directed by the manufacturer. Hematoxylin staining was measured using at least 5 randomly selected 200 or 400 fields of view after slides were stained for 6 minutes. Two pathologists independently evaluated the protein expression. Statistical analysis Statistical analysis Statistical analysis Page 6/28 Unless otherwise specified, the results were presented as means SEM and analyzed using one-way ANOVA or Student's t-test analysis. The statistical significance level was set at p < 0.05. SPSS 22.0 or GraphPad Prism 7 were used for all statistical analyses. Unless otherwise specified, the results were presented as means SEM and analyzed using one-way ANOVA or Student's t-test analysis. The statistical significance level was set at p < 0.05. SPSS 22.0 or GraphPad Prism 7 were used for all statistical analyses. 3. Results To look into the relationship between macrophages and lung cancer. The Cancer Immunome Atlas (https://tcia.at/) is an online database used to assess the macrophage distribution in TME. According to an analysis of macrophage distribution in various cancer, macrophages were highly enriched in lung adenocarcinoma (LUAD) samples (Fig. 1A). In LUAD, M2 macrophages made up the largest fraction of immune cells (Fig. 1B). TAMs are distinguished by specific surface molecules, such as the Mannose Receptor CD206, which related to angiogenesis and cancer metastasis [37]. Lung adenocarcinoma specimens were collected to better understand the distribution of M2 macrophages in the LUAD. M2 macrophages were found to be more prevalent in metastatic lung adenocarcinoma specimens (n = 59) than in non-metastatic lung adenocarcinoma specimens (n = 67) (Fig. 1C, D). Furthermore, high M2 macrophages infiltration was linked to a poor prognosis (Fig. 1E). Overall, macrophages are the most common immune subgroup in lung adenocarcinoma, and lung adenocarcinoma with high M2 macrophages infiltration is more prone to metastasis. 3.2 M2-polarized macrophages enhance NSCLC cells migration and invasion It is reported that tumour-associated macrophages extensively regulate tumour progression and metastasis of a variety tumours [38]. Typically, tumour-associated macrophages exhibit an M2-like phenotype. To explore the impact of M2 macrophages on lung adenocarcinoma, we first successfully constructed an M1/M2 polarized macrophage model in vitro using THP-1 monocytes (Fig. 2A). In contrast to M1-polarized macrophages, M2-polarized macrophages showed increased expression of CD206, CD163, Arg-1 (M2 macrophages-associated marker), along with diminished level of HLA-DRα, TNF-α, iNOS (M1 macrophages-associated marker), which was confirmed by flow cytometry and qPCR assays (Fig. 2B, C). The induced M2 macrophages were then co-cultured with NSCLC cells for 24h, or NSCLC was pretreated with M2 conditioned medium. We found that both treatments markedly enhanced the migration and invasion abilities of H1299 and A549 cells (Fig. 2D, E). Therefore, we demonstrated in vitro that M2-type macrophages assist the progression and metastasis of NSCLC. 3.3 M2 macrophage-derived exosomes enhance NSCLC cells migration and invasion Page 7/28 After conditioned medium pretreatment with M2 macrophages, we found that A549 and H1299 cells migrated and invaded substantially more. Therefore, we wondered if substances derived from M2 macrophages were responsible for the remarkable effect. Accumulated evidences suggested that exosomes derived from tumour cells or tumour-associated stomal cells involve in tumour metastasis [39]. To investigate whether M2-exos are related to cancer metastasis, we extracted exosomes from M2 macrophages culture medium and subsequently treated NSCLC cells in vitro with these exosomes. Exosomes were validated by Western blot analysis using exosome-specific markers, TSG101, CD63, Alix as well as the negative marker calnexin (Fig. 3A). In addition, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to quantify particle size and morphology (Fig. 3B, C). To exposed whether M2-exos can be internalized by NSCLC cells, we pre-treated H1299 cells with PKH26- labeled exosomes for 12h. Confocal fluorescence imaging revealed that M2-exos tagged with PKH26 were highly absorbed by H1299 cells (Fig. 3D). After conditioned medium pretreatment with M2 macrophages, we found that A549 and H1299 cells migrated and invaded substantially more. Therefore, we wondered if substances derived from M2 macrophages were responsible for the remarkable effect. Accumulated evidences suggested that exosomes derived from tumour cells or tumour-associated stomal cells involve in tumour metastasis [39 We hypothesize that M2-exos promote NSCLC cells migratory and invasive capacities. Therefore, H1299 cells and A549 cells were cocultured with M2-macrophage-derived medium (M2-CM), M2-exos, and M2- CM depleted exosomes (M2-CM dexo), respectively. Transwell assays were employed to assess cancer cells' migration and invasion ability. As expected, M2-exos notably increased the migration and invasion of NSCLC cells, but had little impact on cell proliferation (Fig. 3E-H). Collectively, our results revealed that M2 macrophages enhanced the mobility and aggressiveness of NSCLC cells, which were predominantly dependent on exosomes. 3.4 Exosomal ITG αvβ3 derived from M2 macrophages is a key player in mediating NSCLC metastasis According to the evidence presented above, exosomes released by M2 macrophages deliver certain components to NSCLC cells, enhancing cancer cells motility and invasion. Previous research demonstrated that NSCLC cell-derived exosomes played a key role in mediating tumour metastasis through targeting integrin signaling pathways. Integrins, a heterodimeric transmembrane receptor family capable of regulation intercellular interactions with the extracellular matrix (ECM), have a key impact in the regulation in a range of tumor cell behaviors such as proliferation, adhesion, migration, invasion and survival. Thus, we assumed that integrin might played a key role in M2-exos in mediating NSCLC metastasis. We discovered that M2-exo treatment significantly increased the protein expression levels of ITG αv and β3 in H1299 and A549 cells in a concentration-dependent manner. However, there was no significant change in the mRNA levels of ITG αvβ3 (Fig. 4A, B). Therefore, the increased protein expression of ITG αv and β3 in A549 and H1299 cells is not endogenous. We investigated the expression of ITG av and β3 in M2-exos to investigate whether exosomes mediate direct intercellular transmission of ITG αvβ3. The Western blot experiment revealed that ITG αv and β3 were considerably more abundant in M2-exos than in M2 macrophage cell lysate (Fig. 4C). Furthermore, colocalization of ITG αvβ3 and exosomes was seen in A549 and H1299 cells cocultured with M2-exos, indicating that ITG αvβ3 was transported from M2 macrophages to NSCLC cells via exosomes (Fig. 4D). In conclusion, these results revealed that M2-exos enriched ITG αvβ3 meaningfully and could be directly transferred to NSCLC cells. Page 8/28 Page 8/28 To ascertain whether M2-exos-generated ITG αvβ3 on mediating NSCLC cells metastasis, M2-exos were pre-incubated with or without anti-ITG αvβ3 blocking antibody (ITG αvβ3 Ab), and subsequently cocultured with NSCLC cells to detect their migration and invasion abilities. In this investigation, an IgG blocking Ab (IgG Ab) was used to evaluate the specificity of ITG αvβ3 Ab. As shown, when contrasted to the M2-exos + IgG Ab group, the M2-exos + ITG αvβ3 Ab group effectively prevented the invasion and migration of H1299 and A549 cells (Fig. 4E, F). To further confirm the critical role of exosomal ITG αvβ3 derived from M2-macrophages, ITG αv and β3 expression were suppressed in H1299 cells employing two distinct shRNAs, and western blot assays were used to confirm the knock-efficacy (Fig. 4G). 3.5 M2 macrophage-derived exosomal ITG αvβ3 promotes NSCLC metastasis in vivo To investigate whether M2-exos and its component ITG αvβ3 prime NSCLC lung metastasis in vivo, we constructed A549 cells stably expressing luciferase gene (A549luc), followed by treated with M2-exos, M2- exos + IgG Ab, M2-exos + ITG αvβ3 Ab and PBS. Then, A549luc cells with different treatment were injected into caudal veins of male nude mice, and various treatment interventions were performed as illustrated in scheme (Fig. 5A). There was no notable variation in body mass between the groups throughout the experiment (Fig. 5B). When the M2-exos and M2-exos + IgG antibody groups were compared to the control group, we discovered a substantial increase in lung metastases. When compared to the M2-exos and M2- exos + IgG group lung metastasis was considerably reduced in the M2-exos + ITG αvβ3 Ab group (Fig. 5C, D). These results suggested that ITG αvβ3 was indeed the main effector molecule mediating M2-exos to promote tumor metastasis. Histologic investigation revealed that M2-exos dramatically enhanced the metastatic nodules in lung, but blocking exosomal ITG αvβ3 would inhibit this effect (Fig. 5E). These results implied that M2 macrophage-derived exosomal ITG αvβ3 could be transmitted to cancer cells and increased cancer migration and invasion in vivo. 3.6 ITG αvβ3 improve tumour metastasis via activating the FAK signaling pathway. 3.4 Exosomal ITG αvβ3 derived from M2 macrophages is a key player in mediating NSCLC metastasis Moreover, the down-regulation of ITG αv and β3 protein expression in H1299 cells significantly repressed their capacities of migration and invasion, and M2-exos treatment can salvage this inhibitory effect (Fig. 4H, I) g 3.7 Integrin αvβ3 related to NSCLC metastasis and poor 3.7 Integrin αvβ3 related to NSCLC metastasis and poor prognosis 3.6 ITG αvβ3 improve tumour metastasis via activating the FAK signaling pathway. Exosomes have been proven in numerous studies to have a vital function in signal transduction [40–42]. However, the involvement of transportable ITG αvβ3 from M2-exos in the NSCLC migratory and invasive signaling pathway remains unknown. To further explore the relevant molecular mechanisms, we constructed A549 and H1299 cells overexpressing ITG αvβ3 (Fig. 6A, B). We found that A549-ITG αvβ3 and H1299-ITG αvβ3 had dramatically improved migration and invasion abilities (Fig. 6C). We also carried out wound-healing assay. The horizontal mobility of A549-ITGαvβ3 and H1299-ITG αvβ3 was higher than that of the control group, as expected (Fig. 6D). These results further suggested that ITG αvβ3 could be a key effector molecule promoting tumor metastasis. Focal adhesion kinase (FAK) is a non- receptor kinase that is primarily responsible for adhesion signaling and cell migration, but it can also promote cell survival in the absence of stress [43]. Many studies have shown that integrins primarily Page 9/28 Page 9/28 trigger the FAK signaling pathway, regulating various biological function [44, 45]. Therefore, we further investigated whether ITG αvβ3 delivered by M2-exos activates the downstream FAK/p-FAK signaling pathway of NSCLC cells. We discovered that p-FAK protein expression was considerably increased in A549 cells after M2-exos treatment compared to the control group, while p-FAK protein expression was down-regulated after pre-incubation with M2-exo and ITG αvβ3 blocking antibody (Fig. 6E). More importantly, FAK inhibitor treatment significantly offset the increased migration and motility of tumor cells induced by M2-exos. These results further demonstrated that ITG αvβ3 promoted tumor metastasis by activating FAK signaling pathway (Fig. 6F). Taken together, these results indicated that exosomal ITG αvβ3 derived from M2 macrophages is essential for migration and invasion of NSCLC cells. Mechanicall intercellularly transferred exosomal ITG αvβ3 primarily activated the FAK signaling pathway to execute biological functions. 3.7 Integrin αvβ3 related to NSCLC metastasis and poor prognosis We revealed that M2 macrophage-derived exosomes mediate ITG αvβ3 transmission to increase NSCLC migration and invasion in vitro and in vivo models. In order to verify the reliability of this conclusion in real data. We collected 126 lung adenocarcinoma specimens, including 59 metastatic cases and 67 non- metastatic cases (Table 1). Immunohistochemistry was used to determine the levels of ITG αv and β3 expression in lung cancer specimens. We discovered that lung adenocarcinomas with metastasis had greater levels of ITG αv and β3 expression than those without metastasis (Fig. 7A, B). Then the specimens were divided into high or low ITG αv group and ITG β3 group, based on immunohistochemical scores. We found that the rate of metastasis was higher in the high ITG αv and high ITG β3 groups than in the correspondingly low score groups, respectively (Fig. 7C). This suggested that high expression of ITG αv and ITG β3 in tumour cells could indicate a poor prognosis. Therefore, we analyzed metastasis- free survival in each group. When compared to the low expression group, the metastasis-free survival rate of the high expression group was lower than expected (Fig. 7D, E). Overall, these clinical evidences suggested that high expression of ITG αv and ITG β3 was associated with a poor prognosis. Page 10/28 Table 1 correlation between metastasis and clinical pathology characteristics in Lung cancer Variable NO. metastasis X2 P Valve non-meta meta Age < 60 59 32(54.2%) 27(45.8%) 0.924 0.3 > 60 67 42(62.7%) 25(37.3%) Gender Female 57 35(61.4%) 22(23.5%) 0.307 0.6 Male 69 39(56.5%) 30(43.5%) Smoking non-smoking 79 46(58.2%) 33(41.8%) 0.022 0.9 smoking 47 28(59.6%) 19(40.4%) ITG β3 low expression 62 40(64.5%) 22(35.5%) 6.306 0.012 high expression 64 27(42.2%) 37(57.8%) ITG αv low expression 66 41(62.1%) 25(37.9%) 4.455 0.035 high expression 60 26(43.3%) 34(56.7%) 4. Discussion Currently, the vast majority of cancer-related deaths (about 90%) are caused by metastatic disease [46]. Tumour metastasis is a complex biological process involving multiple cascade steps, and there are still many unexplained mechanisms [47]. A mass of studies have shown that TME enrich a variety of immunosuppressive cells, which have a vital role in mediating the invasiveness of primary tumours and the ability to metastasize to distant sites through direct contact with cancer cells or paracrine pathways [48, 49]. Macrophages are the main tumour-infiltrating leukocytes in almost all cancers. They can be "domesticated" by tumour cells to polarize toward M2-like macrophages phenotype, therefore supporting tumour progression [49]. Many studies have reported that the invasion and metastasis of NSCLC is closely related to its microenvironment [50]. However, the link between the metastasis of NSCLC and M2 macrophages and the underlying molecular mechanism have not yet been clarified. Page 11/28 It is reported that TAMs in the TME can be differentiated from bone marrow-derived macrophages, or tissue colonized macrophages, according on where the tumor is located in the body [49]. In NSCLC, the tumour-promoting TAMs are mainly derived from bone marrow macrophages [51]. Therefore, in this study, we simulated the physiological effects of TAMs in vivo by inducing the polarization of acute leukemia monocytes THP-1 to M2 macrophages in vitro. M2 macrophages have been shown to highly express genes such as CD163, CD206, Arg-1, and IL-10, while low expression of M1 macrophage markers, such as HLA-DRα, iNOS and TNF-α. In this study, we successfully induced M2 macrophages in vitro using THP-1 cell line. More importantly, CD206+ M2 macrophages have the characteristics of significantly promoting the migration and invasion phenotype of NSCLC. A study performed by Lee et al. has shown that TAMs in metastatic tumours are mainly M2 macrophage phenotypes [52]. Many studies have reported that M2 macrophages released a wide range of chemokines, cytokines to enhance tumour invasion and metastasis [53]. Nevertheless, the molecular mechanism by which they interact with tumor cells is unknown. More and more studies have shown that exosomes produced from tumour-associated stromal cells have a significant role in mediating intercellular communication [32]. For example, exosomes derived from tumour-associated fibroblasts promoted the metastasis of colorectal cancer cells and chemotherapy resistance [39]. Similarly, M2-exos were confirmed to increase the metastasis of colorectal cancer cells [32]. In addition, Wu et al. 4. Discussion found that M2 macrophages-derived exosomes delivered integrin αMβ2 to hepatocellular carcinoma cells and activated the expression of MMP9, thereby promoting the invasion and metastasis of cancer cells [54]. Consistently, our research has shown that exosomes secreted by M2 macrophages possess the ability to enhance the metastasis of NSCLC cells in vivo and in vitro. Exosomes are made up of a range of biologically active components, including proteins, RNA, DNA and lipids [27, 55]. Importantly, exosomal contents derived from different cell types are unique [56]. Our study found that M2-exo showed a significant enrichment of ITG αvβ3. ITG αvβ3 is a marker of tumour angiogenesis, and its expression on tumour cells is related to cancer progression, drug resistance and EMT [57]. In addition, Wettersten et al. showed that ITG αvβ3 has a significant positive correlation with TAMs markers in various cancers [58]. Additionally, the enrichment of ITG αvβ3 was found in prostate cancer cell-derived exosomes, which could promote the migration phenotype of non-tumourigenic cells through intercellular deliver of exosome [59]. In this study, we demonstrated that M2-exos-mediated invasion and metastasis of NSCLC cells are dependent on ITG αvβ3. Exogenous blocking of ITG αvβ3 derived from M2-exos prevented the migration and invasion of NSCLC induced by M2-exos. Studies so far have shown that exosomes are important mediators of intercellular signal transduction. However, the mechanism of ITG αvβ3 transferred by M2-exo in driving the migration and invasion of NSCLC is still unclear. FAK is a major regulator of growth factor receptor and integrin-mediated signals, which controls the basic processes of normal cells and cancer cells through kinase activity and scaffold function [60]. FAK activity and expression are upregulated in many cancers, and are usually relevant to poor clinical outcomes, implying that FAK could be used to predict tumour progression [60]. In this study, Page 12/28 Page 12/28 we found that M2-exos ITG αvβ3 mainly facilitated the invasion and metastasis of NSCLC through activating the FAK signal transduction. 5. Conclusions Our research showed that M2-exos were rich in ITG αvβ3, which could be directly transferred to NSCLC cells, resulting in accelerated migration and invasion in NSCLC. ITG αvβ3 facilitated NSCLC invasion and metastasis by increasing the phosphorylation of FAK. Blocking exosomal ITG αvβ3 weakened the potential of M2-exos to increase NSCLC cells migration and invasion. Therefore, our study supported that ITG αvβ3 as a biomarker of TAMs activation in NSCLC. In addition, blocking the ITG αvβ3-FAK signal transduction pathway may be a promising treatment to control the metastasis of NSCLC. However, more research is still needed to further clarify its potential mechanism. In addition, our future research will need to isolate TAMs from human specimens for further verification Abbreviations NSCLC: Non-small-cell lung cancer Page 13/28 Page 13/28 TNF-α: Tumor necrosis factor-alpha HLA-DRα: Human leukocyte antigen-DRα iNOS: Inducible nitric oxide synthase TSG101: Tumor susceptibility gene 101 NTA: Nanoparticle tracking analysis TEM: Transmission electron microscopy M2-CM: M2-macrophage-derived medium ECM: Extracellular matrix EMT: Epithelial-Mesenchymal Transition. EMT: Epithelial-Mesenchymal Transition. EMT: Epithelial-Mesenchymal Transition. Consent for publication Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests The authors have no relevant or potential conflicts of interest to declare. Competing interests The authors have no relevant or potential conflicts of interest to declare. Funding This work was supported by National Key R&D Program of China (2021YFE0202000), National Natural Science Foundation of China (81773888). Funding This work was supported by National Key R&D Program of China (2021YFE0202000), National Natural Science Foundation of China (81773888). Authors' contributions LF and FW conceived of the study. LF and JZ designed it. LH, JZ, CS, SW, CY and ML carried out the experiments. LH and JZ analyzed and interpreted the data. LH and JZ drafted the manuscript with comments from all authors. FW and LF reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable Declarations Ethics approval and consent to participate This study involves human subjects and was approved by the institutional research ethics committee of Sun Yat-sen University Cancer Center (SL-B2022-139-01). Subjects gave informed consent to participate in the study before taking part. 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CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma. HNSC, head and neck squamous cell carcinoma. PAAD, pancreatic adenocarcinoma. OV, ovarian serous cystadenocarcinoma. STAD, stomach adenocarcinoma. CRC, colorectal cancer. BLCA, bladder urothelial carcinoma. LUSC, lung squamous cell carcinoma. LUAD, lung adenocarcinoma. UCEC, uterine corpus endometrial carcinoma. SKCM, skin cutaneous melanoma. B. In LUAD specimens, the average fraction of immunological subpopulations (n = 574). C, D. Represents images and statistical results of CD206 immunohistochemical staining in samples with metastasis and without metastasis. E. The relationship between CD206 expression and metastasis-free survival in LUAD patients. There were 59 cases of low expression and 56 cases of high expression. Data are shown as the mean ± SEM. **P < 0.001. Page 20/28 Figure 2 M2-polarized macrophages enhance NSCLC cells migration and invasion. A. Schematic di induced M1 or M2 macrophages from THP-1 monocytes. B. HLA-DR/CD206 flow cytomet were used to identify M1 or M2 macrophages. C. The gene expression levels of M1 and M macrophages were validated by real-time PCR assays. D, E. The migration and invasion ca cancer cells were determined using transwell assays after A549 and H1299 cells were co-c Page 21/28 Figure 2 M2-polarized macrophages enhance NSCLC cells migration and invasion. A. Schematic diagram of induced M1 or M2 macrophages from THP-1 monocytes. B. HLA-DR/CD206 flow cytometry markers were used to identify M1 or M2 macrophages. C. The gene expression levels of M1 and M2 macrophages were validated by real-time PCR assays. D, E. The migration and invasion capacity of cancer cells were determined using transwell assays after A549 and H1299 cells were co-cultured with Figure 2 Page 21/28 M2-polarized macrophages enhance NSCLC cells migration and invasion. A. Schematic diagram of induced M1 or M2 macrophages from THP-1 monocytes. B. HLA-DR/CD206 flow cytometry markers were used to identify M1 or M2 macrophages. C. Figures A549 and H1299 cells were pre-treated for 24 hours with PBS (control), M2 CM, M2-exo, and M2-CM dexo, and the capacity of cancer cells to migrate and invade was determined using transwell assays. The left panel displays representative photos, whereas the right panel displays migrating cell counts. H. MTT tests were used to determine the growth rate of A549 and H1299 cells after treatment using different doses of M2 exos. All data are presented as means ± SEM. P < 0.05, P < 0.01, * P < 0.001. M2 macrophage-derived exosomes enhance NSCLC cells migration and invasion. A. Western blot of exosomal markers in M2 macrophages whole cell lysates and M2-exos. B. Nanoparticle tracking analysis of M2-exos. C. Transmission electron microscope imaging of M2-exos. D. The uptake of DiI-labeled M2- exos by H1299 cells detected by confocal microscopy. Label the cytoskeleton and nucleus of H1299 with FITC-phalloidin and DAPI, respectively (Scale bar: 20μm). E, F. A549 and H1299 cells were pre-treated for 24 hours with PBS (control), M2 CM, M2-exo, and M2-CM dexo, and the capacity of cancer cells to migrate and invade was determined using transwell assays. The left panel displays representative photos, whereas the right panel displays migrating cell counts. H. MTT tests were used to determine the growth rate of A549 and H1299 cells after treatment using different doses of M2 exos. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Figures The gene expression levels of M1 and M2 macrophages were validated by real-time PCR assays. D, E. The migration and invasion capacity of cancer cells were determined using transwell assays after A549 and H1299 cells were co-cultured with Page 21/28 Page 21/28 M2 macrophages or pretreated with conditioned media from M2 macrophages for 24 hours. The left panel shows representative photos, while the right panel shows migrating cell counts. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. M2 macrophages or pretreated with conditioned media from M2 macrophages for 24 hours. The left panel shows representative photos, while the right panel shows migrating cell counts. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Figure 3 Figure 3 Figure 3 Figure 3 Page 22/28 M2 macrophage-derived exosomes enhance NSCLC cells migration and invasion. A. Western blot of exosomal markers in M2 macrophages whole cell lysates and M2-exos. B. Nanoparticle tracking analysis of M2-exos. C. Transmission electron microscope imaging of M2-exos. D. The uptake of DiI-labeled M2- exos by H1299 cells detected by confocal microscopy. Label the cytoskeleton and nucleus of H1299 with FITC-phalloidin and DAPI, respectively (Scale bar: 20μm). E, F. A549 and H1299 cells were pre-treated for 24 hours with PBS (control), M2 CM, M2-exo, and M2-CM dexo, and the capacity of cancer cells to migrate and invade was determined using transwell assays. The left panel displays representative photos, whereas the right panel displays migrating cell counts. H. MTT tests were used to determine the growth rate of A549 and H1299 cells after treatment using different doses of M2 exos. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. M2 macrophage-derived exosomes enhance NSCLC cells migration and invasion. A. Western blot of exosomal markers in M2 macrophages whole cell lysates and M2-exos. B. Nanoparticle tracking analysis of M2-exos. C. Transmission electron microscope imaging of M2-exos. D. The uptake of DiI-labeled M2- exos by H1299 cells detected by confocal microscopy. Label the cytoskeleton and nucleus of H1299 with FITC-phalloidin and DAPI, respectively (Scale bar: 20μm). E, F. Figure 4 ITG αvβ3 is abundant in M2 exosomes and plays an important role in NSCLC metastasis. A. After treatment with different concentrations M2 exos, Western blot analysis of ITG β1, β3, β4, β5, and ITG α2, αv, α6 protein levels in A549 and H1299 cells. B. Real-time PCR assays were used to examine the mRNA expression levels of ITG αv and β3 in H1299 cells after treatment with various concentrations M2-exos. C. Western blot of ITG αvβ3 protein levels in M2 macrophages whole cell lysates and M2-exo. D. Representative images of internalization of M2-exo (red) containing ITG αvβ3 (green) in H1299 cells (Scale bar: 20 μm). E, F. A549 and H1299 cells were pre-treated with PBS (control), M2-exo, M2-exo+anti- IgG blocking Ab and M2-exo+anti-ITG αvβ3 blocking Ab for 24 h, and the migration and invasion capacity of cancer cells were detected using transwell assays. The left panel shows representative photos, while the right panel shows migrating cell counts. (scale bar, 150μm). G, H. the efficacy of ITG αv/β3 silencing by shRNAs was confirmed by Western blot experiment. I. Transwell assays evaluated the migration and invasion ability of normal H1299 cells or ITG αv- and β3-silenced counterparts treated with or without M2- exo. The left panel shows representative photos, while the right panel shows migrating cell counts (scale bar, 150μm). All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Page 23/28 Figure 5 Exosomes secreted from M2 macrophages promote NSCLC cells lung metastasis in vivo. A. Schemat diagram illustrating the experimental process. A549Luc cells were cocultured with PBS, M2 exos, M2- exo+IgG Ab and M2-exo+ITG αvβ3 Ab, and injected into the caudal veins of male nude mice (6 mice fo Page 24/28 Figure 5 Exosomes secreted from M2 macrophages promote NSCLC cells lung metastasis in vivo. A. Schematic diagram illustrating the experimental process. A549Luc cells were cocultured with PBS, M2 exos, M2- exo+IgG Ab and M2-exo+ITG αvβ3 Ab, and injected into the caudal veins of male nude mice (6 mice for each group). Every four days, PBS, M2-exo, M2-exo+IgG Ab, and M2-exo+ ITG αvβ3 Ab were administered into matching mice. B. A line graph depicting the effects on body weight of mice over the course of an Page 24/28 Exosomes secreted from M2 macrophages promote NSCLC cells lung metastasis in vivo. A. Schematic diagram illustrating the experimental process. Figure 4 A549Luc cells were cocultured with PBS, M2 exos, M2- exo+IgG Ab and M2-exo+ITG αvβ3 Ab, and injected into the caudal veins of male nude mice (6 mice for each group). Every four days, PBS, M2-exo, M2-exo+IgG Ab, and M2-exo+ ITG αvβ3 Ab were administered into matching mice. B. A line graph depicting the effects on body weight of mice over the course of an Page 24/28 Page 24/28 experiment (n = 7). C, D. Representative images and fluorescence intensity statistics of in vitro lung fluorescence imaging of each group. E. Hematoxylin and eosin (H&E) staining for A549 cells' lung metastases. Top scale bar, 400μm; bottom scale bar, 200μm. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. experiment (n = 7). C, D. Representative images and fluorescence intensity statistics of in vitro lung fluorescence imaging of each group. E. Hematoxylin and eosin (H&E) staining for A549 cells' lung metastases. Top scale bar, 400μm; bottom scale bar, 200μm. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. experiment (n = 7). C, D. Representative images and fluorescence intensity statistics of in vitro lung fluorescence imaging of each group. E. Hematoxylin and eosin (H&E) staining for A549 cells' lung metastases. Top scale bar, 400μm; bottom scale bar, 200μm. All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Figure 6 Figure 6 Page 25/28 Page 25/28 ITG αvβ3 enhances the migration and invasion of NSCLC cells through FAK signaling pathway. A, B. Western blot and qPCR were employed to detect the protein and mRNA levels of ITG αv and ITG β3 in A549 and H1299 overexpressing ITG αvβ3 cells. C, D. Transwell and wound healing experiments were used to evaluate the migration and invasion properties of A549 and H1299 overexpressing ITG αvβ3. The scale is 200μm. E, F. After 24 hours of pretreatment with PBS (control), M2-exo, M2-exo+IgG Ab, or M2- exo+ ITG αvβ3 Ab, the levels of FAK/p-FAK protein expression and cancer cell migration and invasion abilities were measureed employing Western blot assays and transwell assays, respectively. Representative images are displayed in the left section and statistical results are shown in the right section (scale bar, 150μm). All data are presented as means ± SEM. Figure 4 Comparison of metastatic rates among lung adenocarcinomas with high and low level of ITG α V and β3 expression. D, E. Relationship between ITG αv and β3 expression Figure 4 * P < 0.05, P < 0.01, * P < 0.001. ITG αvβ3 enhances the migration and invasion of NSCLC cells through FAK signaling pathway. A, B. Western blot and qPCR were employed to detect the protein and mRNA levels of ITG αv and ITG β3 in A549 and H1299 overexpressing ITG αvβ3 cells. C, D. Transwell and wound healing experiments were used to evaluate the migration and invasion properties of A549 and H1299 overexpressing ITG αvβ3. The scale is 200μm. E, F. After 24 hours of pretreatment with PBS (control), M2-exo, M2-exo+IgG Ab, or M2- exo+ ITG αvβ3 Ab, the levels of FAK/p-FAK protein expression and cancer cell migration and invasion abilities were measureed employing Western blot assays and transwell assays, respectively. Representative images are displayed in the left section and statistical results are shown in the right section (scale bar, 150μm). All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. ITG αvβ3 enhances the migration and invasion of NSCLC cells through FAK signaling pathway. A, B. Western blot and qPCR were employed to detect the protein and mRNA levels of ITG αv and ITG β3 in A549 and H1299 overexpressing ITG αvβ3 cells. C, D. Transwell and wound healing experiments were used to evaluate the migration and invasion properties of A549 and H1299 overexpressing ITG αvβ3. The scale is 200μm. E, F. After 24 hours of pretreatment with PBS (control), M2-exo, M2-exo+IgG Ab, or M2- exo+ ITG αvβ3 Ab, the levels of FAK/p-FAK protein expression and cancer cell migration and invasion abilities were measureed employing Western blot assays and transwell assays, respectively. Representative images are displayed in the left section and statistical results are shown in the right section (scale bar, 150μm). All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Representative images are displayed in the left section and statistical results are shown in the right section (scale bar, 150μm). All data are presented as means ± SEM. * P < 0.05, P < 0.01, * P < 0.001. Page 26/28 Figure 7 Figure 7 A high integrin αvβ3 level concerned with NSCLC metastasis and a poor prognosis. A, B. Representative images and statistical analysis of ITG αv and ITG β3 expression levels in metastatic and non-metastatic lung adenocarcinoma specimens. C. Figure 7 A high integrin αvβ3 level concerned with NSCLC metastasis and a poor prognosis. A, B. Representative images and statistical analysis of ITG αv and ITG β3 expression levels in metastatic and non-metastatic lung adenocarcinoma specimens. C. Comparison of metastatic rates among lung adenocarcinomas with high and low level of ITG α V and β3 expression. D, E. Relationship between ITG αv and β3 expression Page 27/28 Page 27/28 levels and metastatic - free survival, respectively. F. Schematic diagram of exosomal integrin αvβ3 from M2 macrophage facilitates tumour cell metastasis via the FAK signaling pathway. levels and metastatic - free survival, respectively. F. Schematic diagram of exosomal integrin αvβ3 from M2 macrophage facilitates tumour cell metastasis via the FAK signaling pathway. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementarymaterial.docx Page 28/28
https://openalex.org/W2949387222	http://doc.rero.ch/record/326625/files/Bardelli_PP_2018.pdf	English	null	A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	12,844	A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity Marco BardelliID1☯, Karl FrontzekID2☯, Luca Simonelli1, Simone HornemannID2, Mattia Pedotti1, Federica Mazzola1, Manfredi CartaID2, Valeria EckhardtID2, Rocco D’Antuono1, Tommaso VirgilioID1, Santiago F. Gonza´lez1, Adriano AguzziID2, Luca VaraniID1 Marco BardelliID1☯, Karl FrontzekID2☯, Luca Simonelli1, Simone HornemannID2, Mattia Pedotti1, Federica Mazzola1, Manfredi CartaID2, Valeria EckhardtID2, Rocco D’Antuono1, Tommaso VirgilioID1, Santiago F. Gonza´lez1, Adriano AguzziID2, Luca VaraniID1 1 Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland, 2 Institute of Neuropathology, University of Zurich, Zurich, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * adriano.aguzzi@usz.ch (AA); luca.varani@irb.usi.ch (LV) OPEN ACCESS OPEN ACCESS Citation: Bardelli M, Frontzek K, Simonelli L, Hornemann S, Pedotti M, Mazzola F, et al. (2018) A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity. PLoS Pathog 14(10): e1007335. https://doi.org/10.1371/ journal.ppat.1007335 Editor: Surachai Supattapone, Dartmouth Medical School, USA, UNITED STATES Received: March 29, 2018 Accepted: September 13, 2018 Published: October 1, 2018 Copyright: © 2018 Bardelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Bardelli M, Frontzek K, Simonelli L, Hornemann S, Pedotti M, Mazzola F, et al. (2018) A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity. PLoS Pathog 14(10): e1007335. https://doi.org/10.1371/ journal.ppat.1007335 Editor: Surachai Supattapone, Dartmouth Medical School, USA, UNITED STATES Received: March 29, 2018 Accepted: September 13, 2018 Published: October 1, 2018 Copyright: © 2018 Bardelli 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. Citation: Bardelli M, Frontzek K, Simonelli L, Hornemann S, Pedotti M, Mazzola F, et al. (2018) A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity. PLoS Pathog 14(10): e1007335. https://doi.org/10.1371/ journal.ppat.1007335 Editor: Surachai Supattapone, Dartmouth Medical School, USA, UNITED STATES Received: March 29, 2018 Accepted: September 13, 2018 Published: October 1, 2018 Copyright: © 2018 Bardelli 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. Abstract Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy. RESEARCH ARTICLE Citation: Bardelli M, Frontzek K, Simonelli L, Hornemann S, Pedotti M, Mazzola F, et al. (2018) A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity. PLoS Pathog 14(10): e1007335. https://doi.org/10.1371/ journal.ppat.1007335 Introduction Prions are the causative agent of sporadic, hereditary and iatrogenic forms of transmissible spongiform encephalopathies, which afflict humans and broad spectrum of mammals and are invariably fatal[1–3]. Whereas Bovine Spongiform Encephalopathy (the most prevalent prion disease in the 1990s, also known as “Mad Cow” disease) has been largely defeated, Chronic Wasting Disease, which affects deer, elk and moose, remains prominent in parts of the US, Canada, South Korea and has recently reached Norway[4]. These findings are raising renewed concerns about the contamination of the food chain. Transmission of infectious animal mate- rial to humans causes variant Creutzfeldt-Jakob disease (vCJD). A common Met/Val polymor- phism at codon 129 of the PRNP gene is assumed to be important in susceptibility of humans to prion infections, with homozygous individuals (Met/Met and Val/Val) being overrepre- sented in collectives of CJD patients[5]. The recent discovery of a case of vCJD in a 36-year- old man producing both M129 and V129 variants of PrP, which is much more frequent in the population and is thought to conduce to a disease developing more slowly, has led to sugges- tion that we might be facing a new wave of vCJD cases [6]. Human prion diseases continue to be intractable and are poorly understood at the molecu- lar level. It is firmly established that conversion of cellular prion protein (PrPC) into a toxic, self-replicating form (scrapie, PrPSc) leads to the formation of aggregates [2,7]. How such aggregates induce toxicity, however, is largely unknown. Antibodies against PrP have been proposed as a valid therapeutic strategy against prion dis- eases, much like antibodies targeting the amyloid-β protein are showing promise in clinical tri- als against Alzheimer’s disease (AD) [8,9]. Furthermore, anti-PrP antibodies were shown to be protective in preclinical models of AD [10]. On the other hand, recent literature reports [11] have highlighted potential safety issues, since an epitope-dependent subset of anti-PrP anti- bodies have been found to cause prion mediated neurodegeneration. PrP has a structured globular domain (GD), whose general architecture is highly conserved amongst mammals, and an unstructured N-terminal region, often referred to as flexible tail (FT). Several antibodies against the globular domain were shown to elicit neuronal toxicity [11,12]. The exact epitope on the GD, rather than the binding affinity or other properties, appears to be the main determinant of toxicity. One such neurotoxic antibody with intriguing properties, POM1, was extensively characterized [11,13]. Neuroprotective bispecific anti-prion antibody design, data collection and analysis, decision to publish, or preparation of the manuscript. design, data collection and analysis, decision to publish, or preparation of the manuscript. neurotoxic antibodies cause the formation of soluble PrP oligomers that cause toxicity on PrP expressing cell lines; these are not formed in the presence of prion protective antibod- ies. We suggest that these soluble species might play a role in prion toxicity, similarly to what is generally agreed to happen in other neurodegenerative disorders. Competing interests: The authors have declared that no competing interests exist. Author summary Antibody immunotherapy is considered a viable strategy against prion disease. We previ- ously showed that antibodies against the so-called globular domain of Prion Protein (PrP) can cause PrP dependent neurotoxicity; this does not happen for antibodies against the flexible tail of PrP, which therefore ought to be preferred for therapy. Here we show that simultaneous targeting of both globular domain and flexible tail by a bispecific, combina- tion of a toxic and a non-toxic antibody, results in stronger protection against prion toxic- ity, even if the bispecific is administered when prion pathology is already conspicuous. We hypothesize that neurotoxicity arises from binding to specific “toxicity triggering sites” in the globular domain. We designed our bispecific with two aims: i) occupying one such site and preventing prion or other factors from docking to it and ii) binding to the flexible tail to engage the region of PrP necessary for neurotoxicity. We also show that Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: LV is grateful for support by Swiss National Science Foundation (grant 310030_166445 and grant 157699), Synapsis Foundation -Alzheimer Research Switzerland ARS- and Lions Club Monteceneri. KF is funded by a research grant from the Theodor and Ida Herzog- Egli foundation and a "Rising Star“ grant by Ono Pharmaceuticals. The funders had no role in study 1 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Design of scPOM-bi, a bispecific POM1-POM2 antibody We fused the toxic antibody POM1 with POM2, capable of preventing its toxicity if adminis- tered before POM1, by joining their variable regions in single chain format with a (GGGGS)x3 linker commonly utilized in non-natural antibodies [19,20]. We have had considerable success in using this format to produce bispecific antibodies against various targets. Although the order of the variable fragments and linker size might affect binding and efficacy, computa- tional simulations showed that the chosen design, with the POM1 moiety preceding POM2 and the chains arranged as VL-VH-VH-VL, was compatible with binding to PrP. Indeed, the resulting bispecific nanobody, scPOM-bi, was produced in E. coli and characterized to be func- tional, monomeric and folded with a melting temperature of 75 ˚C (S1 Fig). We did not, there- fore, explore the production of alternative constructs. Computational docking and molecular dynamics simulations, based on the available experimental structures of POM1 in complex with PrP globular domain [21] and POM2 bound to a FT derived peptide [22], indicate that the size and orientation of scPOM-bi is compatible with bivalent engagement of PrP (Fig 1). Neuroprotective bispecific anti-prion antibody toxicity and compounds that rescue POM1-induced toxicity also alleviate PrPSc-induced toxic- ity [15,16]. Overall, the observations indicate that antibody binding to specific sites in the glob- ular domain triggers a toxic process, mediated by the FT, which converges with that initiated by infectious prions. However, toxic prion antibodies do not generate prion infectivity [17]. The above leads us to propose that the binding of POM1 to a “toxicity triggering site” might emulate the docking of PrPSc (or other toxic factors) to the GD. According to this hypothesis, a molecule that occupies the POM1 binding site in the GD might prove beneficial by preventing interaction with PrPSc and, consequently, toxicity. Here we designed a bispecific antibody formed by the POM1 variable region, which binds the GD, and the POM2 variable region, which binds to the FT and was shown to prevent POM1-mediated toxicity. We show here that the POM1-POM2 bispecific single chain anti- body (called scPOM-bi), a combination of the toxic POM1 and non-toxic POM2 antibodies, is capable of preventing prion toxicity in COCS even when given 21 days post infection, when signs of prion pathology are already visible[18]. POM1 forms soluble PrP-containing oligo- mers that cause toxicity to PrP expressing cells. By contrast, delivery of scPOM-bi prevents for- mation of soluble oligomers. scPOM-bi shows increased protection in comparison to the isolated POM2 or to a mixture of POM1 and POM2, despite having similar binding affinity, suggesting that simultaneous targeting of globular domain and flexible tail might represent an optimized strategy for immunotherapy of prion diseases. Introduction POM1 binds with low nanomolar affinity to a discontinuous epitope comprising the α1-α3 region of the GD. PrP expressing mice and cerebellar organotypic cultured slices (COCS) exposed to POM1 show rapid neuro- toxicity [11]. Intriguingly, toxicity was prevented by a deletion of the octapeptide repeats in the FT, even if this deletion did not affect the POM1 epitope. Antibodies against the FT, such as POM2, were also capable of preventing POM1-mediated toxicity, but only if administered before POM1. POM1 and bona fide prions exert similar toxic effects such as neuronal cell loss, astrogliosis, microgliosis and spongiform change. PrPSc, the proteinase K-resistant form of prion protein, is used as a surrogate marker for prions and believed to be the infectious agent[14]. In both POM1 and prions, metabotropic glutamate receptors play an important role in downstream PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 2 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 scPOM-bi binds with high affinity to globular domain and flexible tail of PrP Surface plasmon resonance (SPR) assays showed that scPOM-bi binds PrP with low nanomo- lar affinity, stronger than that of its individual single chain components due to avidity effects, resulting in slower dissociation, when the two antigen binding sites are engaged (Fig 3). Indeed, scPOM-bi was found to bind to both a PrP construct lacking the FT and to one lacking the GD with affinity similar to that of its individual components, scPOM1 and scPOM2 (Fig 3), confirming that both paratopes of scPOM-bi correctly recognize their cognate epitopes. scPOM-bi avidity can arise from two binding modes: simultaneous engagement of the GD and FT binding sites on a single PrP molecule (intramolecular) or binding of the POM1 site on one PrP molecule and the POM2 site on another (intermolecular). Both options are structur- ally allowed according to molecular dynamics simulations (Fig 1). SPR assays performed with different quantities of immobilized PrP indicate that intermolecular binding is available to scPOM-bi (see S1 Text and S3 Fig for details). scPOM-bi protects from prion toxicity when administered late after prion infection When infected with prions, COCS faithfully mimic prion pathology and are easily accessible to pharmacological manipulation [23]. In contrast to POM1, chronic treatment with scPOM-bi for 21 days did not produce observable toxicity in COCS despite comprising the toxic POM1 moiety (Fig 2A). Furthermore, simultaneous addition of the individuals POM1 and POM2 to COCS resulted in neurotoxicity, indicating that the bispecific has different properties than the simple sum of its parts. We added scPOM-bi to COCS from PrPC-overexpressing Tga20 mice infected with either RML6 prions (RML6 = passage 6 of the Rocky Mountain Laboratory strain, mouse-adapted scrapie prions) or non-infectious brain homogenate (NBH) as a con- trol. 45 days post infection (dpi), immunohistochemical staining for NeuN, identifying PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 3 / 22 Neuroprotective bispecific anti-prion antibody Fig 1. A bispecific immunotweezer formed by a combination of toxic (POM1) and non-toxic (POM2) antibodies. (A) The single chain variable domains of POM1 and POM2 are joined by a flexible linker to yield the bispecific scPOM-bi, schematic representation. B) Computational molecular dynamics model of scPOM-bi in complex with mPrP; intra- molecular (left and S1 Movie) and inter-molecular (right and S2 Movie) binding modes are shown. They are both structurally accessible to scPOM-bi. https://doi org/10 1371/journal ppat 1007335 g001 Fig 1. A bispecific immunotweezer formed by a combination of toxic (POM1) and non-toxic (POM2) antibodies. (A) The single chain variable domains of POM1 and POM2 are joined by a flexible linker to yield the bispecific scPOM-bi, schematic representation. B) Computational molecular dynamics model of scPOM-bi in complex with mPrP; intra- molecular (left and S1 Movie) and inter-molecular (right and S2 Movie) binding modes are shown. They are both structurally accessible to scPOM-bi. https://doi.org/10.1371/journal.ppat.1007335.g001 https://doi.org/10.1371/journal.ppat.1007335.g001 neurons, showed widespread neuronal degeneration in the presence of RML but not NBH. By contrast, treatment with scPOM-bi prevented RML-induced neurotoxicity even when admin- istered at 21 dpi (Fig 2B and S2 Fig). The anti-FT antibody POM2 did not afford similar pro- tection levels at 21 dpi, despite being used at 5-fold higher molarity than scPOM-bi (Fig 2B) and having comparable binding affinity for PrP (Fig 3). PrPSc levels, detected by proteinase K- digestion of tissue inoculated with RML, remained constant in prion-infected Tga20 COCS treated with scPOM-bi for 21days (Fig 2C), suggesting that neuroprotection is not primarily mediated by reduced amounts of PrPSc in RML infected COCS. https://doi.org/10.1371/journal.ppat.1007335.g001 Neuroprotective bispecific anti-prion antibody Fig 2. The bispecific scPOM-bi antibody protects against prion infection even when administered 21 days post infection (dpi). (A) Chronic treatment with scPOM-bi for 21 days did not produce observable toxicity in COCS, contrary to POM1. Furthermore, simultaneous addition of the individuals POM1 and POM2 to COCS resulted in neurotoxicity, indicating that the bispecific has different properties than the simple sum of its parts Area staining of neuronal nuclei by NeuN is shown on the y axis (lower values correlate with toxicity). Column 3 () is from a different experiment with a related negative control on which the data was normalized to. (B) scPOM-bi prevents RML induced neurotoxicity even when added 21 dpi (top). Despite similar binding affinity for PrP, POM2-IgG does not achieve similar protection at 21 dpi even at 5 fold higher concentration (bottom). COCS inoculated with non- infectious brain homogenate (NBH) are used as control; the images show NeuN and DAPI staining of COCS, scale bar = 500 μm. p<0.01, p<0.001, n.s. = not significant, one-way ANOVA with Dunnett’s post-hoc test. Upper panel: n = 9 biological replicates (1 COCS = 1 biological replicate) for all treatment groups except for RML alone (n = 8). Lower panel: n = 9 biological replicates for all treatment groups. Images of all biological replicates depicted in S2 Fig. (C) Western blot shows the presence of PK resistant material in COCS inoculated with RML. Addition of scPOM-bi 21 days after prion inoculation of Tga20 COCS did not show conceivable reduction of PrPSc. https://doi org/10 1371/journal ppat 1007335 g002 Fig 2. The bispecific scPOM-bi antibody protects against prion infection even when administered 21 days post infection (dpi). (A) Chronic treatment with scPOM-bi for 21 days did not produce observable toxicity in COCS, contrary to POM1. Furthermore, simultaneous addition of the individuals POM1 and POM2 to COCS resulted in neurotoxicity, indicating that the bispecific has different properties than the simple sum of its parts Area staining of neuronal nuclei by NeuN is shown on the y axis (lower values correlate with toxicity). Column 3 () is from a different experiment with a related negative control on which the data was normalized to. (B) scPOM-bi prevents RML induced neurotoxicity even when added 21 dpi (top). Despite similar binding affinity for PrP, POM2-IgG does not achieve similar protection at 21 dpi even at 5 fold higher concentration (bottom). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 scPOM-bi inhibits the formation of partially PK resistant soluble PrP oligomers POM1 binds the GD and was shown to have neurotoxic effects mediated by the FT [11]. Solu- ble oligomers may be the toxic species responsible for Alzheimer’s and other amyloidosis PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 4 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 COCS inoculated with non- infectious brain homogenate (NBH) are used as control; the images show NeuN and DAPI staining of COCS, scale bar = 500 μm. p<0.01, p<0.001, n.s. = not significant, one-way ANOVA with Dunnett’s post-hoc test. Upper panel: n = 9 biological replicates (1 COCS = 1 biological replicate) for all treatment groups except for RML alone (n = 8). Lower panel: n = 9 biological replicates for all treatment groups. Images of all biological replicates depicted in S2 Fig. (C) Western blot shows the presence of PK resistant material in COCS inoculated with RML. Addition of scPOM-bi 21 days after prion inoculation of Tga20 COCS did not show conceivable reduction of PrPSc. https://doi.org/10.1371/journal.ppat.1007335.g002 5 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Neuroprotective bispecific anti-prion antibody [24–28] and the smallest infectious unit of prions may also be oligomeric [29]. We thus used dynamic light scattering (DLS) to compare the aggregation properties of toxic and protective antibodies. Single species of size compatible with the monomeric forms of uncomplexed recombinant mouse Prion Protein (mPrP) or antibodies were observed by DLS. Addition of the toxic anti- body POM1 to mPrP in vitro, instead, caused the formation of soluble oligomers with a radius of approximately 200nm (Fig 4A). As a reference, the monomeric scPOM1:GD complex has an elliptical shape of ~7x5 nm according to the available x-ray structure[13]. The radius value is derived from interpretation of DLS data using a spherical model which may or may not be appropriate for these molecules. This, however, does not affect the following qualitative and comparative analysis of the results. When POM1 was added in vitro to a truncated version of mPrP, ΔmPrP(residues 90–230), lacking the N-terminal FT, only monomeric species were found in solution. The disordered region of PrP, in other words, was required for the formation of POM1-induced soluble aggre- gates just like POM1-induced neurotoxicity is abrogated in the absence of the FT. Notably, the soluble oligomers were also not formed when mPrP was bound by the non-toxic POM2. Upon addition of scPOM-bi to mPrP, instead, the presence of soluble oligomers are detected at the first observed time point (~5 minutes after complex formation) but these disappear with time (Fig 4A). It was previously shown that POM1-induced toxicity is inhibited by the prior incubation of PrP with POM2 [15]. Similarly, soluble oligomers were not formed when POM1 was added to a pre-formed POM2:mPrP complex (Fig 4A). By contrast, addition of POM2 to pre-formed POM1:mPrP complexes was not capable of preventing toxicity or eliminate the presence of soluble oligomers (Fig 4A). In contrast to POM2, scPOM-bi was able to eliminate soluble oligomers even when added 5 minutes after the formation of a POM1:mPrP complex, when DLS showed that soluble oligo- mers were already present (Fig 4A). The difference was not due to the presence of two binding sites in scPOM-bi since the bivalent IgG versions of POM1 and POM2 behaved like their sin- gle chain counterpart. Since DLS can only detect the presence of species in solution, we used PAGE/Western blot quantification to investigate the presence of insoluble aggregates. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Neuroprotective bispecific anti-prion antibody Fig 3. The bispecific antibody scPOM-bi binds simultaneously to GD and FT of PrP with high affinity. SPR sensorgrams for binding of scPOM-bi to truncated PrP constructs lacking FT (A) or GD (B) indicate that both antigen bi di i f POM bi l h i Th bi ifi ib d (E) h d ffi i h i Fig 3. The bispecific antibody scPOM-bi binds simultaneously to GD and FT of PrP with high affinity. SPR sensorgrams for binding of scPOM-bi to truncated PrP constructs lacking FT (A) or GD (B) indicate that both antigen binding sites of scPOM-bi correctly engage their target. The bispecific antibody (E) had a stronger affinity than its individual components (C-D) due to avidity resulting in a slower dissociation. The fitting of the experimental data used to calculate the binding constants is in grey. Values for the above plus the full IgG versions of POM1 and POM2 are summarized in (F). https://doi.org/10.1371/journal.ppat.1007335.g003 Fig 3. The bispecific antibody scPOM-bi binds simultaneously to GD and FT of PrP with high affinity. SPR sensorgrams for binding of scPOM-bi to truncated PrP constructs lacking FT (A) or GD (B) indicate that both antigen binding sites of scPOM-bi correctly engage their target. The bispecific antibody (E) had a stronger affinity than its individual components (C-D) due to avidity resulting in a slower dissociation. The fitting of the experimental data used to calculate the binding constants is in grey. Values for the above plus the full IgG versions of POM1 and POM2 are summarized in (F). https://doi.org/10.1371/journal.ppat.1007335.g003 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 6 / 22 Neuroprotective bispecific anti-prion antibody scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showe mers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic mbinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or in s comparable to monomeric forms (blue) were detected in solution when POM1 was in c cking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Sim when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable mers generated by POM1. DLS data is shown for 3 time points after complex formation. ( , scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:m Fig 4. scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showed the presence of soluble oligomers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic antibody to recombinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or inhibit toxicity. Smaller species comparable to monomeric forms (blue) were detected in solution when POM1 was in complex with ΔmPrPC 90- 230, lacking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Similarly small species were found when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable of removing the soluble oligomers generated by POM1. DLS data is shown for 3 time points after complex formation. (n = 5 for scPOM1: mPrP, scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:mPrP then scPOM1 and Fig 4. scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showed the presence of soluble oligomers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic antibody to recombinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or inhibit toxicity. Smaller species comparable to monomeric forms (blue) were detected in solution when POM1 was in complex with ΔmPrPC 90- 230, lacking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Similarly small species were found when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable of removing the soluble oligomers generated by POM1. DLS data is shown for 3 time points after complex formation. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 After formation of the mPrP: Ab complexes, part of the sample was analyzed with DLS and the remaining was subjected to 5’ centrifugation at 20’000 x g. The resulting supernatant was analyzed with PAGE to quantify the amount of mPrP and Ab present in solution (Fig 4B). mPrP or antibodies alone neither formed aggregates over the observed time course (up to 1h, DLS analysis) nor precipitated. When the toxic POM1 antibody bound mPrP or ΔmPrP90-230, ~40% of the amount of mPrP and antibody was detected in the soluble fraction after centrifugation (Fig 4B); however oligo- mers were not formed with the truncated form of PrP, which does not cause neurotoxicity when bound to POM1. Similar levels were detected when POM2 was added after formation of the POM1:PrP complex, which is known to cause cell toxicity. By contrast, little to no mPrP or antibody was present in solution in the non-toxic combina- tions such as the scPOM-bi and POM2 complexes or when POM2 was added before POM1. In contrast to POM2, however, the bispecific was able to eliminate the POM1 induced soluble oligomers when added after formation of the POM1:mPrP complex. Again, the difference was not due to the presence of two binding sites in scPOM-bi since the full IgG versions of POM1 and POM2 behaved like their single chain counterpart. In order to further characterize oligomers and insoluble aggregates through an independent method we measured the size of mPrP:Ab complexes by confocal microscopy. Briefly, mPrP and antibody were mixed in a test tube and, after 20 minutes, analyzed by DLS to characterize PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 7 / 22 Neuroprotective bispecific anti-prion antibody scPOM1:mPrP then scPOM-bi) (B) DLS can only detect soluble material. To investigate the presence of insoluble aggregates we formed the mPrP:Ab complexes in vitro, centrifuged them and analyzed the resulting supernatant with PAGE/Western blot. Soluble material was only detected in toxic combinations (POM1:mPrP or POM1:mPrP followed by POM2, red and orange). The percentage of mPrP and antibody in solution (normalized against isolated PrP or antibody) is shown; data from quantification of band intensity on SDS-PAGE (images in S5 Fig—n = 7 for all samples tested). (C) In order to characterize both soluble oligomers and insoluble aggregates we formed the mPrP:Ab complexes in vitro and deposited the resulting material on microscopy slides. Confocal microscopy indicates that toxic antibody combinations (e.g. POM1:mPrP or POM1:mPrP followed by POM2, red and orange) generate species with smaller average size than protective antibody combinations. The surface area of the detected species is reported on the y axis, the horizontal line represents the average. Differences can also be appreciated by visual inspection of the confocal microscopy images (S4 Fig—scPOM1:mPrP n = 166, scPOM2:mPrP n = 1136, scPOM-bi:mPrP n = 204, scPOM1:mPrP then scPOM2 n = 444, scPOM2:mPrP then scPOM1 n = 74 and scPOM1:mPrP then scPOM-bi n = 1767). D) The soluble oligomers generated by POM1 showed increased resistance to in vitro degradation by proteinase K at 2μg/ml (red). Such resistance was abolished when POM1 bound a mPrP construct lacking the FT (light red) or in non-toxic antibodies (shades of blue). Data from quantification of PK resistant bands on western blot, normalized against isolated PrP (images in S6 Fig—scPOM1:mPrP n = 5, scPOM1:ΔPrP n = 3, scPOM2:mPrP n = 4, scPOM-bi:mPrP n = 4). https://doi.org/10.1371/journal.ppat.1007335.g004 https://doi.org/10.1371/journal.ppat.1007335.g004 the aggregation state. At that point the material was deposited on microscopy slides without centrifugation or other clarification steps. The lowest resolvable structure in laser scanning confocal microscopy images in our experimental settings is diffraction limited at ~120nm. Objects of smaller size are detected as points but the size and diameter cannot be properly resolved or measured. The toxic POM1:mPrP complexes formed smaller particles with average area of ~2.5 μm2. Statistically larger particles with an average of ~6 μm2 were detected for the non-toxic POM2 and scPOM-bi combinations (Fig 4C). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 (n = 5 for scPOM1: mPrP, scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:mPrP then scPOM1 and Fig 4. scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showed the presence of soluble oligomers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic antibody to recombinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or inhibit toxicity. Smaller species comparable to monomeric forms (blue) were detected in solution when POM1 was in complex with ΔmPrPC 90- 230, lacking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Similarly small species were found when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable of removing the soluble oligomers generated by POM1. DLS data is shown for 3 time points after complex formation. (n = 5 for scPOM1: mPrP, scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:mPrP then scPOM1 and PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 8 / 22 Addition of the scPOM-bi:mPrP material to cells 10 minutes after complex formation resulted in toxicity, albeit lower than with scPOM1. However, toxicity was not significant if the material was added 60 minutes after complex formation. (n = 4 for all samples tested). https://doi.org/10.1371/journal.ppat.1007335.g005 Neuroprotective bispecific anti-prion antibody Fig 5. scPOM-bi does not induce toxicity on CAD5 expressing PrPC in comparison to scPOM1. The percentage of PI positive cells for different mPrP:antibodies complexes (A) or antibodies alone (B) on CAD5 PrPC (left) and on CAD5 Prnp-/- (right) are shown; each sample was added to cells after 10’ or 60’ of incubation at RT. The scPOM1:mPrP soluble oligomers caused toxicity in PrPC-expressing CAD5 cells but not in PrPC knock-out (PrP-/-) CAD5. No toxicity was detected when scPOM1 was in complex with the truncated ΔmPrP90-230 lacking the FT. Addition of the scPOM2:mPrP complex resulted in no toxicity, as well. Addition of the scPOM-bi:mPrP material to cells 10 minutes after complex formation resulted in toxicity, albeit lower than with scPOM1. However, toxicity was not significant if the material was added 60 minutes after complex formation. (n = 4 for all samples tested). https://doi.org/10.1371/journal.ppat.1007335.g005 Fig 5. scPOM-bi does not induce toxicity on CAD5 expressing PrPC in comparison to scPOM1. The percentage of PI positive cells for different mPrP:antibodies complexes (A) or antibodies alone (B) on CAD5 PrPC (left) and on CAD5 Prnp-/- (right) are shown; each sample was added to cells after 10’ or 60’ of incubation at RT. The scPOM1:mPrP soluble oligomers caused toxicity in PrPC-expressing CAD5 cells but not in PrPC knock-out (PrP-/-) CAD5. No toxicity was detected when scPOM1 was in complex with the truncated ΔmPrP90-230 lacking the FT. Addition of the scPOM2:mPrP complex resulted in no toxicity, as well. Addition of the scPOM-bi:mPrP material to cells 10 minutes after complex formation resulted in toxicity, albeit lower than with scPOM1. However, toxicity was not significant if the material was added 60 minutes after complex formation. (n = 4 for all samples tested). Fig 5. scPOM-bi does not induce toxicity on CAD5 expressing PrPC in comparison to scPOM1. The percentage of PI positive cells for different mPrP:antibodies complexes (A) or antibodies alone (B) on CAD5 PrPC (left) and on CAD5 Prnp-/- (right) are shown; each sample was added to cells after 10’ or 60’ of incubation at RT. The scPOM1:mPrP soluble oligomers caused toxicity in PrPC-expressing CAD5 cells but not in PrPC knock-out (PrP-/-) CAD5. No toxicity was detected when scPOM1 was in complex with the truncated ΔmPrP90-230 lacking the FT. Addition of the scPOM2:mPrP complex resulted in no toxicity, as well. Furthermore, treatment of the mPrP:Ab complexes formed in vitro with 2μg/mL of Protein- ase K showed that species resistant to low PK concentrations were more abundant when mPrP was bound by the toxic POM1 rather than POM2, scPOM-bi or when POM1 bound a PrP construct lacking the FT (Fig 4D). Overall, the above indicates that protective antibodies prevented the formation of soluble, partially PK resistant oligomers whose formation requires the FT and is correlated with POM1 induced neurotoxicity. The protective scPOM-bi bispecific antibody, but not POM2, was capa- ble of eliminating pre-formed POM1-induced soluble oligomers. In order to test if the POM1-dependent soluble oligomers are indeed toxic, we first formed them in vitro by mixing mPrP with the various antibodies and then added the material to CAD5 cells; after 48 hours the cellular toxicity was evaluated with standard propidium iodide staining and FACS analysis (Fig 5). The scPOM1:mPrP soluble oligomers caused toxicity in PrPC-expressing CAD5 cells but not in PrPC knock-out (PrP-/-) CAD5. No toxicity was detected, instead, when scPOM1 was in complex with the truncated ΔmPrP90-230 lacking the FT. Addition of the scPOM2:mPrP complex resulted in no toxicity, as well. Intriguingly, addi- tion of the scPOM-bi:mPrP material to cells 10 minutes after complex formation resulted in toxicity, albeit lower than with scPOM1. However, toxicity was not significant if the material was added 60 minutes after complex formation. DLS shows that scPOM-bi forms soluble olig- omers, much like POM1, in the initial minutes after complex formation (possibly because some of the bispecific engages mPrP only with the POM1 moiety, thus acting just like POM1) but these disappear with time. There is, in other words, correlation between the presence of soluble oligomers and cellular toxicity. By contrast, addition of the isolated scPOM-bi to CAD5 cells resulted in no toxicity, differently from POM1. A possible interpretation is that formation of toxic scPOM-bi soluble oligomers requires local high concentration of mPrP which is available in vitro but not in cells. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 9 / 22 Neuroprotective bispecific anti-prion antibody reaching implications, including the possibility that autoimmunity to PrP may lead to neuro- degenerative diseases. At the molecular level, it suggests that binding to specific sites in the GD can trigger changes in PrP leading to toxicity. POM1, an antibody binding to the α1-α3 region of the GD, is acutely neurotoxic [11,17,31,32] and is thought to phenocopy the binding of PrPSc or other factors to a toxicity- triggering site of PrPC. According to this hypothesis, occupation of the POM1 docking site on PrP may be beneficial against prion diseases. We thus produced a bispecific antibody (termed scPOM-bi) by fusing the single chain versions of POM1, with the intent of blocking the toxic- ity triggering site in the GD, and POM2, which binds the FT and prevents POM1-induced toxicity. Indeed, scPOM-bi not only lacked neurotoxicity, despite containing the toxic POM1 moi- ety, and in contrast to simultaneous addition of POM1 and POM2 to COCS, but also protected organotypic brain cultures from prion-induced neurodegeneration. Neuroprotection was evi- dent even when scPOM-bi was administered 21 days post infection, when signs of prion pathology were already evident, suggesting that molecular tweezers may form the basis for a rational therapy of prion and possibly other neurodegenerative diseases. scPOM-bi afforded stronger neuroprotection than POM2, suggesting that the concomitant blockade of GD and FT is particularly effective against prion toxicity. POM1, but not POM2 or scPOM-bi, caused recombinant mPrP to form soluble oligomers resistant to low concentration of proteinase K. Soluble oligomers were not formed when POM1 bound to an N-terminally truncated construct lacking the FT, ΔmPrP90-230. There is a correlation between oligomer formation and toxicity: neurotoxic antibodies or combinations (such as POM1) formed soluble oligomers, whereas those that were unable to generate them (such as POM2 or the bispecific tweezer described here) were innocuous or even protective in ex vivo cultured brain slices. Toxicity was detected when the POM1 induced soluble oligomers were formed in vitro from isolated, recombinant mPrP and antibody and subsequently added to PrP-expressing CAD5 cells. There was no toxicity, instead, in knock-out CAD5 lacking PrP or upon addition of the complexes formed by the protective antibodies. The bispecific is pecu- liar: soluble oligomers were detected by DLS 10 minutes after formation of the scPOM-bi: mPrP complex, although less abundant than those formed by POM1. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Discussion The discovery that anti-PrP antibodies can block prion pathogenesis in vivo [30] has suggested that such antibodies might be exploited as therapeutics against human prion diseases. How- ever, caution must be exercised as some antibodies directed against the GD of PrP can trigger PrP-mediated cellular neurotoxicity in the absence of prions [12,31]. This finding has far- PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 10 / 22 Toxicity, lower than with POM1, was detected upon addition of these species to CAD5 cells. By contrast, DLS showed that scPOM-bi soluble oligomers disappear over time just like toxicity disappeared if we waited 60 minutes before adding the scPOM-bi:mPrP complex to CAD5 cells. Curiously, no toxicity was detected if scPOM-bi alone was added to CAD5 cells, whereas POM1 is toxic in this condition. A plausible explanation is that transient, soluble oligomers are only formed if scPOM-bi and mPrP are both present at high concentration (10μM in our assay), whereas the local PrP concentration in CAD5 cells is presumably lower. It is also worth noting that treatment with isolated POM1 causes apparently lower toxicity than treatment with the same amount of POM1:PrP soluble oligomers, further suggesting that the oligomers are relevant for toxicity. The above observations indicate that the induction of oligomeric PrP forms may play a role in POM1 toxicity. Since the antibodies that prevent oligomer formation were protective not only against POM1 but also against prion infection, we suggest that these soluble oligo- mers might also be involved in prion mediated toxicity. This interpretation is in agreement with the conjecture that soluble aggregates are the primary toxic species driving diverse protei- nopathies such as Alzheimer’s and Parkinson’s disease. scPOM-bi and other prion protective antibodies may steer PrP from a toxic oligomerization to a non-toxic aggregation pathway. The fact that formation of PK resistant material is not inhibited by scPOM-bi further corrobo- rates the idea that toxicity, or protection, is mediated by a different downstream event. There is evidence for similar mechanisms in alpha-synuclein and Aβ, where non-toxic aggregates of PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 11 / 22 Neuroprotective bispecific anti-prion antibody Fig 6. The bispeficic antibody scPOM-bi prevents the formation of soluble PrP oligomers and protects from prion neurotoxicity even when administered late after infection. Addition of POM1 antibody (top), but not POM2 or scPOM-bi, to PrPC generates soluble, pK resistant PrP oligomers (red) whose presence correlates with toxicity (top); subsequent addition of the neuroprotective scPOM-bi, but not POM2, eliminates them in favor of larger, non-toxic aggregates (blue). Small soluble oligomers might also be responsible for prion induced toxicity (bottom) similarly to other amyloidosis. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 CAD5 PrPC and Prnp-/- In order to assess PrPC-dependent toxicity of the soluble oligomers in vitro we generated Prnp knock-out versions (Prnp-/-) of the murine neuroblastoma cell line CAD5 by CRISPR/Cas9. CAD5 is a subclone of the central nervous system catecholaminergic cell line CAD showing particular susceptibility to prion infection [38,42]. To avoid expression of aberrant or trun- cated versions of PrPC or deletion of the splice acceptor site that would lead to pathological overexpression of Doppel (Dpl) mRNA [43], we used single-stranded guide RNA (sgRNA) cloned into the MLM3636 plasmid aiming at a protospacer adjacent motif (PAM) site in the signal peptide of Prnp (S7A Fig). Cells were co-transfected with MLM3636 and the hCas9 plas- mid followed by transient antibiotic selection. Subsequently, 7 clones were manually picked, expanded and subjected to further characteri- zation. Cells were lysed and PrPC levels were measured by POM1/POM2 sandwich ELISA. Herein, 7 CAD5 Prnp-/- candidate clones #A6, #C2, #C6, #C12, #H7, #H9 and #H12 all showed near-zero PrPC levels comparable to the established Prnp-/- cell line HPL (p>0.05, one-way ANOVA with Dunnett’s post-hoc test, S7B Fig) [44]. 5 clones were further assessed on western blot, where no detectable PrPC levels could be observed (S7C Fig), suggesting a successful knock-out of PrPC in all 5 Prnp-/- candidate clones. DNA was extracted from expanded cells of clones #C2 and #C12 and the mutagenized region was sequenced by PCR amplification of the open reading frame (ORF) of Prnp. Amplified products were cloned into the pCR-Blunt II-TOPO plasmid (Invitrogen) and 10 colonies per clone were sequenced by Sanger sequenc- ing. Herein, #C2 showed four different mutations, i.e., three deletions and one insertion, while #C12 showed two different deletions proximal to the PAM (S7D Fig). These results indicate multiploidy to be more likely in #C2 than in #C12, hence all further experiments are per- formed with the CAD5 Prnp-/- clone #C12. For CRISPR/Cas9-aided generation of CAD5 knock-out cells, mouse Prnp sgRNA was designed using the web-based tools http://crispr.mit.edu/ and http://zifit.partners.org/ZiFiT/ CSquare9GetOligos.aspx (last access on May 15th 2017). The sgRNA expression plasmid MLM3636 was a gift from Keith Joung (Addgene plasmid # 43860, www.addgene.org). Cerebellar organotypic slice cultures (COCS) Preparation of COCS was undertaken as described elsewhere [39]. Briefly, 350 μm thick COCS were prepared from 9–12 day old Tga20 or ZH3 pups [40]. Inoculation of COCS was per- formed with 100 μg brain homogenate per 10 slices from terminally sick prion-infected (RML6) or NBH from CD1 mice, diluted in 2 mL physiological Grey’s balanced salt solution [41]. The infectious brain homogenate was added to the free-floating COCS for 1 h at 4˚C then washed, and 5–10 slices were placed on a 6-well PTFE membrane insert. Antibodies were first added 1, 10 or 21 days post-infection then re-added with every medium change (three times a week). Neuroprotective bispecific anti-prion antibody PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 scPOM-bi might be able to eliminate them just as it does with the POM1-induced oligomers whereas POM2 might not, which would explain why only the bispecific is neuroprotective even at late administration (21 days post infection, dpi). https://doi org/10 1371/journal ppat 1007335 g006 Fig 6. The bispeficic antibody scPOM-bi prevents the formation of soluble PrP oligomers and protects from prion neurotoxicity even when administered late after infection. Addition of POM1 antibody (top), but not POM2 or scPOM-bi, to PrPC generates soluble, pK resistant PrP oligomers (red) whose presence correlates with toxicity (top); subsequent addition of the neuroprotective scPOM-bi, but not POM2, eliminates them in favor of larger, non-toxic aggregates (blue). Small soluble oligomers might also be responsible for prion induced toxicity (bottom) similarly to other amyloidosis. scPOM-bi might be able to eliminate them just as it does with the POM1-induced oligomers whereas POM2 might not, which would explain why only the bispecific is neuroprotective even at late administration (21 days post infection, dpi). https://doi.org/10.1371/journal.ppat.1007335.g006 size and conformation different from those of toxic soluble oligomers were found [33–35]. Distinct tauopathies are linked to different molecular conformers of aggregated Tau, as well [36]. Furthermore, PrPSc conformers of different size, compatible with what we observed by DLS, and shape were found in strains with different properties and infectivity[37,38]. The scPOM-bi bispecific antibody, but not POM2, achieved the elimination of existing olig- omers from a solution of pre-formed POM1:mPrP complexes. scPOM-bi was also more effec- tive than POM2 at blocking prion mediated toxicity. One possibility is that, by bridging across two PrP molecules, scPOM-bi might favor the elongation of preexisting soluble oligomers, leading to larger, non-toxic species (Fig 6). However POM2-IgG can also bridge across two PrP molecules and yet it is less protective than scPOM-bi and cannot eliminate the POM1-in- duced soluble aggregates despite comparably high affinity. Engagement of the globular domain appears to be important, either by inhibiting the binding of other molecules to a toxicity trig- gering site or by locking PrP GD in a non-toxic conformation. Indeed, simultaneous targeting of GD and FT by the bispecific antibody described here resulted in more potent protection, even when given at late timepoints, than simple targeting of the FT. We conclude that the strategy delineated here may be exploited for the development of effective immunotherapeutics against prion and possibly other diseases. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 12 / 22 Enzyme-linked immunosorbent assay (ELISA) For measuring PrPC levels from cell lysates, ELISA was performed as described previously [32]. Herein, 96-well MaxiSorp polystyrene plates (Nunc) were coated with 400 ng/ml POM1 (or POM19) in PBS at 4˚C overnight. Plates were washed three times in PBS + 0.1% Tween-20 (0.1% PBS-T) and blocked with 80 μl per well of 5% skim milk in 0.1% PBS-T for 1.5 h at room temperature. Blocking buffer was discarded and samples and controls were added dissolved in 1% skim milk in 0.1% PBS-T for 1 h at 37˚C. Recombinant, full-length murine PrPC (rmPrP23- 230) was used as positive control, 0.1% PBS-T was used as negative control. Biotinylated POM2 was used to detect PrPC (200 ng/ml in 1% skim milk in 0.1% PBS-T). Neuroprotective bispecific anti-prion antibody to clone the double-stranded DNA Oligomers into the MLM3636 plasmid, using the following reaction: This reaction was put on a thermocycler using the following conditions: 37˚C, 5 minutes 16˚C, 10 minutes (10 cycles with these two steps) 37˚C, 15 minutes 37˚C, 15 minutes 80˚C, 5 minutes 80˚C, 5 minutes The ligated plasmid MLM3636(sgRNAmPrnp) was subsequently transformed into DH5α chemically competent E. coli cells (Invitrogen) and plasmid purification was undertaken using Plasmid Maxi Kit (Qiagen). CAD5 cells were co-transfected using the MLM3636(sgRNAmPrnp) plasmid and the hCas9 plasmid (hCas9 was a gift from George Church, Addgene plasmid # 41815, [46]) dissolved in Lipofectamine 2000 (Invitrogen). After selection of transfected cells with Geneticin (Invitrogen), single colonies were picked and expanded. For sequencing, DNA was extracted from cells using DNeasy Blood & Tissue Kit (Qiagen). PCR amplification with Q5 high-fidelity DNA polymerase was undertaken using the primers Prn-ko F1 (5’—TGC AGG TGA CTT TCT GCA TTC TGG—3’) and P10 rev (5’—GCT GGG CTT GTT CCA CTG ATT ATG GGT AC—3’). After PCR clean-up using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), blunt-end PCR fragments were cloned into Zero Blunt TOPO PCR Clon- ing Kit (Thermo Fisher Scientific) and Sanger Sequencing (Microsynth) was performed to identify mutated Prnp sequences. Western Blot and ELISA was undertaken to confirm Prnp-/- as described below. Unless mentioned otherwise, clone #C12 was used for all experiments. CAD5 PrPC and Prnp-/- For annealing of single-stranded DNA oligomers of sgRNA for subsequent cloning into the MLM3636 plasmid the following ligation reaction was prepared: 10 μl Oligo4 F [100 μM] (5’- ACA CCG CAG TCA TCA TGG CGA ACC TG—3’), 10 μl Oligo4 R [100 μM] (5’—AAA ACA GGT TCG CCA TGA TGA CTG CG—3’), 10 μL of NEB Buffer 2.1 (New England Bio- labs), 70 μl ddH2O. Reaction mix was heated for 4 min at 95˚C on a heating block ThermoStat (Eppendorf), then the heating block was turned off and the reaction was allowed to proceed for 30 min on the block and was then put at 4˚C. Golden Gate assembly [45] was used in order PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 13 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Proteinase K digestion (COCS) COCS were washed twice in PBS and scraped off the slice culture membrane using 10 μL PBS per slice. Slice cultures were homogenized using a TissueLyser LT (Qiagen) small bead mill at 50 Hz for 2 minutes. For determination of PrPSc, RML6 (1 μl of 10% w/v brain homogenate or 2 μg protein per lane) and slice culture homogenates (20 μg protein per lane) of Tga20 COCS were digested with 25 μg mL-1 proteinase K (Roche) at a final volume of 20 μL in PBS for 30 minutes at 37˚C. For inactivation of proteinase K, 7 μL of 4x NuPAGE LDS sample buffer (Thermo Fisher Scientific) was added and samples were boiled at 95˚C for 5 minutes. Equal sample volumes were loaded on Nu-PAGE Bis/Tris precast gels (Life Technologies) and West- ern blotting was performed using the monoclonal anti-PrP antibody POM1 as described else- where[32]. Immunohistochemical stainings and NeuN morphometry 45 days post infection, prior to fixation, COCS were rinsed twice in PBS, fixed in 4% parafor- maldehyde for 2 days at 4˚C, washed twice in PBS and incubated in blocking buffer (0.05% Triton X-100 vol/vol, 0.3% goat serum vol/vol in PBS) for 1 hour at room temperature. NeuN stainings were performed for 3 days at 4˚C with an Alexa-488 conjugated mouse anti-NeuN antibody (Life Technologies) at 1.6 μg/mL in blocking buffer and incubated. After washing for 15 min in PBS, cell nuclei were made visible by a 30 min incubation with DAPI (1 μg/mL) in PBS at room temperature. Slices were then washed again twice in PBS for 15 minutes and mounted with fluorescent mounting medium (DAKO) on a glass slide. NeuN morphometry of COCS was undertaken on images taken on a fluorescent microscope (BX-61, Olympus) at identical exposure times through custom written scripts for the image analysis software cell^P (Olympus) as previously described [11]. Neuroprotective bispecific anti-prion antibody CAD5 PrPC or Prnp-/- were cultured with 20mL Corning Basal Cell Culture Liquid Media—DMEM and Ham’s F-12, 50/50 Mix supplemented with 10% FBS, Gibco MEM Non- Essential Amino Acids Solution 1X, Gibco GlutaMAX Supplement 1X and 0,5mg/mL of Geneticin in T75 Flasks ThermoFisher at 37C 5% CO2. 16 hours before treatment, cells were split into 96wells plates at 25000cells/well in 100μL. Complexes of PrP:Antibodies (1:1 PrP:Ab ratio for monovalent binding, 2:1 PrP:Ab ratio for bivalent binding) or antibodies alone were formed at 5μM final concentration, in 20mM HEPES pH 7,2 150mM NaCl. After 10’ or 60’ upon complex formation, 100μL of each sample, including buffer alone or unrelated antibodies as controls, were added to CAD5 cells, in duplicates. After 48 hours, cells were washed two times with 100μL MACS buffer (PBS + 1% FBS + 2mM EDTA) and resuspended in 100μL MACS buffer. 30” before FACS measurements PI (1μg/mL) was added to cells. Measurements were performed using BD LSRFORTESSA. Statistics: percentage of PI positive cells were plotted in columns as mean with SD. 2way ANOVA test with Tukey test was performed comparing each samples ( p<0.05, p<0.005, p<0.0005, p<0.00001). CAD5 toxicity assay Quantification of toxicity on CAD5 either expressing or lacking PrPC induced by different antibodies alone or in complex with mPrP/ΔmPrP(90–230) was measured as percentage of PI positive cells using Flow Cytometry. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 14 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Neuroprotective bispecific anti-prion antibody was then subjected to 100ns of fully atomistic molecular dynamics simulations (MD) to obtain an energetically favorable and stable conformation. In all cases, the system was initially set up and equilibrated through standard MD protocols: proteins were centered in a triclinic box, 0.2nm from the edge, filled with SPCE water model and 0.15M Na+Cl- ions using the AMBER99SB-ILDN protein force field. Energy minimiza- tion was performed in order to let the ions achieve a stable conformation. Temperature and pressure equilibration steps, respectively at 298˚K and 1 Bar, of 100ps each were then per- formed before performing 300ns molecular dynamics simulations with the above mentioned force field. MD trajectory files were analyzed after removal of Periodic Boundary Conditions. The stability of each simulated complex was verified by root mean square deviation, radius of gyration and visual analysis. Protein production Recombinant mouse PrP full length (23–230), PrP lacking FT (90–230), globular domain only (120–230) or Flexible tail only (23–120) were expressed and purified from E.coli as previously described[47,48]. scPOM1, scPOM2 and scPOM-bi sequences were codon optimized, cloned in frame into a pET21a plasmid (Novagen) and expressed in E.coli Rosetta (scPOM-bi) or Rosetta pLysS (for scPOM1 and scPOM2) cells. Bacterial cells were grown in 2XYT media plus Ampicillin (100μg/mL) and chloramphenicol (34μg/mL) at 25˚C (scPOM1 and scPOM2) or 37˚C (scPOM-bi) until OD600 reached 0.6, then induced with 0.5mM IPTG and harvested 16 (scPOM1 and scPOM2) or 3 (scPOM-bi) hours post-induction. Bacterial pellets were soni- cated with 50mM MES pH6.5 100mM NaCl and 0.5mM DTT (50mL per liter of colture) and centrifuged at 16500rpm (rotor ss34) for 30’ at 4˚C. The pellet was then washed with sonica- tion buffer plus 0.5% of Triton-X100 and then with sonication buffer to remove excess of Tri- ton-X100. The pellet was solubilized in 50mM MES pH 6.5 1M NaCl 6M Guanidine-HCl (Buffer A). Following addition of 0.2% PEI and centrifugation to remove DNA contamination, 60% ammonium sulfate was added to the supernatant to remove traces of PEI. After centrifu- gation the pellet was resuspended in Buffer A, filtered and loaded on pre-equilibrated HisTrap Column (GE healthcare) with Buffer A. The column was washed with at least 5 volumes of Buffer A and eluted with Buffer A plus 500mM Imidazole. Antibody containing fractions were diluted 20 times into refolding buffer (20 mM phosphate buffer pH 10.5, 100 mM NaCl, 200 mM arginine, 1mM Glutathione Reduced and 0.1mM Glutathione Oxidized). scPOM1, scPOM2 and scPOM-bi were finally purified on a Superdex-75 size exclusion column (GE) in PBS buffer. The elution and dynamic light scattering profiles of all proteins were consistent with monomeric species. Full IgG POM1 and POM2 monoclonal antibodies were produced and purified as described previously[32]. Computational modelling and molecular dynamics of scPOM-bi The structure of scPOM-bi used in the computational simulations was modeled on the experi- mental structures of the POM1:PrP124-225 complex (PDB code 4H88) and POM2:octarepeat- peptide (PDB code: 4J8R) complexes. After initial superposition of the POM1 and POM2 moi- ety on the structure of one (for intramolecular binding models) or two (for intermolecular binding models) PrP molecules, the linker joining the two scFv was manually built. The system PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 15 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 Neuroprotective bispecific anti-prion antibody performed with Bio-Rad ProteOn Manager software (version 3.1.0.6). In the PrP dilution experiments used to evaluate avidity effects, serial dilution (1:100, 1:200, 1:500 and 1:1000) of NHS/EDAC compounds were used for GLC chip surface activation in order to limit the amount of immobilized protein. Precipitation assays Quantification of soluble PrP either alone or in complex with different antibodies was per- formed using SDS-PAGE and either comassie staining (for PrP alone and in complex with scFv) or western blot (for PrP/IgG complexes). The precipitation assays were run in parallel to DLS assays in the conditions indicated above. Samples were centrifuged for 2 minutes at 21000g at 4˚C. 25μL of supernatant was collected, mixed with equal volume of sample buffer and loaded on polyacrylamide gel (4% Stacking– 12% running). For comassie staining, SDS-PAGEs were left 10 minutes in 2.5g/L Comassie Brilliant Blue G-250 (Sigma) 40% Metha- nol and 10% Glacial Acetic Acid and then destained using 70% ddH20, 20% Methanol and 10% Glacial Acetic Acid for 1hour at least. Gels were then acquired using ImageQuant LAS 4000 (GE Healthcare) according to standard procedures. For western blot, proteins from SDS-PAGE were transferred onto PVDF membranes, blocked in TBS-Tween20 10% Milk for 10 minutes at RT and probed with an antibody against PrP (POM19 mouse IgG 1μg/mL in TBS-Tween20 for 16 hours at 4˚C) that does not compete with either scPOM1, scPOM2 and scPOM-bi. The primary antibody was detected using a goat anti-Mouse-HRP conjugated anti- body (1:10000 in TBS-Tween20 for 1hour at RT, from ThermoFisher) and developed using Pierce ECL Western Blotting Substrate (ThermoFisher). Chemiluminescence from PrP spe- cific bands were acquired using a ImageQuant LAS 4000 (GE Healthcare) using High Resolu- tion for sensitivity, 1/60 or 1/100 sec exposure time and Precision as exposure type. Quantification of PrP was then performed using Multi Gauge Software (from FujiFilm) with standard protocol, normalizing all samples to PrP Alone control bands. At least 3 independent replicates of the experiments were performed. Statistics: all experiments are shown as mean with SEM. Quantification of PrP was then performed using Multi Gauge Software (from FujiFilm) with standard protocol, normalizing all samples to PrP Alone control bands. At least 3 independent replicates of the experiments were performed. Statistics: all experiments are shown as mean with SEM. Dynamic Light Scattering (DLS) assays The size of PrP either alone or in complex with different antibodies was estimated by Dynamic Light Scattering (DLS) at 25˚C using a “DynaPro—NanoStar” instrument (WYATT) in 10μL at a concentration of 5μM. The PrP:antibody ratio was 1:1 for monovalent binding (e.g. scFv) and 2:1 for bivalent binding species (e.g. full IgG). Readings were taken 2, 5, 10, 20 and 30 min- utes after complex formation. When evaluating addition of a second antibody (e.g. forming a PrP/scPOM1 complex and then adding scPOM-bi), PrP and the first antibody were pre-mixed for 5 minutes and then the second antibody was added. Each time point measurement was per- formed by 10 repetitions of 5 seconds signal integration. A Rayleigh sphere model was used for size estimation. At least 3 repetitions of the same experiment were performed on different, freshly prepared samples. Before complex formation, all samples were dialyzed together in 20mM HEPES pH 7.2 150mM NaCl, centrifuged at 21000g and filtered with 0.22μm filters before measurement. Statistics: all experiments are shown as mean with standard error of the mean (SEM). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 SPR binding assays The binding properties of the complexes between scPOM1, scPOM2, either in single chain or IgG version, and scPOM-bi on different mPrP constructs were analyzed at 25˚C on a ProteOn XPR-36 instrument (Bio-Rad) using 20mM HEPES pH 7.2 150mM NaCl 3mM EDTA and 0.005% Tween-20 as running buffer. 50nM solutions of PrP constructs were immobilized on the surface of a GLC sensor chip through standard amine coupling. Serial dilution of antibod- ies in the nanomolar range were injected at a flow rate of 100 μL/min (contact time 6 minutes); dissociation was followed for 5 minutes. Analyte responses were corrected for unspecific bind- ing and buffer responses by subtracting the signal of both a channel were no PrP was immobi- lized and a channel were no antibody was added. Curve fitting and data analysis were PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 16 / 22 Neuroprotective bispecific anti-prion antibody ratio for monovalent binding (e.g. scFv) and 2:1 for bivalent binding species (e.g. POM-bi) as for DLS assays. After 10’ of incubation at 25C, 2μL of each complex was added to glass micros- copy slides (Thermo Fisher Scientific) and covered with coverslip. The same samples were also subjected to DLS analyses, in parallel. Images were acquired using a Leica TCS SP5 confocal microscope using sequential acquisition settings to visualize aggregates containing labelled antibodies. For each mPrP:Ab complex, 4 fields of view of 246 μm x 246 μm were acquired with a 63X/1.4 NA oil immersion objective. Images were analysed using IMARIS software (Bit- plane). To estimate particles size surfaces were generated in software based on the fluorescent signal from Alexa647 dye (segmentation parameters: surface grain size 0.01 μm, intensity threshold set at 50). Statistics: all particles were shown in dot plot graph as mean with SD. Mann-Whitney test was performed ( p<0.05, p<0.01, p<0.001, p<0.0001). Ethics statement All animal experiments were conducted in strict accordance with the Rules and Regulations for the Protection of Animal Rights (Tierschutzgesetz and Tierschutzverordnung) of the Swiss Bundesamt fu¨r Lebensmittelsicherheit und Veterina¨rwesen BLV. Body weights were measured weekly. All animal protocols and experiments performed were specifically approved for this study by the responsible institutional animal care committee, namely the Animal Welfare Committee of the Canton of Zu¨rich (permit numbers Versuchstierhaltung 123, ZH139/16 and 90/2013). All efforts were made to minimize animal discomfort and suffering. Supporting information Supporting information S1 Text. SPR analysis of scPOM-bi binding to PrP. (DOCX) S1 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with one molecule mPrP. (MPG) S2 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with two molecules mPrP. (MPG) S1 Fig. Thermal denaturation of scPOM-bi measured by CD spectroscopy, indicating a melting temperature of 75˚C. (TIF) S2 Fig. Fluorescent micrographs of all biological replicates. (A) scPOM-bi; (B) POM2 IgG. Scale bar = 500 μm. (TIF) S3 Fig. Values of association (ka, left) and dissociation constants (kd, right) for scPOM1 (yellow), POM1-IgG (red) and scPOM-bi (blue) at different concentrations of PrP, mea- sured by SPR. The dilution of PrP on the sensor chip is reported. The dissociation constant, but not the association, is affected by PrP dilution, indicating that intermolecular avidity effects are present in POM1 IgG and scPOM-bi. See S1 Text for further details. (TIF) S4 Fig. Representative confocal microscopy images of PrP:Ab oligomers and aggregates (scale bar = 10μm). See main text (Fig 4) for quantification and methods for experimental details. Briefly, complexes between recombinant mPrP and antibodies were formed in vitro Confocal analyses of PrP/Ab Oligomers Antibodies were labelled with Alexa Fluor 647 NHS Ester (Thermo Fisher Scientific) in PBS carbonate pH 8.3 with a 1:2 Ab:Dye ratio; unbound dyes were removed using Size Exclusion Chromatography. Complexes between mPrP and Abs were generated at 5μM in 10μL with 1:1 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 17 / 22 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1007335 October 1, 2018 S1 Text. SPR analysis of scPOM-bi binding to PrP. (DOCX) S1 Text. SPR analysis of scPOM-bi binding to PrP. (DOCX) S1 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with one molecule mPrP. (MPG) S1 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with one molecule mPrP. (MPG) S2 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with two molecules mPrP. (MPG) S1 Fig. Thermal denaturation of scPOM-bi measured by CD spectroscopy, indicating a melting temperature of 75˚C. (TIF) S2 Fig. Fluorescent micrographs of all biological replicates. (A) scPOM-bi; (B) POM2 IgG. Scale bar = 500 μm. (TIF) S3 Fig. Values of association (ka, left) and dissociation constants (kd, right) for scPOM1 (yellow), POM1-IgG (red) and scPOM-bi (blue) at different concentrations of PrP, mea- sured by SPR. The dilution of PrP on the sensor chip is reported. The dissociation constant, but not the association, is affected by PrP dilution, indicating that intermolecular avidity effects are present in POM1 IgG and scPOM-bi. See S1 Text for further details. (TIF) S4 Fig. Representative confocal microscopy images of PrP:Ab oligomers and aggregates (scale bar = 10μm). See main text (Fig 4) for quantification and methods for experimental details Briefly complexes between recombinant mPrP and antibodies were formed in vitro S2 Movie. Fully atomistic molecular dynamics simulation of 30 ns of the model of scPOM- bi in complex with two molecules mPrP. (MPG) S1 Fig. Thermal denaturation of scPOM-bi measured by CD spectroscopy, indicating a melting temperature of 75˚C. (TIF) S2 Fig. Fluorescent micrographs of all biological replicates. (A) scPOM-bi; (B) POM2 IgG. Scale bar = 500 μm. (TIF) S2 Fig. Fluorescent micrographs of all biological replicates. (A) scPOM-bi; (B) POM2 IgG. Scale bar = 500 μm. (TIF) S3 Fig. Values of association (ka, left) and dissociation constants (kd, right) for scPOM1 (yellow), POM1-IgG (red) and scPOM-bi (blue) at different concentrations of PrP, mea- sured by SPR. The dilution of PrP on the sensor chip is reported. The dissociation constant, but not the association, is affected by PrP dilution, indicating that intermolecular avidity effects are present in POM1 IgG and scPOM-bi. See S1 Text for further details. (TIF) S4 Fig. Representative confocal microscopy images of PrP:Ab oligomers and aggregates (scale bar = 10μm). See main text (Fig 4) for quantification and methods for experimental details. Acknowledgments We thank Robyn Grace Holden for graphic assistance and Diego Morone for microscope tech- nical support. Neuroprotective bispecific anti-prion antibody and the material deposited on microscopy slides without centrifugation or other purification steps. Species of different size are apparent when mPrP is in complex with toxic (POM1) or non toxic antibodies. (TIF) S5 Fig. Precipitation assays confirmed that the toxic scPOM1:mPrP complex generates soluble oligomers containing both PrP and antibody (A). No soluble material was present in the com- plexes between mPrP and scPOM2 (B), scPOM-bi or when scPOM-bi was added after scPOM1 (A). See main text (Fig 4) for quantification and methods for experimental details. Briefly, after formation of the mPrP:Ab complexes in vitro the samples were centrifuged at 20’000 x g. The amount of mPrP and Ab present in the resulting supernatant was estimated with PAGE/Western Blot. The quantity of soluble material is reported as percentage of soluble mPrP or Ab alone, which do not precipitate or form aggregates over the observed time frame. The variability is due to the experimental set up and to the fact that we are analyzing transient, non-homogeneous species that are likely to change over time. Such variability does not affect the statistical significance of the measures. S6 Fig. Low concentration PK resistance assay. mPrP:Ab complexes were formed in vitro and 2μg/mL of Proteinase K were added. The presence of PK resistance species was assessed by western blot. An increased amount of species resistant to PK was detected in the scPOM1: mPrP complexes, but only if the flexible tail was present, which correlates to toxicity and pro- tection assays. See main text (Fig 4) for quantification and statistics. (TIF) S7 Fig. Generation of a stable CAD5 Prnp-/- cell line. (A) Design of sgRNA for CRISPR/Cas9 mediated generation of CAD5 Prnp-/- cells. A PAM in the coding sequence of the signal pep- tide was chosen. (B) ELISA of 7 candidate CAD5 Prnp-/- clones showed similar PrPC levels compared to the established Prnp-/- cell line HPL (p>0.05, one-way ANOVA with Dunnett’s post-hoc test, all clones versus HPL), 5 of which were further assessed by PrPC western blot, confirming lack of PrPC expression (C). (D) Sanger sequencing of PCR amplified Prnp ORF showed n = 4 different mutations in #C2 and n = 2 different mutations, labelling according to (A). The splice acceptor site is unaffected in both of the constructs. (TIF) S1 Text. SPR analysis of scPOM-bi binding to PrP. (DOCX) Briefly, complexes between recombinant mPrP and antibodies were formed in vitro S4 Fig. Representative confocal microscopy images of PrP:Ab oligomers and aggregates (scale bar = 10μm). See main text (Fig 4) for quantification and methods for experimental details. 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Aguzzi A, Weissmann C (1997) Prion research: the next frontiers. Nature 389: 795–798. Neuroprotective bispecific anti-prion antibody Methodology: Marco Bardelli, Mattia Pedotti, Rocco D’Antuono, Tommaso Virgilio, Santiago F. Gonza´lez, Luca Varani. Project administration: Adriano Aguzzi, Luca Varani. Writing – original draft: Marco Bardelli, Karl Frontzek, Adriano Aguzzi, Luca Varani. Writing – review & editing: Marco Bardelli, Karl Frontzek, Adriano Aguzzi, Luca Varani. Methodology: Marco Bardelli, Mattia Pedotti, Rocco D’Antuono, Tommaso Virgilio, Santiago F. Gonza´lez, Luca Varani. Methodology: Marco Bardelli, Mattia Pedotti, Rocco D’Antuono, Tommaso Virgilio, Santiago F. Gonza´lez, Luca Varani. Project administration: Adriano Aguzzi, Luca Varani. Writing – original draft: Marco Bardelli, Karl Frontzek, Adriano Aguzzi, Luca Varani. Writing – review & editing: Marco Bardelli, Karl Frontzek, Adriano Aguzzi, Luca Varani. 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https://openalex.org/W4393978631	https://www.intechopen.com/citation-pdf-url/89250	English	null	NMDA Receptors in Health and Disease	Physiology	2,024	cc-by	12,572	Abstract NMDA receptors (NMDARs) are a subtype of ionotropic glutamate receptors that mediate excitatory neurotransmission and synaptic plasticity in the brain. NMDARs play important roles in various normal brain functions such as learning, memory, and cognition, but also contribute to the pathogenesis of several develop- mental, neurological, and psychiatric disorders. Alterations in NMDARs can result in either hypo- or hyperfunction of NMDARs, which can impair neuronal viability, synaptic efficacy, and network oscillations. In this review, we summarize the current knowledge on the involvement of NMDA receptors in Alzheimer’s disease, autism spectrum disorder, epilepsy, and schizophrenia. We also highlight the potential therapeutic strategies that target NMDAR modulation and dysfunction in these disorders. Keywords: NMDA receptors, Alzheimer’s disease, autistic spectrum disorder, epilepsy, schizophrenia Chapter Yue-Qiao Huang 2. NMDARs in health NMDARs are involved in various processes of the synapse, such as its formation, modification, learning, memory, and cognitive functions [2]. NMDARs are different from AMPARs and kainate receptors in that they need both glycine (or D-serine in the body) and glutamate to be activated. They allow calcium to enter the cell and are blocked by magnesium when the cell is at rest [2]. NMDARs can detect the timing of pre-synaptic glutamate release and post-synaptic membrane depolarization, which makes them unique among the receptors. q g p NMDARs are composed of four subunits, each of which belongs to one of three families: GluN1, GluN2, and GluN3. GluN1 is the obligatory subunit that forms the core of the receptor and binds glycine as a co-agonist. GluN2 subunits (A–D) deter- mine the pharmacological and biophysical properties of the receptor and bind gluta- mate as the main agonist. GluN3 subunits (A and B) modulate the receptor function and can also bind glycine. The subunit composition of NMDARs varies depending on the brain region, cell type, and developmental stage, resulting in a diversity of recep- tor subtypes and complexes that have distinct roles in synaptic plasticity, learning, memory, and neurological disorders [2]. y g Each subunit of NMDARs has a similar structure that consists of four main domains: the amino-terminal domain (ATD/NTD), the ligand-binding domain (LBD), the transmembrane domain (TMD), and the carboxyl-terminal domain (CTD) [2]. The ATD/NTD is located at the extracellular side of the receptor and is involved in subunit assembly, allosteric modulation, and receptor trafficking. The LBD is also extracellular and contains the binding sites for glutamate and glycine, as well as for various modulators such as zinc, magnesium, polyamines, protons, and neurosteroids. The TMD spans the membrane four times (TM1, TM2, TM3, and TM4) and forms the ion channel pore that allows the flux of sodium, potassium, and calcium ions upon receptor activation. The CTD is intracellular and interacts with various signaling molecules and scaffolding proteins that regulate the receptor localization, function, and coupling to downstream pathways. p g p y Each NMDAR is composed of two GluN1 subunits and two GluN2 or GluN3 subunits, which can be either identical (diheteromers) or different (triheteromers). The subunit composition of NMDARs affects their biophysical and pharmacological properties, such as their affinity to co-agonists (glycine and glutamate), sensitivity to modulators (zinc and magnesium), and interaction with other drugs. 1. Introduction Glutamate receptors are a diverse group of proteins that play a crucial role in the nervous system. They are responsible for mediating the majority of excitatory neurotransmission in the brain. Glutamate receptors can be broadly classified into two main types: ionotropic and metabotropic [1, 2]. Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that allow ions to flow across the cell membrane in response to the binding of glutamate [2]. They can be further divided into three main subtypes: N-methyl-D-aspartate receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and kainate receptors [2]. Each subtype has distinct properties and roles in synaptic transmission and plasticity. Metabotropic glutamate receptors (mGluRs), on the other hand, are G protein-coupled receptors that activate intracellular signaling pathways in response to glutamate [3]. Both iGluRs and mGluRs are involved in various physiological processes, such as neuronal development, synaptic plasticity, learning, and memory [2, 3]. 1 1 Cell Communication and Signaling in Health and Disease 2. NMDARs in health NMDARs also have different expression patterns and functions in different brain regions and developmental stages. NMDARs are not static but rather undergo dynamic changes in their trafficking, organization, diffusion, and surface expression. These processes are regulated by various factors, such as protein-protein interactions, post-translational modifications, extracellular matrix components, and synaptic activity [4–7]. y y The term diheteromer or triheteromer refers to the number of distinct subunits in an NMDAR, not the total number of subunits, which is always four. The most com- mon triheteromer in the adult forebrain is GluN1/GluN2A/GluN2B, which constitutes a major synaptic NMDAR population. Other triheteromers, such as GluN1/GluN2A/ GluN2C, GluN1/GluN2A/GluN2D, and GluN1/GluN2B/GluN2D, have also been reported to function in specific brain regions. NMDARs that contain GluN3 sub- units are considered atypical and unconventional. GluN3A can form triheteromers with GluN1 and GluN2A or GluN2B in neurons, or with GluN1 and GluN2C in oligodendrocytes. These receptors are thought to play a role in synapse pruning and destabilization [2, 5]. 2 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 NMDARs are crucial for long-term synaptic plasticity induction at different synapses and for cognitive functions. Animal models showed that non-competitive NMDAR antagonists (such as PCP, ketamine, and MK-801) induced cognitive impair- ment similar to schizophrenia [8]. Likewise, in healthy humans, NMDAR inhibition caused cognitive and behavioral dysfunction [9]. For instance, ketamine induced both positive and negative schizophrenia symptoms [10]. Genetically, GluN1 gene global knock-out caused neonatal death [11], confirming the importance of NMDARs. Specific GluN1 knock-out in the hippocampal CA1 region showed that NMDARs are essential for memory formation consolidation [12]. GluN1 ablation in the hippocam- pal CA3 region impaired spatial memory retrieval [13]. Moreover, GluN1 deletion in parvalbumin-positive interneurons disrupted hippocampal synchrony, spatial representations, and working memory [14]. Therefore, the evidence supports the roles of NMDARs in synaptic plasticity and cognition. y p p y g NMDARs have implications for various neurological and psychiatric disorders, depending on whether they are overactivated or underactivated by different factors. When NMDARs are overactivated, they can cause excitotoxicity, which is the harm- ful overstimulation of neurons by glutamate. This can happen in situations such as stroke, trauma, hypoxia, ischemia, and neurological diseases such as Alzheimer’s disease (AD), epilepsy, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS). When NMDARs are underactivated, they can disrupt synaptic plasticity and cognitive functions, and lead to problems such as memory loss, schizo- phrenia, and autism. 2. NMDARs in health In the following sections, we explore the roles of NMDARs in AD, autism spectrum disorder (ASD), epilepsy, and schizophrenia. For other disease states that are not covered here, readers can refer to some excellent recent review articles [15–20]. 3.1.1 Introduction AD is a chronic brain disorder that gradually erodes cognitive functions, memory, and behavior. It is the leading cause of dementia among older people and a major public health challenge [21]. The exact mechanisms of AD are not fully understood, but several factors contribute to its onset and progression. These include genetic variations, accumu- lation of amyloid-beta (Aβ) plaques, formation of tau tangles, inflammation of brain cells, oxidative damage, impaired blood flow, reduced acetylcholine levels, and environmental influences [21]. Currently, there is no cure for AD, but some therapies can help to reduce the symptoms and slow down the cognitive deteriora- tion. These include drugs that inhibit acetylcholinesterase or block NMDARs, antioxidants, anti-inflammatory agents, and lifestyle modifications such as diet, exercise, and cognitive training [21]. g y antioxidants, anti-inflammatory agents, and lifestyle modifications such as diet, exercise, and cognitive training [21]. 3.1.3.1 NMDAR trafficking Firstly, Aβ oligomers affect synapses by altering the trafficking and localization of NMDARs, which are crucial for learning and memory. Aβ oligomers can impact on both synaptic and extrasynaptic NMDARs, but they preferentially impair NMDARs containing the GluN2B subunit, which are more vulnerable to excessive calcium influx and excitotoxicity [26]. Research has demonstrated that Aβ oligomers can suppress long-term potentiation (LTP) [27–29], enhance the induction of long-term depression (LTD) [30, 31], and trigger the internalization of AMPA and NMDA receptors [32–35]. Thus, Aβ1–42 has been found to decrease the surface expression of GluN2B-containing NMDARs in cortical neurons [34, 36]. Aβ oligomers can also bind to the EphB2 receptor (Eph receptor B2) and trigger its proteasome-dependent degradation, leading to reduced surface expression and phosphorylation of GluN2B- containing NMDARs [37]. This results in reduced calcium influx into active spines, impaired induction of LTP, enhanced induction of LTD, and increased spine loss. These effects of Aβ oligomers on NMDARs contribute to cognitive impairment and neurodegeneration in AD. 3.1.3 NMDARs in AD Both Aβ and tau can interact with NMDARs, which are essential for synaptic plasticity and memory formation. Aβ can interact with both synaptic and extrasynap- tic NMDARs, but it preferentially impairs the trafficking and localization of NMDARs containing the GluN2B subunit, which are more- localized at extrasynaptic sites and vulnerable to excessive calcium influx and excitotoxicity. Aβ also interferes with the signaling pathways downstream of NMDARs, such as by inhibiting CaMKII activity, reducing CREB phosphorylation, activating calcineurin and caspase-3, and inducing tau hyperphosphorylation. These effects impair synaptic plasticity and promote den- dritic degeneration. Moreover, Aβ can bind to other receptors that modulate NMDAR function, such as mGluR5, PrPc, and α7 nicotinic receptors, leading to further synap- tic dysfunction and loss. We will now discuss in more detail how Aβ and tau proteins can interact with NMDARs, and how NMDARs can interact with Aβ and tau in return. 3.1.2 Amyloid and tau hypotheses Both Aβ and tau proteins are normally present in the brain, but they can undergo pathological changes that lead to their accumulation and aggregation [21, 22]. 3 3 Cell Communication and Signaling in Health and Disease Aβ is a peptide that is derived from the cleavage of a larger protein called amyloid precursor protein (APP). Aβ can form insoluble aggregates that deposit as plaques in the brain. These plaques are believed to impair neuronal function and cause neuro- degeneration [23]. Tau is a protein that binds to microtubules, which are essential for intracellular transport and neuronal structure. Tau can become hyperphosphorylated, meaning that it has too many phosphate groups attached to it. Hyperphosphorylated tau can detach from microtubules and form intracellular aggregates called neurofi- brillary tangles. These tangles are believed to disrupt neuronal transport and cause cell death [24]. Both Aβ and tau have been implicated in the cognitive impairment and memory loss that are hallmarks of AD. Both Aβ and tau have been shown to interact with NMDARs, which are essential for synaptic plasticity and memory formation [24, 25]. 3.1.3.3 Tau and NMDARs Tau can interact with NMDARs, influencing their function and vice versa. Pathological tau can lead to synaptic loss and dysfunction, as evidenced by reduced spine density and impaired LTP in tau P301L transgenic mice rTg4510 [43]. The mechanisms behind tau synaptotoxicity are still being investigated, but Fyn kinase is a potential mediator. Fyn kinase, located at postsynaptic densities, can modulate tau-dependent synaptic and cognitive dysfunction. Tau binds to Fyn and enhances its interactions and stabilizing effects with NMDARs, which is required for LTP induc- tion [44]. Deleting tau in mice alters Fyn localization in postsynaptic compartments and reduces NMDAR-dependent excitatory toxicity in response to Aβ [45]. Inhibiting Fyn kinase also reduces tau aggregation, suggesting that tau-Fyn interactions can exacerbate tau pathology in an AD mouse model [46]. Furthermore, tau can bind to Fyn and induce Fyn phosphorylation in the brain of an AD patient. Phosphorylated Fyn enhances interactions between NMDAR and the postsynaptic scaffolding component, PSD95, which can increase excitatory glutamate sensitivity and thereby exacerbate Aβ excitotoxicity [45]. Finally, Aβ can trigger an influx of calcium through NMDARs, which then activates GSK3β. This can lead to excessive phosphorylation of tau protein, causing it to mislocalize to dendrites [47]. 3.1.3.2 NMDAR signaling Besides impairing NMDAR expression and localization, Aβ oligomers may interfere with the signaling pathways that depend on or converge on NMDARs. 4 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 Aβ oligomers can also form ion-permeable channels in cellular membranes, allowing calcium influx into neurons and affecting synaptic plasticity [38–40]. Aβ oligomers can cause overactivation of extrasynaptic NMDARs (eNMDARs), which are located outside the synaptic cleft and have different subunit composition and function from synaptic NMDARs (sNMDARs). eNMDAR activation by Aβ oligomers leads to excessive calcium influx and oxidative stress, which in turn activate various enzymes, such as calpain, caspase, Cdk5, GSK-3β, Fyn, and CaMKII, that can phosphorylate tau, cleave dynamin, disrupt cytoskeleton, and cause endocytosis of AMPARs and NMDARs [41]. These events result in synaptic depression, spine elimination, mitochondrial dysfunction, and neuronal death. On the other hand, sNMDAR activation by physiological glutamate stimulation is beneficial for neuronal survival, synaptic plasticity, and memory formation [42]. Therefore, Aβ oligomers shift the balance between eNMDARs and sNMDARs toward the former, leading to synaptic dysfunction in AD. 3.2.1 Introduction ASD is a condition related to brain development that impacts how a person perceives and socializes with others, causing problems in social interaction and com- munication. The term “spectrum” in ASD refers to the wide range of symptoms and severity that can vary from person to person [48]. ASD is believed to be caused by a combination of genetic and environmental factors that affect synaptic plasticity and connectivity in the brain. Many genes associated with ASD encode proteins that regulate synaptic plasticity and connectiv- ity at different levels [48]. Mutations in these genes can disrupt the balance between synaptic excitation and inhibition, leading to abnormal brain development and func- tion. Environmental factors, such as prenatal exposure to infections, toxins or stress, and others can also modulate the effects of genetic mutations on ASD risk [48]. 3.1.4 Clinical implications and summary A better understanding of NMDARs in AD may lead to more effective therapy. NMDAR antagonists are drugs that reduce NMDAR activity and may help to treat AD symptoms and slow down disease progression. The only drug of this class approved for AD is memantine, which has a weak and fast binding to NMDARs, allowing it to prevent excessive stimulation without interfering with normal function. Memantine can enhance cognition and delay functional decline in patients with moderate to severe AD, especially when used together with cholinesterase inhibitors, another class of drugs that increase acetylcholine levels in the brain. However, memantine does not work for mild AD or for preventing AD onset, and its effects are small and inconsis- tent among patients. g p Therefore, more research is needed to develop better and more specific NMDAR modulators that can target the different types and components of NMDARs, as well as their interactions with Aβ and other factors related to AD development. Some 5 Cell Communication and Signaling in Health and Disease promising candidates are NR2B-selective antagonists, glycine site modulators, and allosteric modulators. These drugs may have better outcomes than memantine in terms of strength, selectivity, safety, and effectiveness. However, more clinical studies are required to verify their usefulness and optimal dose for AD patients. q y p p In summary, NMDARs are involved in various aspects of AD, but their exact role is still unclear. AD is characterized by the accumulation of amyloid-beta and tau proteins, which can affect NMDAR activity and lead to neuronal death, synaptic dys- function, and spine loss. However, the effects of amyloid-beta and tau on NMDARs are not uniform, as they depend on the oligomeric state and subcellular localization of these proteins. Further research is required to understand how NMDAR dysfunction contributes to AD and to identify more effective treatments. 3.2.3 NMDAR gene mutations in ASD Mutations in genes that encode NMDAR subunits have now been firmly linked to ASD, which directly support the role of NMDARs in ASD. Two of the commonly mutated NMDAR genes in ASD are GRIN2B and GRIN2A, which encode the GluN2B and GluN2A subunits respectively. GluN2B and/or GluN2A is essential for the proper localization and function of NMDARs in the dendrites of cortical neurons. Dendrite morphology and dynamics are crucial for neuronal connectivity and information processing in the brain. Mutations in GRIN2B can disrupt the normal development and maintenance of dendritic arbors, leading to aberrant neuronal circuitry and impaired synaptic plasticity. Similarly, mutations in GRIN2A can also lead to altered synaptic signaling and neuronal development [51]. In other words, mutations in both GRIN2B and GRIN2A can interfere with how neurons grow, connect, and com- municate with each other. This can lead to a range of neurodevelopmental problems, including ASD [52]. g Some mutations cause truncation or loss-of-function of the GluN2B subunit, while others alter its amino acid sequence or affect its splicing or expression levels. These mutations can affect the assembly, trafficking, stability, or activity of NMDARs, resulting in either reduced or enhanced NMDAR signaling. The effects of these muta- tions on neuronal morphology and function may depend on the timing, location, and severity of the mutation, as well as on the interactions with other genetic or environ- mental factors [51, 52]. g Some mutations cause truncation or loss-of-function of the GluN2B subunit, while others alter its amino acid sequence or affect its splicing or expression levels. These mutations can affect the assembly, trafficking, stability, or activity of NMDARs, Some examples of mutations in GRIN2B that have been reported in ASD patients are: • A single-base substitution (c27172-2A > G) at the canonical 3′ splice site of exon 10 causes a premature stop codon at position 724 of GluN2B (GluN2B 724 t). This mutation does not prevent the formation of functional NMDARs, but rather reduces their surface expression and calcium currents. It also reduces its traffick- ing to dendrites and synapses. The mutation also interferes with spine density and morphology, resulting in fewer and smaller spines [53, 54]. • A de novo missense mutation that causes a premature stop at position 373 of GluN2B (c.1119G > A p.(Trp373). 3.2.2 Genetic mutations in ASD Recent advances in genomics have enabled the identification of many mutations that are associated with ASD [49]. For example, FMR1, MECP2, and ATRX are single genes that control gene expression and chromatin structure. Other mutations affect multiple genes that interact in common pathways or networks. For instance, many genes that encode proteins involved in synaptic function and plasticity, such as SynGAP, SHANK3, NRXN1, and NLGN3, have been found to be mutated in ASD. These genes modulate the formation and strength of synapses, which are essential for learning, memory, and behavior. Still, another example is the group of genes that regulate actin dynamics, such as ACTN4, SWAP-70, and SRGAP3, which have been shown to alter the morphology and localization of dendritic spines, the sites of synapses [49]. The role of NMDARs in ASD is supported by multiple lines of evidence. Firstly, several ASD-associated genes encode proteins that interact with or regulate NMDARs. Mutations in these genes, such as SYNGAP1, FMR1, SHANK2, and SHANK3, can affect the expression, trafficking, or function of NMDAR subunits, leading to altered synaptic strength and plasticity [48]. Secondly, mutations in other genes like PTEN, MECP2, and NLGN3, which are also associated with ASD, can alter the signaling pathways downstream of NMDAR activation [48]. These pathways, including mTOR, ERK, and WNT, play crucial roles in neuronal development and connectivity. Lastly, subtle changes in NMDAR expression, 6 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 trafficking, or function have been observed in animal models of ASD [50]. These findings collectively suggest that NMDARs are critical for the proper formation and function of neural circuits, and their dysregulation may contribute to the cognitive and behavioral impairments observed in ASD. 3.2.4 Anti-NMDAR encephalitis and ASD Anti-NMDAR encephalitis is a rare autoimmune disorder that affects the brain and causes various neurological and psychiatric symptoms, such as psychosis, hallucina- tions, personality changes, cognitive impairment, seizures, and movement disorders. It is caused by the production of antibodies that target the NMDARs, which are involved in glutamate neurotransmission and synaptic plasticity. There’s some initial evidence that suggests a potential connection between anti- NMDAR encephalitis and ASD [58]. Some instances of anti-NMDAR encephalitis, especially in children and teenagers, have been reported to show symptoms similar to ASD or have been mistakenly diagnosed as ASD. Additionally, some research has found increased levels of anti-NMDAR antibodies or other signs of autoimmunity in certain individuals with ASD. However, the exact meaning and specificity of these findings are not yet clear. The cause-and-effect relationship between anti-NMDAR encephalitis and ASD is still uncertain, and more studies are needed to understand the mechanisms and implications of this link. GRIN2A variants are also linked to ASD but to a lesser degree than seen with IN2B variants. These mutations may affect the expression and activity of NMDARs in different brain regions or cell types, leading to synaptic dysfunction and behavioral abnor- malities in ASD. Therefore, the NMDAR signaling network could be a central hub to integrate the functions of the genes that are mutated in ASD, as it modulates various aspects of neuronal development and plasticity that are relevant to the disorder. However, the relationship between NMDAR dysfunction and ASD is complex and context-dependent, as different mutations may have different effects on NMDAR function and expression, depending on the subunit composition, brain region, devel- opmental stage, and synaptic state. Moreover, NMDAR signaling interacts with other signaling pathways that are also implicated in ASD, such as mTOR, ERK, GSK3β, and Wnt [49]. Therefore, more studies are needed to elucidate the molecular mechanisms of NMDAR mutations in ASD and to explore the potential therapeutic strategies based on modulating NMDAR function. 3.2.3 NMDAR gene mutations in ASD Rats with this mutation showed reduced social interaction and preference for social novelty, increased anxiety and stereotyped behaviors, impaired spatial learning and memory, and enhanced susceptibility to seizures. The mutation decreased the surface expression of GluN2B and the amplitude of NMDA receptor currents in the hippocampus, a brain region important for learning and memory. These findings suggest that the GluN2B-Trp373 mutation contributes to ASD-associated symptoms by disrupting the function of NMDA receptors [55, 56]. • A mutation in GluN2B (GluN2B R540H) increases NMDAR surface expression slightly compared to wild-type GluN2B. However, it does reduce NMDAR cur- rent density, as well as their sensitivity to glutamate and glycine. The mutation • A mutation in GluN2B (GluN2B R540H) increases NMDAR surface expression slightly compared to wild-type GluN2B. However, it does reduce NMDAR cur- rent density, as well as their sensitivity to glutamate and glycine. The mutation 7 Cell Communication and Signaling in Health and Disease impairs LTP and LTD, but only when expressed in neurons that lack endogenous GluN2A. When GluN2A is present, the mutant subunits co-assemble with GluN2A and produce normal synaptic plasticity [57]. 3.2.5 Clinical implications While there is broad consensus that autism involves some form of glutamatergic dysfunction, the specifics of this dysfunction (i.e., whether it involves an excess or deficiency of glutamate) remain a topic of ongoing research. Evidence suggests that NMDARs may be underactive in ASD. This evidence stems from three primary sources: (1) GRIN gene knockout mice: Mice with knockout mutations in GRIN genes, which encode NMDAR subunits, often display reduced sociability and other autism-like symptoms, (2) GRIN gene mutations in ASD: Many of the GRIN gene mutations identified in individuals with ASD appear to result in NMDAR hypofunc- tion, and (3) Anti-NMDAR encephalitis studies: Investigations into anti-NMDAR encephalitis, a condition characterized by the presence of antibodies against NMDARs, have provided additional insights into the role of these receptors in ASD. In 8 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 summary, while it’s clear that glutamatergic neurotransmission is somehow disrupted in ASD, the exact nature of this disruption is still being explored. Given the observed underactivity of NMDARs in ASD, one strategy is to enhance NMDAR function using NMDAR agonists, such as D-cycloserine [59]. summary, while it’s clear that glutamatergic neurotransmission is somehow disrupted in ASD, the exact nature of this disruption is still being explored. Given the observed underactivity of NMDARs in ASD, one strategy is to enhance NMDAR function using NMDAR agonists, such as D-cycloserine [59]. Interestingly, there is evidence pointing toward an excess of glutamate (and a reduction in GABA) in ASD. Therapies targeting glutamate aim to normalize its neu- rotransmission. Several potential drugs aim to temper glutamate transmission. One approach is to inhibit the release of glutamate, as seen with the drug riluzole. Another strategy is to decrease glutamate signaling through the use of NMDAR antagonists, such as acamprosate (which also antagonizes mGluR5 and GABAB) and memantine [60, 61]. While some studies have shown promising results for glutamate therapy in improving ASD symptoms and cognitive function, more research is needed to estab- lish its safety and efficacy. This includes determining the optimal dosage, duration of treatment, and combination of agents. 3.2.6 Summary In summary, NMDARs have been implicated in ASD. Two of the genes that have been associated with ASDs are GRIN2B and GRIN2A. Mutations in GRIN2B and/or GRIN2A can result in a defective GluN2 protein that does not form functional recep- tors resulting in abnormal synaptic connectivity and function in the brain. Another way that NMDARs could be involved in ASDs is through autoimmunity. Some cases of anti-NMDAR encephalitis have been reported to be associated with ASDs, suggest- ing that autoantibodies could impair glutamate neurotransmission and contribute to ASD pathophysiology. Therapeutic strategies based on a better understanding of the molecular nature of ASD to target NMDARs are undergoing, but more studies are required to confirm the efficacy and safety [61]. 3.3.2 NMDAR gene mutations in epilepsy Mutations in NMDAR genes can affect the structure and function of the NMDARs in various ways, such as altering the binding affinity of glutamate, the channel open- ing and closing kinetics, the receptor biogenesis and trafficking, and the sensitivity to modulators and inhibitors. These changes can have different impacts on synaptic and non-synaptic NMDAR activity, which are important for brain development, plastic- ity, and cognition. Depending on the location and type of mutation, as well as the expression pattern and function of the affected subunit, mutations in NMDAR genes can cause various epilepsy and other neurodevelopmental disorders such as intel- lectual disability, ASD, and schizophrenia [64]. Indeed, epileptic phenotypes have been linked to mutations in the genes GRIN1, GRIN2A, GRIN2B, and GRIN2D. These genes are responsible for the production of the GluN1, GluN2A, GluN2B, and GluN2D subunits of NMDARs respectively [65]. p y Mutations in the GRIN1 gene impair the function of the NMDAR, leading to abnormal neuronal activity and seizures. GRIN1 mutations in patients with epilepsy include duplication mutation, nonsense mutation, and missense mutations, which affect different domains of the GluN1 subunit [65]. These mutations may alter the biophysical properties of the NMDAR, such as channel opening, closing, and desen- sitization [65]. A novel mutation in GRIN1 (c.1923G > A, p.Met641Ile) was found in a child with refractory epilepsy and early-onset epileptic encephalopathy. Laboratory tests show that NMDARs with GluN1-M641I have increased agonist affinity and decreased Mg2+ sensitivity. NMDARs with GluN1-M641I are more responsive to the NMDAR channel inhibitors memantine, ketamine, and dextromethorphan than the normal receptors. The patient’s seizure frequency was significantly reduced by adding memantine to the anti-seizure therapy [66]. One of the most common NMDAR subunit mutations found in epilepsy is the p.Arg518His substitution in the GluN2A subunit, which affects the transmembrane domain of the protein. This mutation was first reported in a patient with Landau- Kleffner syndrome (LKS), a rare form of epileptic amnesic syndrome (EAS) that manifests with acquired aphasia and focal epileptic activity [67]. The p.Arg518His mutation was shown to impair the surface expression and trafficking of GluN2A- containing NMDARs, resulting in reduced NMDAR-mediated currents and synaptic plasticity. The mutation also altered the sensitivity of NMDARs to Mg2+ block and glycine modulation, which may affect the balance between excitation and inhibition in the brain. 3.3.1 Introduction Epilepsy is one of the most common neurological disorders characterized by recurrent seizures, which can affect the quality of life and cognitive function of patients. Epilepsy can be caused by various factors, such as brain injury, genetic muta- tions, or inflammation, that disrupt the balance between excitatory and inhibitory neurotransmission in the brain [62]. One of the major excitatory neurotransmitters in the brain is glutamate, which acts on different types of receptors, including NMDARs. NMDARs play a complex and dynamic role in epilepsy, as they can modulate seizure initiation, propagation, termination, and epileptogenesis. Depending on the subunit composition, location, and activation state of NMDARs, they can have pro-convul- sant or anti-convulsant effects on different types of seizures and epileptic syndromes. Abnormal expression or function of NMDARs can lead to neuronal excitotoxicity, inflammation, and epileptogenesis. Therefore, NMDARs are regarded as a potential target for suppressing epileptogenesis and treating epilepsy [63]. In this section, I will review how NMDARs contribute to the pathophysiology of epilepsy and how target- ing NMDARs can offer novel therapeutic opportunities for epilepsy treatment. g p pp p p y NMDARs are widely distributed in the central nervous system (CNS) and play critical roles in neuronal excitability in the CNS [4]. Both clinical and preclinical studies have revealed that the abnormal expression or function of these receptors can 9 Cell Communication and Signaling in Health and Disease underlie the pathophysiology of seizure disorders and epilepsy. For example, genetic studies have identified mutations in NMDAR subunits, such as GRIN1, GRIN2A, GRIN2B, and GRIN2D, that can cause various forms of epilepsy, such as developmen- tal and epileptic encephalopathies (DEEs), rolandic epilepsy, and infantile spasms. These mutations can alter the biophysical properties, trafficking, or interactions of NMDARs, leading to either gain-of-function or loss-of-function effects [63]. We now review the molecular mechanisms and clinical implications of NMDAR mutations in epilepsy. 3.3.2 NMDAR gene mutations in epilepsy The p.Arg518His mutation has been found in several other patients with EAS disorders, suggesting a common pathogenic mechanism for these syndromes [67]. gg g p g y Mutations in the GRIN2B gene have also been linked to epilepsy [68]. The pathophysiological mechanism underlying the variability of clinical phenotypes in patients with GRIN2B mutations is unclear. A recent study identified a novel GRIN2B mutation (c.3272A > C, p.K1091T) in a patient with epilepsy and intellectual 10 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 disability [69]. This mutation reduces the interaction of GluN2B with PSD-95, a scaffolding protein that stabilizes NMDA receptors at the synapse. The mutation also impairs the surface expression, glutamate sensitivity, and current density of NMDA receptors in HEK 293 T cells and hippocampal neurons. Moreover, p.K1091T mutation decreases the dendritic spine density and excitatory synaptic transmission in hippocampal neurons. These findings suggest that the GRIN2B-K1091T mutation causes a loss-of-function effect on NMDA receptors, which may contribute to the patient’s phenotype [69]. p p yp Finally, changes in the GRIN2D gene can cause different types of epilepsy. Up to now, 11 particular GRIN2D variants have been found in patients with devel- opmental and epileptic encephalopathy (DEE). Out of them, 6 were in the M3 domain (Val667Ile, Leu670Phe, Thr674Lys, Ala675Thr, Ala678Asp, and Met681Ile). Laboratory tests of GRIN2D variants showed that Val667Ile and Leu670Phe variants greatly enhance agonist affinity and channel opening probability, and extend the closing time, leading to increased calcium entry that probably affects the clinical features seen in patients [70–72]. Mutations in the NMDAR gene result in either a gain-of-function (GoF) or loss-of-function (LoF) protein. GoF mutations in NMDAR can lead to an increase in excitatory postsynaptic currents (EPSC), which can disrupt the balance between excitatory and inhibitory discharges in the neuronal network. On the other hand, LoF mutations are thought to diminish inhibitory postsynaptic currents (IPSC) by either reducing the release of GABA from the presynaptic membrane of GABAergic neurons or through a postsynaptic mechanism [64], which can result in epilepsy. However, the exact mechanism through which GoF or LoF mutations in the NMDAR gene cause epilepsy is not fully understood. p p y y To summarize, mutations in NMDAR subunit-encoding genes can cause various forms of epilepsy by altering the structure, function, and regulation of NMDARs. 3.3.2 NMDAR gene mutations in epilepsy These mutations can affect the expression, trafficking, gating, modulation, and signaling of NMDARs, leading to changes in synaptic transmission, plasticity, and excitotoxicity. The functional consequences of these mutations depend on the location, type, and severity of the amino acid substitution, as well as on the genetic background and environmental factors of the patients. Understanding the molecular mechanisms of these mutations may help to identify novel therapeutic targets and strategies for epilepsy. al 3.3.3 NMDAR as a therapeutic target in epilepsy NMDAR antagonists and agonists have been investigated for their potential therapeutic effects on epilepsy, as well as their possible adverse effects. Some NMDAR antagonists, such as ketamine, memantine, and efavirenz, are FDA-approved drugs for other indications. However, there is evidence from clinical trials and case reports that these drugs can also reduce seizure frequency and severity in some patients with refractory epilepsy or GRIN mutations. Ketamine, a noncompetitive NMDAR antagonist, has been used in several clini- cal studies and case reports for the treatment of refractory SE (RSE) and super- refractory SE (SRSE) in adults and children, with variable doses and durations. The reported efficacy rates range from 32 to 74%, depending on the timing of administra- tion and the type and duration of SE. However, the evidence on ketamine is still lim- ited by the retrospective and heterogeneous nature of the data, and more prospective and randomized trials are needed to establish its optimal dose, timing, and duration in SE treatment [73]. 11 Cell Communication and Signaling in Health and Disease Memantine, a noncompetitive, open-channel NMDAR antagonist, has been used as an add-on therapy for a girl with refractory epilepsy due to a de novo GRIN2A mutation. The patient had a reduction in seizure frequency [74]. In another random- ized control trail, memantine was effective for nine patients of 27 (33%), compared to only two patients (7%) in the placebo group (P < 0.02). Therefore, memantine appears to be a safe and effective treatment for children with developmental epileptic encephalopathy [75]. p p y Efavirenz is a drug that can increase the level of 24(S)-hydroxycholesterol, a mol- ecule that can restore the function of NMDARs with GluN2A/Grin2a-V685G muta- tion. This mutation causes a loss of function of NMDA receptors. Efavirenz treatment improved the seizure susceptibility, cortical EEG activity, and synaptic currents of GluN2A/Grin2a-V685G mutant mice. These results suggest that efavirenz may be a potential therapeutic option for epilepsy caused by NMDA receptor dysfunction [76]. p p p p p y y p y In addition, there are other strategies to antagonize NMDARs. These include the use of drugs such as amantadine, magnesium sulfate, remacemide, and MK-801 in epilepsy [77]. These examples illustrate that some NMDAR blockers can reduce seizure frequency and severity in some patients or animal models with refractory epilepsy or GRIN mutations. 3.3.3 NMDAR as a therapeutic target in epilepsy However, the mechanisms of action, efficacy, safety, and optimal dosing of these drugs for epilepsy are still unclear and need further investigation [77]. In summary, NMDAR antagonists are promising pharmacological agents that can modulate NMDAR activity for epilepsy treatment. They can have beneficial effects on seizure control, neuroprotection, and neuroplasticity in some patients with refractory epilepsy or GRIN mutations. However, they can also have adverse effects on cognition, mood, and neurotoxicity in some patients or at high doses. Therefore, more research is needed to elucidate the mechanisms of action, efficacy, safety, and optimal dosing of these drugs for epilepsy. Moreover, biomarkers and predictors of response and adverse effects are needed to guide the personalized use of these drugs for epilepsy. 3.3.4 NMDAR interaction with other neurotransmitter systems NMDARs interact with GABA, glutamate, and serotonin to modulate or mediate epileptic phenomena [63, 77]. For example, NMDARs can modulate the function of GABAergic neurons and interneurons, which affects the balance between excitation and inhibition. In epilepsy, glutamate levels are elevated, which can lead to overstim- ulation of NMDARs and cause neuronal hyperexcitability. NMDARs can also interact with other glutamate receptor subtypes, which can modulate their trafficking, expres- sion, and function. In addition, serotonin can modulate the release of glutamate or GABA from presynaptic terminals, which can affect NMDAR activation and synaptic transmission. In epilepsy, serotonin levels are altered, which may influence NMDAR function and epileptic activity. In conclusion: NMDARs play a crucial role in epilepsy by interacting with other neurotransmitter systems. Therefore, understanding these interactions may provide new insights into the pathophysiology and treatment of epilepsy. 3.4.1 Introduction Schizophrenia is a severe mental disorder that affects how people think, feel, and behave. It is characterized by symptoms such as hallucinations, delusions, disor- ganized speech, and cognitive impairment. Schizophrenia affects about 1% of the world’s population and has a significant impact on the quality of life and functioning of the affected individuals and their families [78]. One of the most intriguing aspects of schizophrenia is the involvement of NMDARs in its pathophysiology. NMDARs have been implicated in schizophrenia by various lines of evidence, including genetic studies, pharmacological studies, animal models, and clinical trials. However, the exact role of NMDARs in schizophrenia remains unclear and controversial. The aim of this section is to review the current state of knowledge on the role of NMDARs in schizophrenia and critically evaluate the strengths and limitations of dif- ferent approaches to studying this topic. The section will cover the following aspects: (1) genetic studies linking NMDARs and schizophrenia; (2) pharmacological studies examining the effects of drugs that target NMDARs on schizophrenia symptoms; (3) animal models used to investigate the role of NMDARs in schizophrenia; and (4) clinical trials testing NMDAR-targeting treatments for schizophrenia. The section will conclude with a summary of the main points and a reflection on the potential future directions for research on NMDARs and schizophrenia. 3.3.5 Summary In conclusion, NMDARs play a crucial role in epilepsy by interacting with other neurotransmitter systems. Evidence from genetic studies, clinical trials, animal 12 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 models, and pharmacological research has shown that these receptors and their subunits play crucial roles in excitatory neurotransmission, synaptic plasticity, and the balance between excitation and inhibition in the brain. Mutations in NMDAR subunits can cause various forms of epilepsy, and modulating NMDAR activity with antagonists or agonists can reduce seizure frequency and severity in some patients. However, the expression and function of NMDARs can be influenced by many genetic and environmental factors, and they interact with other neurotransmitter systems in ways that may modulate or mediate the epileptic phenomena. Future studies could focus on identifying new NMDAR subunit mutations associated with epilepsy, inves- tigating the effects of different environmental factors on NMDAR expression and function, exploring the interactions of NMDARs with other neurotransmitter systems in more detail, and testing new NMDAR modulators in preclinical models and clinical trials [63, 77]. models, and pharmacological research has shown that these receptors and their subunits play crucial roles in excitatory neurotransmission, synaptic plasticity, and the balance between excitation and inhibition in the brain. Mutations in NMDAR subunits can cause various forms of epilepsy, and modulating NMDAR activity with antagonists or agonists can reduce seizure frequency and severity in some patients. However, the expression and function of NMDARs can be influenced by many genetic and environmental factors, and they interact with other neurotransmitter systems in ways that may modulate or mediate the epileptic phenomena. Future studies could focus on identifying new NMDAR subunit mutations associated with epilepsy, inves- tigating the effects of different environmental factors on NMDAR expression and function, exploring the interactions of NMDARs with other neurotransmitter systems in more detail, and testing new NMDAR modulators in preclinical models and clinical trials [63, 77]. 3.4.2 Genetics studies Genetic studies have provided evidence for the involvement of NMDARs in schizo- phrenia. Several genes that encode NMDAR subunits or modulate their function have been associated with schizophrenia risk or altered expression in patients. For example, GRIN2A, which codes for the GluN2A subunit of the NMDAR, has been firmly linked to schizophrenia by genome-wide association studies (GWAS) and meta-analyses [79–81]. To a lesser extent of confidence, schizophrenia has also been linked to gene mutations in GRIN2B, GRIN2C, GRIN2D, GRIN3A, and GRIN3B [82–84]. Moreover, these genes show reduced expression in the postmortem brains of schizophrenia patients, suggesting impaired NMDAR signaling. Other genes that interact with NMDARs, such as dysbindin, neuregulin, DISC1 (Disrupted-in-Schizophrenia 1), 13 Cell Communication and Signaling in Health and Disease D-Amino acid oxidase, and RGS4, have also been implicated in schizophrenia by genetic and functional studies. These genes may affect NMDAR trafficking, localiza- tion, or modulation, thereby influencing synaptic plasticity and information process- ing [78, 85, 86]. D-Amino acid oxidase, and RGS4, have also been implicated in schizophrenia by genetic and functional studies. These genes may affect NMDAR trafficking, localiza- tion, or modulation, thereby influencing synaptic plasticity and information process- ing [78, 85, 86]. 3.4.3 Pharmacological studies The observation that drugs that block NMDARs, such as ketamine and phency- clidine (PCP), can induce psychotic symptoms in healthy individuals and exacerbate them in patients with schizophrenia has led to the NMDAR hypofunction hypothesis of schizophrenia [87]. These drugs act as noncompetitive antagonists at the PCP binding site of the NMDAR, preventing the influx of calcium ions and reducing the synaptic transmission mediated by glutamate. The psychotomimetic effects of these drugs are dose-dependent and correlate with the degree of NMDAR blockade [88]. The pharmacological studies that support the NMDAR hypofunction hypoth- esis of schizophrenia have used various approaches, such as measuring the effects of NMDAR antagonists on cognitive functions, neurophysiological parameters, neurochemical markers, and brain imaging in healthy volunteers and patients with schizophrenia [89]. These studies have shown that NMDAR antagonists impair working memory, attention, executive functions, and sensory gating, as well as alter the activity of dopaminergic, serotonergic, and cholinergic systems in the brain [90]. Moreover, these drugs affect the regional cerebral blood flow and glucose metabolism, especially in the prefrontal cortex and the hippocampus, regions that are implicated in the pathophysiology of schizophrenia [91]. The pharmacological studies have also examined the effects of agents that modulate NMDAR function, such as glycine, D-serine, sarcosine, and NMDAR co-agonists, on the symptoms and cognitive deficits of schizophrenia [92]. These agents act by enhancing the activity of NMDARs by binding to the glycine site or by increasing the availability of glycine or D-serine. Some of these agents have shown beneficial effects on negative symptoms and cognitive functions in patients with schizophrenia, especially when combined with antipsychotic drugs [87]. However, the results are not consistent across studies and depend on various factors, such as the dose, duration, and type of treatment, as well as the patient population and outcome measures. p p The pharmacological studies on NMDARs and schizophrenia have provided valuable insights into the role of glutamatergic neurotransmission in the etiology and treatment of this disorder. However, they also have some limitations, such as the lack of specificity of NMDAR antagonists and co-agonists, the complexity of NMDAR subtypes and interactions with other receptors and channels, and the variability of individual responses to these drugs [93]. 3.4.4 Animal models Animal models are useful tools to study the role of NMDAR hypofunction in schizophrenia, as they can mimic some of the behavioral and neurobiological features of the disorder. There are two main approaches to generate animal models of NMDAR hypofunction: pharmacological and genetic. y Pharmacological models involve the administration of NMDAR antagonists, such as ketamine, phencyclidine (PCP), or dizocilpine (MK-801), to induce transient or chronic NMDAR blockade in rodents or non-human primates [94]. These drugs can 14 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 produce positive, negative, and cognitive symptoms of schizophrenia in humans, as well as neurochemical, electrophysiological, and neuroplastic changes in the brain that resemble those observed in schizophrenia patients. However, pharmacological models have some limitations, such as the lack of specificity for NMDAR subtypes, the potential involvement of other receptors or channels, and the variability in dose, route, and duration of administration. Genetic models involve the manipulation of genes that encode NMDAR subunits or modulators, such as NR1, NR2A, NR2B, NR3A, D-serine, glycine transporter 1 (GlyT1), or serine racemase [94, 95]. These genes have been implicated in schizophre- nia by human genetic studies or by their role in regulating NMDAR function. Genetic models can provide more selective and stable NMDAR hypofunction than pharmaco- logical models, as well as insights into the developmental and cell-type specific effects of NMDAR dysfunction. However, genetic models also have some drawbacks, such as the potential compensatory mechanisms, the pleiotropic effects of gene manipula- tion, and the difficulty in replicating human genetic variations in animals [94, 95]. Despite these challenges, animal models of NMDAR hypofunction have contributed to the understanding of the molecular and cellular mechanisms underlying schizo- phrenia pathophysiology, as well as to the identification of novel therapeutic targets for this disorder. 3.4.5 Clinical trials Several clinical trials have tested the efficacy and safety of NMDAR-targeting treatments for schizophrenia, with mixed results. Some of the treatments that have been investigated include: • Glycine modulatory site agonists: These compounds bind to the glycine site on the NMDAR and enhance its function. Examples are glycine, d-serine, d-cycloserine, and sarcosine. These agents have shown some benefits for nega- tive and cognitive symptoms of schizophrenia, but their effects are modest and inconsistent [96–98]. • Kynurenine pathway inhibitors: These compounds inhibit the enzymes that degrade tryptophan into kynurenic acid, a potent NMDAR antagonist. Examples are laquinimod, roquinimex, and JM6. These agents have shown some neuro- protective and anti-inflammatory effects in animal models of schizophrenia, but their clinical efficacy is unclear [99]. • Cystine-glutamate antiporter modulators: These compounds increase the extracellular levels of cystine, which stimulates the cystine-glutamate antiporter to release more glutamate into the synaptic cleft. This enhances the activation of NMDARs and other glutamate receptors. Examples are N-acetylcysteine (NAC) and l-cysteine. These agents have shown some benefits for negative and cognitive symptoms of schizophrenia, as well as reducing oxidative stress and inflammation [100]. • mGluR modulators: These compounds modulate the activity of mGluRs, which are G-protein coupled receptors that regulate glutamate release and synaptic plasticity. Examples are mGluR2/3 agonists (e.g., LY2140023), mGluR2 positive allosteric modulators (PAMs) (e.g., AZD8529), mGluR5 PAMs 15 Cell Communication and Signaling in Health and Disease (e.g., basimglurant), and mGluR4 PAMs (e.g., VU0155041). These agents have shown some benefits for positive, negative, and cognitive symptoms of schizo- phrenia, as well as improving neuroplasticity and neurogenesis [3]. (e.g., basimglurant), and mGluR4 PAMs (e.g., VU0155041). These agents have shown some benefits for positive, negative, and cognitive symptoms of schizo- phrenia, as well as improving neuroplasticity and neurogenesis [3]. The results of these clinical trials suggest that targeting NMDARs may be a prom- ising strategy for treating schizophrenia, but more research is needed to identify the optimal compounds, doses, combinations, and patient populations. In conclusion, the evidence from genetics studies, pharmacological studies, animal models, and clinical trials suggests that NMDARs play a significant role in schizo- phrenia. 3.4.5 Clinical trials A comprehensive understanding of the role of NMDARs in schizophrenia should consider the complexity and diversity of NMDAR function and regulation; the interactions between NMDARs and other neurotransmitter systems; the developmen- tal and environmental factors that influence NMDAR function; and the individual differences among schizophrenia patients in terms of genetics, symptoms, cognition, and response to treatment. 4. Conclusions The NMDAR, a critical component of the central nervous system, has been the subject of extensive research due to its pivotal role in neurological function and disease. In health, the NMDAR plays a crucial role in synaptic plasticity, learning, and memory. It is a key player in the delicate balance of excitation and inhibition that maintains homeostasis in the brain. However, when this balance is disrupted, the consequences can be severe. The NMDAR, a critical component of the central nervous system, has been the subject of extensive research due to its pivotal role in neurological function and disease. In health, the NMDAR plays a crucial role in synaptic plasticity, learning, and memory. It is a key player in the delicate balance of excitation and inhibition that maintains homeostasis in the brain. However, when this balance is disrupted, the consequences can be severe. q In disease states such as AD, autism, epilepsy, and schizophrenia aberrant NMDAR function has been implicated. One of the common themes that emerge from the review of NMDARs in health and disease is the role of genetic mutations in NMDAR subunits or genes that encode proteins that interact with NMDARs. These mutations can affect the expression, trafficking, function, or modulation of NMDARs, resulting in either hypo- or hyperfunction of NMDARs. Depending on the brain region, devel- opmental stage, and synaptic state, these mutations can have different effects on neu- ronal excitability, synaptic plasticity, network oscillations, and cognitive functions. These mutations can also interact with environmental factors, such as infections, toxins, or stress, to modulate the risk and severity of neurological and psychiatric disorders. Therefore, understanding the molecular mechanisms and clinical implica- tions of these mutations is crucial for developing novel diagnostic and therapeutic strategies for these disorders. However, genetic studies only provide a framework to link the genes and proteins in a network. It requires a concerted effort to understand the disease mechanisms at multiple levels, from molecular to cellular to network to behavior. NMDARs are central to this effort, as they are involved in various aspects of neuronal development and plasticity that are relevant to these disorders. q In disease states such as AD, autism, epilepsy, and schizophrenia aberrant NMDAR function has been implicated. 4. Conclusions One of the common themes that emerge from the review of NMDARs in health and disease is the role of genetic mutations in NMDAR subunits or genes that encode proteins that interact with NMDARs. These mutations can affect the expression, trafficking, function, or modulation of NMDARs, resulting in either hypo- or hyperfunction of NMDARs. Depending on the brain region, devel- opmental stage, and synaptic state, these mutations can have different effects on neu- ronal excitability, synaptic plasticity, network oscillations, and cognitive functions. p p y The development of pharmacological agents targeting the NMDAR offers promis- ing avenues for therapeutic intervention. However, the challenge lies in selectively modulating NMDAR activity without disrupting its physiological functions. Looking forward, our journey in understanding the NMDAR is far from over. Future research should focus on elucidating the precise mechanisms of NMDAR regulation and exploring novel therapeutic strategies. The development of drugs with improved specificity for NMDAR subtypes or co-agonist sites could potentially minimize side effects and enhance therapeutic efficacy. 16 NMDA Receptors in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.114003 NMDA Receptors in Health and Disease In conclusion, the NMDAR remains a fascinating subject of study with significant implications for neuroscience and medicine. As we continue to unravel its mysteries, we move closer to our ultimate goal: improving human health through science. Author details Yue-Qiao Huang Department of Pharmaceutical Sciences, School of Pharmacy, Philadelphia College of Osteopathic Medicine Georgia, Suwanee, GA, USA Yue-Qiao Huang Department of Pharmaceutical Sciences, School of Pharmacy, Philadelphia College of Osteopathic Medicine Georgia, Suwanee, GA, USA Address all correspondence to: yueqiaohu@pcom.edu Acknowledgements We wish to acknowledge the financial support from the Philadelphia College of Osteopathic Medicine Chief Research and Science Officer (CRSO) Fund. an update. Neuropharmacology. 2018;142:41-62 [9] Gunduz-Bruce H. The acute effects of NMDA antagonism: From the rodent to the human brain. Brain Research Reviews. 2009;60(2):279-286 [2] Hansen KB, Wollmuth LP, Bowie D, Furukawa H, Menniti FS, Sobolevsky AI, et al. Structure, function, and pharmacology of glutamate receptor ion channels. Pharmacological Reviews. 2021;73(4):298-487 [2] Hansen KB, Wollmuth LP, Bowie D, Furukawa H, Menniti FS, Sobolevsky AI, et al. 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https://openalex.org/W1966587420	https://www.nature.com/articles/srep09560.pdf	English	null	Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria	Scientific reports	2,015	cc-by	11,816	* These authors contributed equally to this work. Correspondence and requests for materials should be addressed to W.R.H. (wolfgang. hess@biologie.uni- freiburg.de) OPEN SUBJECT AREAS: GENE EXPRESSION BACTERIAL TRANSCRIPTION SMALL RNAS TRANSCRIPTOMICS Received 7 January 2015 Accepted 5 March 2015 Published 2 April 2015 2 Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Scha¨nzlestr. 1, 79104 Freiburg, Germany. In all studied organisms, a substantial portion of the transcriptome consists of non-coding RNAs that frequently execute regulatory functions. Here, we have compared the primary transcriptomes of the cyanobacteria Synechocystis sp. PCC 6714 and PCC 6803 under 10 different conditions. These strains share 2854 protein-coding genes and a 16S rRNA identity of 99.4%, indicating their close relatedness. Conserved major transcriptional start sites (TSSs) give rise to non-coding transcripts within the sigB gene, from the 59UTRs of cmpA and isiA, and 168 loci in antisense orientation. Distinct differences include single nucleotide polymorphisms rendering promoters inactive in one of the strains, e.g., for cmpR and for the asRNA PsbA2R. Based on the genome-wide mapped location, regulation and classification of TSSs, non-coding transcripts were identified as the most dynamic component of the transcriptome. We identified a class of mRNAs that originate by read-through from an sRNA that accumulates as a discrete and abundant transcript while also serving as the 59UTR. Such an sRNA/mRNA structure, which we name ‘actuaton’, represents another way for bacteria to remodel their transcriptional network. Our findings support the hypothesis that variations in the non-coding transcriptome constitute a major evolutionary element of inter-strain divergence and capability for physiological adaptation. O f 1 O rganismic diversity as well as differences in metabolic, developmental and physiological capabilities cannot be related to divergent gene content and gene arrangement alone. Instead, differences in the regulation of gene expression and the composition of the transcriptome have been suggested as critical factors1. Accordingly, a substantial share of the transcriptome consists of non-coding and antisense RNAs, many of which have regulatory impact, e.g., in the form of miRNAs2, long non-coding RNAs3 or long natural antisense transcripts4. It is widely accepted that RNA complexity is at the heart of biological complexity5. For prokaryotic organisms, it has long been thought that regulatory and transcriptomic divergence is less relevant because genomic differences, higher mutation rates and horizontal gene transfer provide sufficient means for rapid adaptation to various environments. Moreover, most bacterial genomes are relatively compact and have a large protein-coding fraction, leaving less room for non-coding transcripts. Results The primary transcriptome of Synechocystis 6714 and its comparison to the closely related model Synechocystis 6803. There is not a single genome-wide study of gene expression for Synechocystis 6714 thus far. To enable the double-comparative transcriptomics approach, we used existing information for strain 680319 and generated a matching dataset for strain 6714 employing the same growth conditions, library prefoparation protocols and computational methods. Total RNA was isolated from cells cultured under multiple growth conditions (darkness, high light (HL), cold (15uC) and heat stress (42uC), depletion of iron (-Fe), phosphate (-P), nitrogen (-N) or inorganic carbon (-C), and exponential and stationary growth phases), analysed according to the dRNA-seq protocol7 and used to infer transcriptional units (TUs)27. Following the previously introduced terminology19, we termed a transcriptional unit gTU if it covers one or more annotated genes, aTU if it is antisense to another TU (overlap $20 nt) and nTU if it is free-standing. The classification of a TU can be ambiguous as is the case for excludons28, which cover annotated genes and regions antisense to another gene or TU. Therefore, we provided all possible notations when a TU belonged to several categories. Each TU has a corresponding TSS whose associated read count in the treated library defines the expression level of that TU. The full list of TUs is presented in Table S1 and a genome-wide visualisation is presented in Supporting files S1-4. We defined 4,292 TUs in Synechocystis 6714 compared to 4,091 TUs in Synechocystis 680319. Table 1 summarises the numbers in the different transcript categories for each strain. We found 2,373 (83%) of the 2,854 protein-coding orthologous genes26 transcribed from a gTSS in both strains under at least one of the examined conditions, yielding 2,012 and 1,924 gTUs for Synechocystis 6803 and Synechocystis 6714, respectively. From these, 850 gTUs (.42%) are To address the extent to which bacterial transcriptome organisa- tion and composition is conserved and functionally relevant, here we performed a multi-condition, double-comparative transcriptomic analysis of two closely related strains of the unicellular cyanobacter- ium Synechocystis. In Synechocystis 6803, substantial pervasive tran- scription was reported, with ,64% of all TSSs giving rise to antisense or sRNAs in a genome that is to 87% protein coding8. Recently, we elucidated the response of Synechocystis 6803 to specific envir- onmental conditions and identified more than 4000 transcriptional units, about half of which represent non-coding RNAs19. OPEN However, the discovery of large numbers of sRNAs, including asRNAs6–13, and of their versatile roles in regulatory processes, especially during stress adaptation, have clearly demonstrated the relevance of non-coding RNA in prokaryotes14–16. Genomic comparisons between closely related bacteria have been pivotal in gaining insight into their metabolic potential, regulatory networks and genome evolution. In contrast, the number of inter-strain or inter-species transcriptomic comparisons has remained relatively scarce so far. Differential RNA-seq-type transcriptomic analyses (dRNA-seq7) are especially powerful, as this technique enables the identification of TSSs at a gen- ome-wide scale at single-nucleotide resolution and can easily identify sRNAs as well as transcripts that originate within genes in either orientation. Thus, the detailed information on TSSs provided by dRNA-seq gives deep insight into the transcriptional landscape of an organism. Comparative transcriptomics has proven useful at inferring the dynamics of transcriptional regulation by analysing regulatory responses to different conditions. Such an analysis compared primary transcriptomes of the human pathogen Helicobacter pylori under the mid- logarithmic growth phase versus acid stress conditions, mimicking the host environment7. A comparative analysis of the primary transcriptome of the cyanobacterium Anabaena sp. PCC 7120 revealed more than 10,000 TSSs active during the differentiation of N2-fixing heterocysts, of which .900 TSSs exhibited minimum SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 1 www.nature.com/scientificreports encoded genes, fraction of non-coding DNA and 16S rDNA (99.4% identity)26. Both strains share 2854 protein-coding genes, leaving 829 unique genes in Synechocystis 6803 and 916 in Synechocystis 6714, and have an average nucleotide identity (ANI) of 86.4% (see Table 1). Here, we present genome-wide maps of the TSSs active in Synechocystis 6714 under the same 10 conditions as in the previous analysis of Synechocystis 680319, and identified 4292 transcriptional units (TUs). The genome-wide comparison of these transcripts and the TSS maps in both strains under the different conditions revealed substantial conservation but also specific differences in the respective transcriptional organisation. In the transcriptome comparison we find the presence and expression of non-coding RNAs to constitute the evolutionarily most flexible component and identified a new gen- etic element, which we propose calling actuaton. fold changes (FCs) of eight, suggesting a large number of unidentified regulators of cell differentiation and N2-fixation9. There are very few double-comparative transcriptomic approaches in which the responses of two different but closely related organisms to multiple environmental conditions have been studied. OPEN The com- parison of the primary transcriptomes of pathogenic L. monocyto- genes and non-pathogenic L. innocua species under mid-log and stationary growth phases led to the discovery of 33 sRNAs and 53 asRNAs in L. monocytogenes11. Interestingly, some were not expressed in one of the species, although they were conserved at the DNA level, which indicates the importance of transcriptomic analyses and suggests a possible additional layer of divergence11. Comparative analysis of samples from mid-logarithmic growth stages of three isolates of the human pathogen and one isolate of the chicken pathogen Campylobacter jejuni revealed conserved as well as strain-specific TSSs and detected 15 conserved and 24 strain-specific sRNA candidates17. The comparison of transcriptome profiles of the model cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 (from here: Synechocystis 6803) revealed substantial differences in the transcriptional response to envir- onmental fluctuations18, which in fact may be linked to the relatively large taxonomic distance between the two species, indicated by the 10% divergence in their 16S ribosomal RNA sequences. Results Interestingly, the TSS driving transcription of the anTU antisense to the sigB 59UTR is under environmental control. In both strains, it is high in the cold and –N condition but relatively weak during heat stress (Figure 3A). The most bizarre findings are two iTSSs, one located towards the end of the sigB reading frame and the other located at the end of the downstream, tail-to-tail oriented gene. Additionally, these iTSSs appear to be environmentally regulated, suggesting functional relevance. The very strong iTSS at the end of the sigB reading frame is regulated different from the sigB host gene, supporting it further as a separate transcriptional entity. The transcript originating from this iTSS was identified in Synechocystis 6803 independently before, characterising it as a ,100 nt sRNA on the basis of Northern and tiling microarray analyses34. This observation is consistent with the prediction of a short sRNA originating from a conserved promoter and finishing with a pronounced Rho-independent terminator of transcription (Figure 3B,C). Consequently, it is very interesting to note that not only the complexity of this arrangement within and around the sigB gene has been conserved, but also the modes of regulation. In Synechocystis 6714, the longest TU (TU2897, Table S1) encom- passes 18 genes encoding ribosomal proteins (corresponding to the enterobacterial S10 and spc operons, except for the rps10 gene). The longest TU in Synechocystis 6803 (22 genes) contains mainly genes without an ortholog in 6714 (or in any other cyanobacteria) and has features of a genomic island19,26. This genomic island exists in both strains. In both cases the genes encode glycosyltransferases and gly- coside hydrolases possibly involved in the modification of cell surface properties. However, these genes are of entirely different phylogen- etic origin in the two strains. The median 59 UTR length for Synechocystis 6714 is 54 nt, similar to the 52 nt in Synechocystis 6803 (Supplementary Figure S1), and the median 39 UTR length is, with 128 nt, also only slightly longer than the 118 nt found in strain 6803. Our data provide evidence for 48 leaderless mRNAs in Synechocystis 6714. Interestingly, their expression was distributed over all conditions, and only 6 of them are conserved in Synechocystis 6803 (Table S3). Leaderless mRNAs play a role in stress adaptation in E. coli29 and are generally thought to be restricted to a particular condition, which we did not observe in our data. Results To address the question of whether orthologous transcripts have globally similar expression patterns, we performed a combined clus- tering of all orthologous TUs based on their expression profiles. Interestingly, only about one third of the orthologs were grouped in the same cluster for a relatively low number of clusters (k-means; k 5 10), and this value decreased with increasing k (Figure S2). This shows that the majority of the orthologous transcripts differ substan- tially in their expression pattern. While the expression may differ in one aspect, regulation as a whole may still be conserved to a large degree. Thus, in a second approach, we compared the FCs of ortholog TUs for all pairs of growth conditions. The resulting scatter plot indicates an overall medium correlation (R2 5 0.70) between the FCs of the two strains (Figure 1). Remarkable regulatory differences include the cmpR gTSS, which strongly responds to HL in strain 6714 but is barely active in strain 6803 in this condition (Figure 2). The cmpR gene encodes a LysR-type transcriptional activator for a part of the carbon concentrating mechanism30. Its divergent regulation may affect the expression of its main target, the cmpABCD operon encod- ing a transporter for bicarbonate uptake (Figure 2A, B), and can be linked to specific differences in its promoter organisation, in particu- lar to a single nucleotide exchange in the 210 element (TATAAT R TGTAAT) that renders it less active in strain 6803 (Figure 2D). Another observation concerns the 59UTR of cmpA that accumulates as a very abundant separate short transcript in both strains, most likely caused by premature termination at Rho-independent termi- nators (Figure 2B, C). g g The expression of stress-related genes may be restricted to a single condition in which a TSS shows maximum activity. We analysed TSS activity globally and comparatively by dividing the top number of normalised reads for a gene in one condition by the number of reads from the condition with the second highest number of reads (UEF, unique expression factor19). A high UEF (.5.0) indicates strong induction of the respective TSS under one particular condition. Many of the top-induced TSSs are consistent with the respective stress conditions, e.g., for the phycobilisome degradation protein NblA during -N or for the aforementioned transcriptional regulator CmpR under HL (Table S1). In the previous analysis of Synechocystis 6803, the phosphate and iron stress regulons were studied more closely. Results We assessed the four different TU types for their propensity for differential expression by computing for each TU the maximum FC in every pair of condi- tions. Figure S3A shows that the majority of TUs of all types differ by more than 10-fold in expression in at least one pair of conditions. It is noteworthy that gTUs more frequently show high FCs compared to aTUs and iTUs. Similarly, gTUs also show a tendency for higher expression levels (Figure S3B). For each of the 10 tested conditions, the TU associated with the highest read number, among all TUs that are maximally expressed under the respective condition, is given in Table S2. In three condi- tions, the top ranking TU gave rise to an abundant sRNA (Table S2). The sRNA Ncr0700 (TU3047), with 11,257,987 normalised reads, is the most abundant transcript in strain 6714 and is maximally expressed in darkness, as is its ortholog in strain 680319. Altogether, in four conditions (-P, darkness, 42uC and -N) we found orthologous transcripts to be associated with the highest read num- bers in both strains (Table S2). For two TUs (-C and cold), the ortholog in Synechocystis 6803 either showed highest read levels under a different growth condition or was outperformed by another TU with higher read numbers. For the TUs top ranking in stat. phase and HL, ortholog TUs in 6803 are simply lacking, while for the remaining (-Fe and exp. phase), interesting regulatory differences were detected (see below). Responses to environmental stimuli are largely conserved but also reveal specific regulatory differences. The sigB (sll0306) gene, encoding the RNA polymerase s factor SigB, illustrates the complexity and conservation of transcriptional signals. There are at least four TSSs associated with this gene (Figure 3). In Synechocystis 6803, sigB is induced by a short treatment at 42uC31,32 and possesses a central role in the survival of the cells during short heat stress conditions32,33. Consistent with these facts, we identified a gTSS that is strongly induced upon heat stress. This gTSS is conserved in Synechocystis 6714 and, similar to strain 6803, shows maximum expression under heat stress. Furthermore, in both strains, the sigB gTSS is located antisense to a putative sRNA (TU3431 in 6714), causing a 197 nt (200 nt in 6803) long overlap within the 59UTR of sigB and qualifying the sigB mRNA as a gaTU. Results Several of these non-coding RNAs are important regulators of photosynthetic gene expression, such as the cis-encoded antisense RNAs (asRNAs) IsrR, As1_flv4 and PsbA2R20–22 or the trans-encoded sRNA PsrR123. There are several more non-coding RNAs with an expression that is tightly controlled by environmental conditions19, and therefore are likely to be of similar importance, but their characterisation is pending24. Moreover, information about the conservation and expression of these non-coding RNAs in other strains would be of high interest, but is largely lacking. We defined 4,292 TUs in Synechocystis 6714 compared to 4,091 TUs in Synechocystis 680319. Table 1 summarises the numbers in the different transcript categories for each strain. We found 2,373 (83%) of the 2,854 protein-coding orthologous genes26 transcribed from a gTSS in both strains under at least one of the examined conditions, yielding 2,012 and 1,924 gTUs for Synechocystis 6803 and Synechocystis 6714, respectively. From these, 850 gTUs (.42%) are Synechocystis sp. strain PCC 6714 (from here: Synechocystis 6714) is closely related to Synechocystis 6803 and its genome has recently been sequenced25. Their genomes are similar in size, number of Table 1 \| Genome statistics and numbers of different types of TUs for Synechocystis 6803 and 6714. gTUs were assumed to be conserved when the complete TU arrangement including the covered CDSs was conserved. Alignment positions with regard to the encoded amino acid sequence were used for the determination of conserved aTUs and iTUs. Conserved nTUs were detected via BLASTN and known non- nTU sRNAs were included Synechocystis 6803a Synechocystis 6714 Conservation Genome [kb] 3957 3739 ANI: 86%; 16S: 99.4% Plasmids 7 3 - Protein genes 3683 3770 2854 Non-coding fraction 12.85% 12.47% - Transcriptome characteristics TUs total 4091 4292 1341 gTUs 2012 1924 850 aTUs 3040 3758 168 iTUs 483 424 147 (incl. iTSSs) iTSSs 766 776 nTUs 367 306 221 (incl. known sRNAs) aNumbers for Synechocystis 6803 are taken from the literature19. ANI 5 average nucleotide identity. SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 2 www.nature.com/scientificreports www.nature.com/scientificreports perfectly conserved with regard to the covered genes and their arrangement. Strain-specific gTSSs for genes without any TSS in the other strain existed for 159 and 202 orthologs in Synechocystis 6803 or 6714, respectively. Differential expression is commonly regarded as evidence of the functional significance of a gene because it requires the existence of a regulator and a specific regulatory element. Results See Figure 2 and Table S1 for details about the expression patterns and the annotation of the mentioned genes; HP, hypothetical protein. Figure 1 \| Regulation and conservation. Scatter plot of all pairwise log2 fold changes for the 859 gTUs orthologous between Synechocystis 6803 (x-axis) and 6714 (y-axis). The coordinates of each point resemble the pairwise log2 fold change between a pair of conditions in one strain compared with the corresponding pair of conditions in the other. Some extreme examples are annotated. For iutA, encoding the TonB-dependent ferric siderophore receptor the regulation is conserved with maximum expression in -Fe and no expression in any of the other conditions. For gdbH, the maximum differences in fold changes between Synechocystis 6803 and 6714 appeared for 15uC/42uC, 15uC/stat. phase and 15uC/-C. For cmpR, the maximum difference in fold changes was detected for -C/HL. See Figure 2 and Table S1 for details about the expression patterns and the annotation of the mentioned genes; HP, hypothetical protein. whereas the homologous gene in strain 6803, slr1085, is part of a multicistron not affected by -Fe. phosphate depletion and that the former had just so missed the threshold in the previous analysis. These two regulatory proteins are quite different from each other. Their example illustrates how only by the comparison of the two datasets novel regulatory factors can be identified. Both proteins are widely distributed throughout the cyanobacterial phylum but their involvement in the response to phosphorus depletion was not known so far. In several bacteria, regulatory sRNAs have been characterised that play an important role in the iron stress response36–38. Indeed, we also detected the antisense RNA IsrR in Synechocystis 6714 (Figure 4A, B), which acts as a negative regulator of isiA encoding the iron stress inducible protein A in Synechocystis 680320. In addition, the comparison of possible secondary structures shows 4 base transitions that are consistent with the structural model, suggesting selective force on the structure of IsrR. Moreover, we identified the sRNA IsaR119 with an UEF of 130.9 to be the specifically and by far the most strongly up-regulated nTU under iron depletion (Table S5), supporting its functional relevance within the iron stress regulon. Similar to the analysis of the phosphate stress regulon, we ranked the iron stress-specific TUs according to their maximum UEF values (Table S5). Results The Synechocystis 6803 regulon responding to phosphate depletion encompassed 8 TUs19, which comprise the genes reported before35 as well as newly identified ones, e.g., the phosphate-stress- induced PsiR1 transcript. Our results (Table S4) reveal a high level of conservation for this regulon, with the exception of PsiR1, which is missing in Synechocystis 6714 and two TUs that are not affected by phosphate depletion in strain 6714. Interestingly, we identified with D082_04580 in addition to D082_05330 a second cAMP-binding regulator of the CAP family of transcription factors that belongs into the P-regulon. Upon closer inspection, it turned out that also the two homologs in strain 6803, Slr0607 and Sll0594, are up-regulated upon SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 3 Figure 1 \| Regulation and conservation. Scatter plot of all pairwise log2 fold changes for the 859 gTUs orthologous between Synechocystis 6803 (x-axis) and 6714 (y-axis). The coordinates of each point resemble the pairwise log2 fold change between a pair of conditions in one strain compared with the corresponding pair of conditions in the other. Some extreme examples are annotated. For iutA, encoding the TonB-dependent ferric siderophore receptor the regulation is conserved with maximum expression in -Fe and no expression in any of the other conditions. For gdbH, the maximum differences in fold changes between Synechocystis 6803 and 6714 appeared for 15uC/42uC, 15uC/stat. phase and 15uC/-C. For cmpR, the maximum difference in fold changes was detected for -C/HL. See Figure 2 and Table S1 for details about the expression patterns and the annotation of the mentioned genes; HP, hypothetical protein. www.nature.com/scientificreports www.nature.com/scientificrepo Figure 1 \| Regulation and conservation. Scatter plot of all pairwise log2 fold changes for the 859 gTUs orthologous between Synechocystis 6803 (x-axis) and 6714 (y-axis). The coordinates of each point resemble the pairwise log2 fold change between a pair of conditions in one strain compared with the corresponding pair of conditions in the other. Some extreme examples are annotated. For iutA, encoding the TonB-dependent ferric siderophore receptor the regulation is conserved with maximum expression in -Fe and no expression in any of the other conditions. For gdbH, the maximum differences in fold changes between Synechocystis 6803 and 6714 appeared for 15uC/42uC, 15uC/stat. phase and 15uC/-C. For cmpR, the maximum difference in fold changes was detected for -C/HL. Results In Synechocystis 6803 18 TUs belong into the core iron stress regulon19, compared to 17 TUs in Synechocystis 6714. In both strains, these TUs include 32 protein-coding genes and one sRNA. The fact that the majority of genes responded in a similar fashion in both strains, including the well-characterised iron stress marker gene isiA (Figure 4), suggests a highly conserved iron stress response in the two strains. Differences include the two genes ssr2333 and slr1392, which lack a homolog in Synechocystis 6714. These genes encode a FeoA/FeoB-type ferrous iron transporter that exists in only few cya- nobacteria. On the other hand, there are two additional genes in Synechocystis 6714 that belong into the core iron stress regulon. These are D082_02330, without known function, and D082_21730 encoding a GT1_wbuB_like protein closely related to the GT1 family of glycosyltransferases that is a monocistron in strain 6714 (TU959), Diversity and conservation within the non-coding transcriptome. The number of orthologous genes that are associated with at least one antisense transcript (aTU) in both organisms is 995. However, the corresponding TSS was conserved for 168 aTUs only (Table S6). Many of them likely play an important regulatory role, because they are conserved in terms of sequence, position and sometimes even regulation. Indeed, we detected the antisense RNA IsrR in Synechocystis 6714 in this class (Figure 4). Other examples of conserved asRNAs were found for the ABC transporter subunit SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 4 www.nature.com/scientificreports www.nature.com/scientificreports re 2 \| Differences in the expression of CmpR, a LysR family transcription factor involved in the control of carbon uptake and concentr scriptional organization in the intergenic region between cmpA and cmpR. The color-coded graphs represent the accumulation of prim in the dRNA-seq analysis for the ten compared conditions. From the TSS identification and secondary read coverage (grey), transcripti , red) were inferred. Protein-coding genes are displayed in blue. A gTSS upstream of cmpR in strain 6714, but below the detection limit in st dicated by an arrow. Positions of 32P-labelled probes for Northern verification in panel (B) are marked by asterisked bars. The dashed line holds for the required minimum sequence coverage. (B) Northern verification for cmpA that shows accumulation of a distinct, small trans 9UTR. The signal for the 5S rRNA was used as a loading control. Results (C) Potential RNA terminator hairpins upstream of cmpA between 23 5 first nucleotide of the cmpA start codon). (D) The cmpA/cmpR intergenic region harbouring the promoters for both genes. The und ence was used for the RNA structure prediction in panel (C). The red box indicates putative cmpR binding sites consistent with the conse N TAA described for Synechococcus sp PCC 794230 The entire region is almost identical in both strains except a small number of s Figure 2 \| Differences in the expression of CmpR, a LysR family transcription factor involved in the control of carbon uptake and concentration. (A) Transcriptional organization in the intergenic region between cmpA and cmpR. The color-coded graphs represent the accumulation of primary reads in the dRNA-seq analysis for the ten compared conditions. From the TSS identification and secondary read coverage (grey), transcriptional units (TUs, red) were inferred. Protein-coding genes are displayed in blue. A gTSS upstream of cmpR in strain 6714, but below the detection limit in strain 6803, is indicated by an arrow. Positions of 32P-labelled probes for Northern verification in panel (B) are marked by asterisked bars. The dashed lines mark the thresholds for the required minimum sequence coverage. (B) Northern verification for cmpA that shows accumulation of a distinct, small transcript from the 59UTR. The signal for the 5S rRNA was used as a loading control. (C) Potential RNA terminator hairpins upstream of cmpA between 239 to 2114 (11 5 first nucleotide of the cmpA start codon). (D) The cmpA/cmpR intergenic region harbouring the promoters for both genes. The underlined sequence was used for the RNA structure prediction in panel (C). The red box indicates putative cmpR binding sites consistent with the consensus motif TTA-N7/8-TAA described for Synechococcus sp. PCC 794230. The entire region is almost identical in both strains, except a small number of single nucleotide exchanges, one of which is in the 210 element (boxed 1 green letters) and probably linked to the observed weaker cmpR promoter activity in Synechocystis 6803. Figure 2 \| Differences in the expression of CmpR, a LysR family transcription factor involved in the control of carbon uptake and concentration. (A) Transcriptional organization in the intergenic region between cmpA and cmpR. The color-coded graphs represent the accumulation of primary reads in the dRNA-seq analysis for the ten compared conditions. From the TSS identification and secondary read coverage (grey), transcriptional units (TUs, red) were inferred. Results (A) The sigB gene and loci near to it illustrate the complexity and conservation of TSS arrangements. In accordance with previous findings, the sigB gene in Synechocystis 6803 is induced by a short treatment at 42uC31,32. This regulation is also found in strain 6714. In both strains, the sigB TSS is located antisense to a putative sRNA, qualifying the sigB mRNA as a gaTU. In addition there are two iTSS, one located within the sigB gaTU towards the end of the sigB reading frame and the other one located at the end of the downstream, tail-to-tail oriented genes slr0320/ D082_29700, which encode an Fe-S oxidoreductase. Also these iTSSs appear to be environmentally regulated, suggesting functional relevance. For annotation details see Figure 2A. (B) Details for the location of the rear iTSS in sigB. (C) The consensus structure of the transcript originating at the sigB iTSS supports its classification as an sRNA based on length, lack of a reading frame and high support from the program RNAz58 with an SVM RNA class probability of 0.99. genes sll0484/D082_06780 or the ccmK genes (sll1028/D082_28020). The latter encode the carbon dioxide-concentrating mechanism protein K homolog 2. Therefore, the maximum expression of these ccmK-aTUs under N-depletion points to a possible silencing function when nitrogen is limited (Supporting File S1). Thus, a single mutation led to the activation of this antisense pro- moter in strain 6803, or alternatively, disrupted it in strain 6714. Our verification experiments show that in addition to the known co- induction of psbA2 and psbA2R in Synechocystis 6803, the asRNA is expressed more highly under conditions that lead to oxidative stress (Figure 5B), possibly extending the known functional role of psbA2R22. Thus, a single mutation led to the activation of this antisense pro- moter in strain 6803, or alternatively, disrupted it in strain 6714. Our verification experiments show that in addition to the known co- induction of psbA2 and psbA2R in Synechocystis 6803, the asRNA is expressed more highly under conditions that lead to oxidative stress (Figure 5B), possibly extending the known functional role of psbA2R22. In contrast, we found in Synechocystis 6714 no evidence for the conservation of asRNAs to the psbA genes, which sustain high levels of expression of psbA2 and psbA3 in Synechocystis 680322. Results Protein-coding genes are displayed in blue. A gTSS upstream of cmpR in strain 6714, but below the detection limit in strain 6803, is indicated by an arrow. Positions of 32P-labelled probes for Northern verification in panel (B) are marked by asterisked bars. The dashed lines mark the thresholds for the required minimum sequence coverage. (B) Northern verification for cmpA that shows accumulation of a distinct, small transcript from the 59UTR. The signal for the 5S rRNA was used as a loading control. (C) Potential RNA terminator hairpins upstream of cmpA between 239 to 2114 (11 5 first nucleotide of the cmpA start codon). (D) The cmpA/cmpR intergenic region harbouring the promoters for both genes. The underlined sequence was used for the RNA structure prediction in panel (C). The red box indicates putative cmpR binding sites consistent with the consensus motif TTA-N7/8-TAA described for Synechococcus sp. PCC 794230. The entire region is almost identical in both strains, except a small number of single nucleotide exchanges, one of which is in the 210 element (boxed 1 green letters) and probably linked to the observed weaker cmpR promoter activity in Synechocystis 6803. SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 5 www.nature.com/scientificreports Figure 3 \| Conserved complex TSS arrangements. (A) The sigB gene and loci near to it illustrate the complexity and conservation of TSS arrangements. In ccordance with previous findings, the sigB gene in Synechocystis 6803 is induced by a short treatment at 42uC31,32. This regulation is also found in strain 714. In both strains, the sigB TSS is located antisense to a putative sRNA, qualifying the sigB mRNA as a gaTU. In addition there are two iTSS, one located within the sigB gaTU towards the end of the sigB reading frame and the other one located at the end of the downstream, tail-to-tail oriented genes slr0320/ D082_29700, which encode an Fe-S oxidoreductase. Also these iTSSs appear to be environmentally regulated, suggesting functional relevance. For nnotation details see Figure 2A. (B) Details for the location of the rear iTSS in sigB. (C) The consensus structure of the transcript originating at the sigB TSS supports its classification as an sRNA based on length, lack of a reading frame and high support from the program RNAz58 with an SVM RNA class robability of 0.99. Figure 3 \| Conserved complex TSS arrangements. Results Despite the very high (97%) nucleotide sequence conservation between the psbA genes of both strains, corresponding asRNAs were not detectable in Synechocystis 6714 (Figure 5A, B). On a closer inspection, two nuc- leotide polymorphisms were identified, from which a G-to-A trans- ition within the 210 element of the TSS of the asRNA PsbA2R is a likely gain-of-function mutation in Synechocystis 6803 (Figure 5C). Trans-encoded sRNAs are by far the most promising class of non- protein regulators. Therefore, we checked conservation and regu- lation of these transcripts revealing 69 intergenic transcripts (nTUs) that are conserved and predicted as nTU in both strains. Additionally, 48 nTUs from Synechocystis 6714 and 91 nTUs from Synechocystis 6803 were conserved but classified differently in the other strain (Table S7). Furthermore, in both strains, 12 known SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 6 6 www.nature.com/scientificreports \| Two conserved sRNAs in the iron stress-response of Synechocystis. (A) Transcriptional organization and expression of the isiA locus in ystis 6803 and 6714. For annotation details see Figure 2. In both strains, isiA is highly inducible in the -Fe condition (brown graph), while the rR is inversely regulated, suggesting a conserved function. (B) Verification experiment for the presence of IsrR and for the iron stress-dependent tion of the isiA mRNA and a separate sRNA from its 59UTR. The 5S rRNA was used as a loading control. (C) Consensus structure of the IsrR s predicted by RNAz58. Figure 4 \| Two conserved sRNAs in the iron stress-response of Synechocystis. (A) Transcriptional organization and expression of the isiA locus in Synechocystis 6803 and 6714. For annotation details see Figure 2. In both strains, isiA is highly inducible in the -Fe condition (brown graph), while the asRNA IsrR is inversely regulated, suggesting a conserved function. (B) Verification experiment for the presence of IsrR and for the iron stress-dependent upregulation of the isiA mRNA and a separate sRNA from its 59UTR. The 5S rRNA was used as a loading control. (C) Consensus structure of the IsrR homologs predicted by RNAz58. The same type of analysis revealed conservation of 147 iTSSs in 141 out of 360 iTSS-containing ortholog pairs (Table S8). Some of these conserved iTSSs appear very strong and are in genes of known relevance. Results The mRNAs of the very closely related genes psbA2 and 3 are positively regulated by light in both strains, whereas the asRNAs PsbA2R and PsbA3R22 originating from a TSS 19 nt upstream of the start codon (TU12 and TU1885) are specific for Synechocystis 6803. (B) The absence of PsbA2R in Synechocystis 6714 was verified by Northern blots. The 5S rRNA was used as a loading control (the psbA2 membrane is shown). (C) Alignment of the promoter sequences for PsbA2R and PsbA3R with the corresponding regions in Synechocystis 6714 indicate only two mismatches between the two strains. Of these, a single transition TATAAT R TATGAT within the 210 element (boxed) is likely involved in the activation/deactivation of the aTSS. For orientation, the psbA start codon on the reverse strand is shown in bold, and the direction of translation is indicated by the arrow bar. Figure 5 \| Example for a functional asRNA that is not conserved. (A) Transcriptional organization and expression of the psbA2 locus in Synechocystis 6803 and 6714. The mRNAs of the very closely related genes psbA2 and 3 are positively regulated by light in both strains, whereas the asRNAs PsbA2R and PsbA3R22 originating from a TSS 19 nt upstream of the start codon (TU12 and TU1885) are specific for Synechocystis 6803. (B) The absence of PsbA2R in Synechocystis 6714 was verified by Northern blots. The 5S rRNA was used as a loading control (the psbA2 membrane is shown). (C) Alignment of the promoter sequences for PsbA2R and PsbA3R with the corresponding regions in Synechocystis 6714 indicate only two mismatches between the two strains. Of these, a single transition TATAAT R TATGAT within the 210 element (boxed) is likely involved in the activation/deactivation of the aTSS. For orientation, the psbA start codon on the reverse strand is shown in bold, and the direction of translation is indicated by the arrow bar. (slr1212/uirS or pixA) and two response regulator genes (slr1213/ uirR or nixB and the PatA-type regulator slr1214/lsiR or nixC), which encode a UV-A-activated signaling system that is required for nega- tive phototaxis in Synechocystis 680341,42, is, except csiR1, fully syn- tenic between the two strains (Figure 6A). As we show here, the previously reported expression and accumulation of CsiR1 in Synechocystis 680319 results from integration of a sequence down- stream of an otherwise conserved promoter element (Figure 6B). Results Among them are the iTUs originating within the sigB gene (Figure 3), in the gene rre39 (slr1588/D082_10840, regulatory com- ponent of sensory transduction system Ssp2/Rre39), and in the ntcA gene, which encodes the major regulator of nitrogen metabolism. sRNAs were part of gTUs or aTUs, e.g., caused by a downstream located and co-transcribed gene lacking a gTSS. When these known sRNAs are included, the number of conserved sRNAs and sRNA candidates increases to 221. We found both, the sequences (87.8% average identity) as well as the expression of many of these sRNAs conserved, pointing to their potential functional relevance. This is illustrated by the above mentioned sRNAs IsaR1 and Ncr0700, which are maximally expressed during iron depletion or in the dark, as well as PsrR1 (Photosynthesis regulatory RNA 1), which is up-regulated under high-light treatment or CO2 depletion19,34 and controls the expression of several genes encoding photosynthetic proteins23, and by the sRNA NsiR4, which is strongly induced upon nitrogen depletion (Table S1 and Figure S4). sRNAs were part of gTUs or aTUs, e.g., caused by a downstream located and co-transcribed gene lacking a gTSS. When these known sRNAs are included, the number of conserved sRNAs and sRNA candidates increases to 221. We found both, the sequences (87.8% average identity) as well as the expression of many of these sRNAs conserved, pointing to their potential functional relevance. This is illustrated by the above mentioned sRNAs IsaR1 and Ncr0700, which are maximally expressed during iron depletion or in the dark, as well as PsrR1 (Photosynthesis regulatory RNA 1), which is up-regulated under high-light treatment or CO2 depletion19,34 and controls the expression of several genes encoding photosynthetic proteins23, and by the sRNA NsiR4, which is strongly induced upon nitrogen depletion (Table S1 and Figure S4). Actuatons: sRNA cassettes driving gene expression. We noticed that some known sRNAs were classified as gTUs because a downstream located gene in sense orientation lacks a specific gTSS and is apparently co-transcribed with the sRNA due to incomplete termination of transcription (Table 2). An example of this SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 7 www.nature.com/scientificreports Figure 5 \| Example for a functional asRNA that is not conserved. (A) Transcriptional organization and expression of the psbA2 locus in Synechocystis 6803 and 6714. Results Such an sRNA/mRNA structure, which we named ‘actuaton’, constitutes a means of remodeling the transcriptional network. This concept is strongly supported by TSS/sRNA cassettes that are fused to different genes or are specific for one of the inves- tigated strains and hence provide a different expression pattern. Examples include the Ncr0700 sRNA that originates from a free- standing TU in Synechocystis 6714, whereas it became part of a chimeric TU in strain 6803 due to rearrangement by transposition. Results In Synechocystis 6714, the intergenic region between uiR and lsiR mea- sures only 205 bp, while it spans 535 bp in Synechocystis 6803. Although the sequence was integrated downstream of the -10 ele- ment, it has a clear influence on the general expression level, which is lower in strain 6714. Moreover, whereas the expression of that TSS seems rather constitutive under these tested conditions in strain 6803, it appears very low or even absent in darkness and in stationary phase cells of strain 6714 (Figure 6A, Supporting File S1). arrangement is constituted by the sRNAs Yfr2b and Yfr2c. These belong to an sRNA family of unknown function that is widely distributed among cyanobacteria, the genes of which occur in different genetic arrangements and in copy numbers from one to nine39. There are three members of the Yfr2 family in Synechocystis 6803, which accumulate as sRNAs of 80, 65 and 70 nt40. All three are conserved in strain 6714 and present in the same genomic context, with yfr2a as a free-standing gene, and yfr2b/c linked to the respective orthologous protein-coding genes in the two strains (Table 2). g p g g We noticed at least 10 cases in which very abundant sRNAs became part of such a chimeric precursor transcript and therefore give rise to the respective mRNAs, which otherwise have no distinct TSS (Table 2). Such an sRNA/mRNA structure, which we named ‘actuaton’, constitutes a means of remodeling the transcriptional network. This concept is strongly supported by TSS/sRNA cassettes that are fused to different genes or are specific for one of the inves- tigated strains and hence provide a different expression pattern. Examples include the Ncr0700 sRNA that originates from a free- standing TU in Synechocystis 6714, whereas it became part of a chimeric TU in strain 6803 due to rearrangement by transposition. Evolutionary events such as gain or loss of an sRNA within or close to a promoter may directly affect other genes. The sRNA CsiR119 illustrates this effect (Figure 6). CsiR1 originates from the uirS-lsiR region in Synechocystis 6803 but is not present in strain 6714. However, the uirS-lsiR region encompassing a cyanobacteriochrome We noticed at least 10 cases in which very abundant sRNAs became part of such a chimeric precursor transcript and therefore give rise to the respective mRNAs, which otherwise have no distinct TSS (Table 2). SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 Discussion TU3300 SyR5 TU1772 D082_15700 Georg et al., 200934 O-antigen polymerase and TPR domain; conserved arrangement TU3575 Yfr2c TU341 D082_02910 Voß et al., 200940 protease of the Abi (CAAX) family; conserved arrangement TU2628-2629 SyR9 TU610–611 D082_05310 Kla¨hn et al., 201445 aldehyde deformylating oxygenase; conserved arrangement in the two strains TU2885 Yfr2b TU2652 D082_23050 Voß et al., 200940 glutamine amidotransferase; conserved arrangement TU643 Ncr0280 NA NA Kopf et al., 201419 integrin subunit alpha; Ncr0280 and Slr1028 unconserved in Synechocystis 6803 TU905 CsiR1 TU19 D082_00130 Kopf et al., 201419 two-component response regulator LsiR or NixC; flanking genes are conserved but not csiR1 TU3599 Ncr1680 TU1242 D082_10970 Kopf et al., 201419 probable transport protein; conserved arrangement TU1715 Ncr0700 TU3047 none Kopf et al., 201419, this paper, Figure S4 transposase in Synechocystis 6803, free-standing gene in 6714; sequence of Ncr0700 is 91% identical in a 195 nt long overlap TU87 Ncr0020 TU2357 D082_20640 Kopf et al., 201419 ribonuclease E; conserved arrangement TU3332 Ncr1575 TU1031 D082_08870 Kopf et al., 201419, this paper, Figure 2B cmpA; conserved arrangement TU2592 Ncr1265 TU5109 NA Kopf et al., 201419 lipid A disaccharide synthase LpxB; Ncr1265 exists in 11 copies in strain 6803, representing a likely RNA-OUT transcript from an ISY523-type transposase gene. It is a free standing TU on plasmid pSYLA in Synechocystis 6714. TU1556 - TU228 D082_02000 Kopf et al., 201419, Du¨hring et al., 200620; this paper, Figure 4 isiA 59UTR sll0815 sll0737 sll1477 sll0208 slr0199 slr1028 slr1214 slr0753 ssr2227 slr1129 slr0040 slr0015 sll0247 y From the conserved protein-coding genes between Synechocystis 6803 and 6714, we found 83% to be expressed in both, but for only 45%, the transcriptional organisation was also conserved. In line with previous findings from Listeria11 and enterobacteria43, 53% of sRNAs and sRNA candidates, but only 12% of the iTSSs and 4% of the aTSSs were conserved between the two Synechocystis strains. The lower degree of conservation of asRNAs raises the question of whether they result from pervasive transcription, the functional relevance of which is the scope of a lively debate43,44. Lack of conservation is often seen as evidence for a lack of function, used e.g., for bacterial antisense transcripts in a comparison between two different enter- obacteria43. If this argument is turned around, our identification of 168 highly conserved cis-asRNAs with regard to sequence, position and regulation, likely identified important regulatory players. Indeed, one of them is the functionally well-characterised asRNA IsrR20. Discussion However, in addition to IsrR there are three more asRNAs with known regulatory function in Synechocystis 6803, As1_flv421, PsbA2R and PsbA3R22, none of which were found in Synechocystis 6714. For As2_flv4, the respective operon is simply lacking26. However, psbA genes are present in both strains and here we iden- tified two base mutations, one of which is within the 210 element as the likely molecular basis for the presence or absence of PsbA2R and PsbA3R asRNAs (Figure 5B, C). Hence, a single point mutation leading to the loss or gain of a functionally relevant asRNA may provide a small but significant physiological advantage that becomes rapidly fixed during only a few generations, as was experimentally demonstrated for the psbA2/PsbA2R sense-antisense pair in Synechocystis 680322. This is even more interesting, as PsbA2R is present only in substoichiometric amounts, is co-regulated with its sense gene and has a very short half life22. The number of asRNAs with a characterised function is still very small compared to the total number of such transcripts. Nevertheless, the existing data indicate that it might be too simple to infer non-functionality of bacterial asRNAs from the lack of conservation. Our data suggest that the majority of these TUs are subject to regulation because they show largely different expression levels under the conditions tested. These differences should mainly reflect changes in promoter activity and be less strongly affected by tran- script stability or the presence of stable degradation products, as we focused here on primary transcripts. This finding is consistent with an earlier report in which, based on microarray datasets, Synechocystis 6803 cultures exposed to only three different condi- tions (high light, CO2 depletion, or darkness) were interrogated with respect to the percentage of transcripts with significant regulation, which was 46.4% for putative trans-acting sRNAs, similar as for mRNAs (43%)8. An exciting feature of the transcriptomic adaption identified here are the genetic elements that we named actuatons. A hallmark of these elements is that they are followed by a protein-coding gene in sense direction that lacks its own gTSS. Nevertheless, the dominating RNA species is an sRNA that accumulates as an abundant and dis- crete transcript and therefore constitutes a clearly separate entity. Therefore, an actuaton gives rise to an sRNA and at the same time constitutes part of the 59 region of a gene. Discussion With ten different conditions analysed in two closely related cyano- bacteria, this work presents a complex comparative study of micro- bial primary transcriptomes at single-nucleotide resolution. We consider the selected conditions to include some of the most relevant environmental factors for a photosynthetic organism. In particular, high light, darkness, the bioavailable iron and the supply of inorganic carbon are important determinants for the performance of oxygenic Evolutionary events such as gain or loss of an sRNA within or close to a promoter may directly affect other genes. The sRNA CsiR119 illustrates this effect (Figure 6). CsiR1 originates from the uirS-lsiR region in Synechocystis 6803 but is not present in strain 6714. However, the uirS-lsiR region encompassing a cyanobacteriochrome SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 8 www.nature.com/scientificreports photosynthesis and the photosynthetic electron chain. The other chosen conditions, cold and heat stress, the depletion of phosphate or nitrogen and the effects of differential growth phases are also highly relevant but represent factors of general relevance. For these reasons, our data are useful to understand the lifestyle of these pho- tosynthetic microbes and their behaviour in nature. Table 2 \| Actuatons detected in Synechocystis 6803 and 6714 6803 TU ncRNA 6803 first gene 6714 TU 6714 first gene Accumulation of sRNA part shown in Annotation and comments TU1737 Ncl0820 NA NA Kopf et al., 201419 hypothetical protein; only flanking genes Sll0814 and Sll0816 but not Ncl0820 and Sll0815 are conserved. Discussion We found evidence for at least ten such events and show that insertion of an actuaton modifies the expression of the downstream gene. Striking examples include the CsiR1 element (Figure 6), the Yfr2b and Yfr2c sRNA genes, which belong to an sRNA family that is widely distributed among cyanobacteria39, and the sRNA SyR9 forming a variable part of the 9 Figure 6 \| Actuatons in bacterial gene expression. The uirS-lsiR region encompassing a cyanobacteriochrome (slr1212/uirS or pixA) and two respo regulator genes (slr1213/uirR or nixB and the PatA-type regulator slr1214/lsiR or nixC) encodes a UV-A-activated signaling system41,42. This genom region is very similar in both strains with the exception of a proximal TSS upstream of D082_00130 and a more distally located TSS upstream of i ortholog slr1214. The latter gives rise to the sRNA CsiR1, from which read-through occurs into slr1214. (B) Sequence alignment of the intergenic reg between slr1213/D082_00120 and slr1214/D082_00130. Figure 6 \| Actuatons in bacterial gene expression. The uirS-lsiR region encompassing a cyanobacteriochrome (slr1212/uirS or pixA) and two response regulator genes (slr1213/uirR or nixB and the PatA-type regulator slr1214/lsiR or nixC) encodes a UV-A-activated signaling system41,42. This genomic region is very similar in both strains with the exception of a proximal TSS upstream of D082_00130 and a more distally located TSS upstream of its ortholog slr1214. The latter gives rise to the sRNA CsiR1, from which read-through occurs into slr1214. (B) Sequence alignment of the intergenic region between slr1213/D082_00120 and slr1214/D082_00130. Figure 6 \| Actuatons in bacterial gene expression. The uirS-lsiR region encompassing a cyanobacteriochrome (slr1212/uirS or pixA) and two response regulator genes (slr1213/uirR or nixB and the PatA-type regulator slr1214/lsiR or nixC) encodes a UV-A-activated signaling system41,42. This genomic region is very similar in both strains with the exception of a proximal TSS upstream of D082_00130 and a more distally located TSS upstream of its ortholog slr1214. The latter gives rise to the sRNA CsiR1, from which read-through occurs into slr1214. (B) Sequence alignment of the intergenic region between slr1213/D082_00120 and slr1214/D082_00130. mRNA encoding aldehyde deformylating oxygenase45, the key enzyme for cyanobacterial alkane biosynthesis. Even the long 59 UTRs of TUs encompassing otherwise well-characterised genes, such as those encoding RNase E and RNase HII (Table 2), the CmpABCD bicarbonate transporter (Figure 2B) or IsiA (Figure 4B), belong to this class. Methods Bi l i l Biological material and growth conditions. Synechocystis 6714 was purchased from the Pasteur Culture Collection (PCC) in Paris, France. Liquid cultures were grown at 30uC in BG11 medium52 under continuous white light illumination of 50–80 mmol quanta m22s21 and a continuous stream of air to the desired growth phase (OD750 5 0.6–0.8). For the transcriptome analyses, cultures were initially grown under standard conditions and then transferred to ten different conditions: (1) cold stress, 15uC for 30 min; (2) heat stress, 42uC for 30 min; (3) Ci depletion, 150 mL of culture was washed 3 times with 100 mL of carbon-free BG11 and cultured for additional 20 h; (4) dark, no light for 12 h; (5) Fe21 limitation, addition of iron-specific chelator desferrioxamine B (DFB) and cultivation for additional 24 h; (6) high light, 470 mmol q s21 m22 for 30 min; (7) N depletion, 150 mL of culture was washed 3 times with 100 mL of nitrogen-free BG11 and cultured for additional 12 h; (8) P depletion, cultures were washed 3 times with P-free BG11 and further grown for 12 h; (9) stationary phase, cells were grown until an OD750 of 4.1 was reached; (10) exponential phase, cells were harvested at an OD750 of 0.6. Northern Blot analysis. Selected transcripts were verified by Northern hybridization using single-stranded radioactively labelled RNA probes. These probes were generated by in vitro transcription as described57, using templates amplified by PCR and oligonucleotides listed in Table S10. For the analysis, 3 mg of total RNA was separated on 1.5% agarose gels, transferred to Hybond-N nylon membranes by capillary blotting and cross-linked by UV-illumination. The membranes were hybridized with the labelled RNA probes as described57. The signals were visualized using a Personal Molecular Imager FX system with Quantity One software (Bio-Rad). 1. King, M. C. & Wilson, A. C. Evolution at two levels in humans and chimpanzees. Science 188, 107–116 (1975). 2. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009). 3. Nagano, T. & Fraser, P. No-Nonsense Functions for Long Noncoding RNAs. Cell 145, 178–181 (2011). RNA extraction, cDNA synthesis and sequencing. Synechocystis 6714 cultures were harvested by rapid filtration (Pall Supor 800 Filter, 0.8 mm), the filter immediately immersed in 1 ml of PGTX solution53 and frozen in liquid nitrogen. Total RNA was extracted and analysed by gel electrophoresis and Northern blotting as described54. Discussion The latter mechanism was suggested to act as a posttranscriptional mechanism to adjust mRNA levels in a genome-wide fashion48. Thus, the transcription of non-coding RNA in bacteria is pervasive6–13, but not meaningless, because there are many examples that demonstrate that non-coding RNA in bacteria fulfils specific regulatory functions targeting indi- vidual genes15,16, or serves more global functions48. These facts resemble findings for the composition of the eukaryotic transcriptome and the prevalence and functionality of non-coding transcripts49,50. In eukaryotes, comparative analyses suggested the evolution of the non- coding share of the transcriptome at a more rapid pace than the protein-coding fraction and there are spectacular examples on how this has impacted organismic complexity, cellular differentiation and the capability for physiological adaptation51. Read mapping, data normalization and differential expression analysis. A total of 216,592,987 reads were obtained for Synechocystis 6714 from the ten different conditions and the untreated library. The data were deposited in the NCBI Short Read Archive under accession SRP032230. A total of 204,428,620 reads (29,555,678 non- ribosomal reads) were mapped to the genome with segemehl55 using the default parameters. Details about the mapping statistics of the individual libraries are provided in Table S10. The data were normalized in two steps. First, the treated libraries were scaled to library sizes of 100 million reads, and positions with $500 read starts and with a ratio .0.5 between read starts and coverage were defined as true primary positions. Second, library-specific correction factors based on the fraction of counts at true primary positions were applied. The counts for the untreated library were scaled to counts per 100 million. The fold change (FC) was computed as the ratio of the normalised reads. The FC between the condition with maximum number of reads and the condition with the second highest number of reads is defined as the unique expression factor (UEF)19. For statistical support of UEF values, a pairwise analysis of TSS raw counts from all combinations of conditions was performed with Analysis of Sequence Counts (ASC)56. A UEF was considered significant if jFCj $ 2 and p(jFCj $ 2) $ 0.95. Prediction of transcriptional units. Transcriptional units were detected by RNAseg27 as described19. Discussion The change in expression of the mRNA part of an actuaton may result only from the sRNA promoter replacing the original one. In more complex scenarios, it may also result from events causing differential termination, for instance from the attenuation of tran- scription, as can happen with some riboswitches. Actuatons may also be predecessors or derivatives of riboswitches in which the sRNA function is modified to serve as the metabolite-sensing entity regu- lating the expression of the protein-coding part. One particular case of an actuaton is represented by ncr1265 (Table 2). This ncRNA exists in 11 copies in strain 6803, whereas there is only a single free-standing TU of this type in Synechocystis 6714, on plasmid pSYLA. Ncr1265 originates in all cases from an ISY523-type trans- posase gene on the reverse strand close to the start codon, covering it and the ribosome binding site as an asRNA. With these features, Ncr1265 appears as a silencing RNA for the transposase, analogous to the RNA-OUT transcripts in enteric bacteria46. In the arrange- ment with lpxB in strain 6803, the original promoter was disrupted SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 10 www.nature.com/scientificreports by the insertion of the transposase-ncr1265 cassette and there is no other TSS associated with lpxB, hence the mRNA is generated by read-through from ncr1265. resulting cDNA samples were double-stranded with a length of ,150–700 bp. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For the untreated library, we pooled total RNA from samples representing all 10 growth conditions, depleted rRNA using the MICROBExpress kit (Ambion), fragmented the RNA and performed a treatment with T4 polynucleotide kinase. Otherwise, the libraries were processed as described for the treated libraries, except for the TEX treatment. g These findings are in line with the increasing understanding that functional RNA elements possessa certain plasticity14. Other examples include riboswitches in L. monocytogenes, that also act as trans-acting sRNAs with a regulatory function47 or those that control the 59UTR of one gene by transcriptional termination in one condition as well as terminating the upstream gene in another condition12. In addition to regulation, non-coding RNAs in bacteria fulfill more global functions. A process, discovered in Staphylococcus aureus, and possibly evolutio- narily conserved in Gram-positive bacteria uses pervasive antisense transcriptiontoprocess sensetranscripts for upto75% ofall annotated genes by creating double-stranded substrates13. Discussion We termed a transcriptional unit gTU when one or more annotated genes were covered, aTU when it was antisense to annotated genes or TUs (overlap $20 nt), iTU when the TU was located within an annotated gene and nTU when it was freestanding. In ambiguous cases we provided all possible classifications. Thus, a TU starting at an iTSS, covering an annotated gene and extending into an antisense region was termed gaiTU. Based on the double-comparative analysis of the primary tran- scriptomes of two closely related cyanobacteria, we demonstrated not only commonalities but also large and distinct differences in their repertoires of non-coding RNAs and in the regulation of gene expression among them. Several of these differences are of known functional relevance. We conclude that rapid fluctuations in the composition of the non-coding share of the bacterial transcriptome play an underestimated role in bacterial evolution and that pervasive transcription serves specific purposes rather than constituting an accidental activity. Leaderless transcript detection. gTSSs that were mapped to start codons, either the A of AUG or the preceding 10 nucleotides, were subjected to a closer inspection. When N-terminally shorter homologs were found in other bacteria, the transcripts were re- classified as non-leaderless and the corresponding gene models corrected and submitted to Genbank (CP007542.1 - CP007545.1). Ortholog TSS prediction. Orthologs of protein-coding genes in Synechocystis 6803 were defined previously26. Evolutionarily conserved gTSSs needed to be present in both orthologs. For aTSSs and iTSSs, we required the locations within the orthologous genes to differ by at most 10 nt. For nTSSs, we could not make use of prior ortholog information, thus, we used BLASTN with an e-value cut-off of 1e-5 and query coverage of at least 50% to infer evolutionary conservation. Methods Bi l i l For each condition, total RNA from two independent cultures was pooled. For sequence analysis, cDNA libraries were constructed (vertis Biotechnologie AG, Germany) and sequenced on an Illumina HiSeq 2000 machine as previously described9. The dRNA-seq protocol7 distinguishes treated and untreated libraries. In both cases, total RNA was fragmented with ultrasound (four pulses of 30 s at 4uC). For the treated libraries, RNA with a 59 monophosphate was degraded using TerminatorTM 59 phosphate-dependent exonuclease (TEX, Epicentre). The remaining RNA (mainly primary transcripts with 59-PPP) was poly(A)-tailed using poly(A) polymerase. Then, 59-PPP RNA was dephosphorylated using tobacco acid pyrophosphatase (TAP) and ligated to an RNA linker with reverse complementarity to the linker-specific primer Lsp1 (Table S9). First-strand cDNA synthesis was initiated using M-MLV reverse transcriptase and oligo(dT)-adapter primer OdT1 containing a library-specific barcode sequence (Table S9). The cDNA fragments were amplified by 11–12 PCR cycles using linker-specific primer Lsp1 (Table S9). The 4. Magistri, M., Faghihi, M. A., St Laurent III, G. & Wahlestedt, C. Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts. Trends Genet. 28, 389–396 (2012). 5. Licatalosi, D. D. & Darnell, R. B. RNA processing and its regulation: global insights into biological networks. Nat. Rev. Genet. 11, 75–87 (2010). 6. Ja¨ger, D. et al. Deep sequencing analysis of the Methanosarcina mazei Go¨1 transcriptome in response to nitrogen availability. Proc. Natl. Acad. Sci. 106, 21878–21882 (2009). 7. 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Bacteriol. 191, 1330–1342 (2009). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. Additional information The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ydrogenase in Neisseria meningitidis. J. Bacteriol. 191, 1330–1342 y g g 38. Wilderman, P. J. et al. Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc. Natl. Acad. Sci. 101, 9792–9797 (2004). 39. Gierga, G., Voß, B. & Hess, W. R. The Yfr2 ncRNA family, a group of abundant RNA molecules widely conserved in cyanobacteria. RNA Biol. 6, 222–227 (2009). SCIENTIFIC REPORTS \| 5 : 9560 \| DOI: 10.1038/srep09560 12
https://openalex.org/W4282834074	http://nrl.northumbria.ac.uk/id/eprint/49276/10/btac391.pdf	English	null	SPEAR: Systematic ProtEin AnnotatoR	Bioinformatics	2,022	cc-by	2,921	Published by: Oxford University Press Published by: Oxford University Press URL: https://doi.org/10.1093/bioinformatics/btac391 <https://doi.org/10.1093/bioinformatics/btac391> This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/49276/ URL: https://doi.org/10.1093/bioinformatics/btac391 <https://doi.org/10.1093/bioinformatics/btac391> This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/49276/ This version was downloaded from Northumbria Research http://nrl.northumbria.ac.uk/id/eprint/49276/ Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. 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To read and/or cite from the published version of the research, please visit the publisher’s website (a subscription may be required.) Northumbria Research Link Citation: Crown, Matthew, Teruel, Natalia, Najmanovich, Rafael and Bashton, Matthew (2022) SPEAR: Systematic ProtEin AnnotatoR. Bioinformatics, 38 (15). pp. 3827-3829. ISSN 1367-4803 2.2 Variant detection SPEAR aligns consensus input files to SARS-CoV-2 reference gen- ome NC_045512.2 using MUSCLE (Edgar, 2004). Single nucleotide polymorphisms (SNPs) are obtained from the alignment. SPEAR detects indels and multi-nucleotide polymorphisms (MNPs) in the alignment and combines linked events (e.g. SNP followed by dele- tion) into a single VCF row for accurate amino acid (AA) conse- quence description. To this end, we present Systematic ProtEin AnnotatoR (SPEAR), a tool to flag potential VoCs, highlighting samples that show poten- tially elevated immune escape and enhanced infectivity at point of sequencing. SPEAR is a lightweight functional genomic surveillance discovery tool, that utilizes information from protein structure, deep mutational scanning (DMS) and computational molecular biophys- ics to provide comprehensive full protein product annotation for SARS-CoV-2. To whom correspondence should be addressed. Associate Editor: Can Alkan Associate Editor: Can Alkan Received on February 23, 2022; revised on May 23, 2022; editorial decision on June 6, 2022; accepted on June 9, 2022 2.1 Input 2.1 Input SPEAR is written in Python (version 3.10) and utilizes Snakemake (Mo¨lder et al., 2021) for workflow management and parallel job execution. SPEAR is flexible, allowing for single and multi-sample input in the form of either: consensus FASTA sequence, FASTA mul- tiple sequence alignment (MSA) or VCF file(s). Abstract Summary: We present Systematic ProtEin AnnotatoR (SPEAR), a lightweight and rapid SARS-CoV-2 variant annota- tion and scoring tool, for identifying mutations contributing to potential immune escape and transmissibility (ACE2 binding) at point of sequencing. SPEAR can be used in the ﬁeld to evaluate genomic surveillance results in real time and features a powerful interactive data visualization report. Availability and implementation: SPEAR and documentation are freely available on GitHub: https://github.com/m- crown/SPEAR and are implemented in Python and installable via Conda environment. Supplementary information: Supplementary data are available at Bioinformatics online. Bioinformatics, 2022, 1–3 https://doi.org/10.1093/bioinformatics/btac391 Advance Access Publication Date: 13 June 2022 Applications Note Bioinformatics, 2022, 1–3 https://doi.org/10.1093/bioinformatics/btac391 Advance Access Publication Date: 13 June 2022 Applications Note Genome analysis y y 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 unre- stricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Matthew Crown 1, Nata´lia Teruel 2, Rafael Najmanovich 2 and Matthew Bashton 1, 1Hub for Biotechnology in the Built Environment, Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK and 2Department of Pharmacology and Physiology, Universite´ de Montre´al, Montreal, QC H3T 1J4, Canada *To whom correspondence should be addressed. Associate Editor: Can Alkan 1 Introduction The SARS-CoV-2 virus caused a global pandemic with >5.8 mil- lion deaths and more than 412 million infections worldwide at time of writing. During this period there have been several variants of concern (VoCs), with enhanced transmissibility and/or immune escape (Chen et al., 2021; Davis et al., 2021; Elliott et al., 2021; Graham et al., 2021; Kraemer et al., 2021; O’Toole et al., 2021; Twohig et al., 2022; Wang et al., 2021). Currently, these VoCs are defined by health authorities, World Health Organization and/or by lineages, such as PANGO Lineages (Rambaut et al., 2020) or Nextstrain (Hadfield et al., 2018). These designations are reactive, based on a set of novel mutations must be observed as a distinct clade before being first assigned a lineage, and only then can it be labelled as such in sequencing output and the spread of the variant tracked. There is a clear and pressing need to be able to identify evolutionary ingress of potentially problematic variants as they emerge directly from the sequencing data. Especially as we now approach the endemic stage of the pandemic where transmission levels are high, and surveillance and mitigations may no longer be in place. SPEAR integrates existing tools with its own internal annotation and QC processes, to ensure that annotation is informative, particu- larly when dealing with low-quality sequences. A more detailed overview of the implementation is available in Supplementary Data S1 and Figure S1. 2.5 Summary output poor sequence quality and may bias downstream score estimates. SNPs can also be optionally filtered to remove commonly problem- atic positions. SPEAR will provide a per run score summary which sums immune escape and ACE2 interaction scores for each sample. This is the sum of scores for all variants within the sample. A terminal output table of these scores is produced using the Rich python package. SPEAR also provides a Spike protein dropout detection system with user configurable parameters to flag gaps in Spike coverage (>150 bp) due to amplicon dropout, as well as flagging high levels of global N content (>25%), and Ns in the Spike receptor binding domain (RBD) (>12 nt). Dropout detection is critical as missing mutations in Spike cannot be scored and need to be drawn to the user’s attention. SPEAR produces a HTML report with interactive heatmaps of scores for all samples and metrics as well as sortable tables of muta- tions and associated scores, built using Plotly and Bootstrap. This re- port is distributed with all dependencies required for offline viewing. Per sample ORF plots can also be viewed that enable the mutations to be shown against the protein product they reside in along with associated scores. Supplementary Data S3 contain ex- ample HTML reports and ORF plots for the Alpha, Delta, Omicron, BA.1, BA.1.1 and BA.2 lineages. S: p. G142_Y145delinsD to G142D, V143del, Y144del, Y145del SPEAR utilizes the AA and gene annotations from SnpEff for its downstream functional and structural annotations. The full SnpEff annotation is retained in the final VCF file produced for each sam- ple. SPEAR examines the AA variants in the Spike and evaluates these to show potential increases in immune escape relative to that of the original ‘wild-type’ for the different Barns classes (Barnes et al., 2020) of antibody binding epitopes using DMS data (Dong et al., 2021; Greaney et al., 2021a, b; Starr et al., 2021a, b, c; Tortorici et al., 2021). ACE2 binding is assessed using DMS scores available for the Spike RBD (Starr et al., 2020) which show the like- ly impact of each mutation, and Vibrational Difference Scores (VDS) (Teruel et al., 2021) which shows the propensity for the open conformational state (that correlates with infectivity). Scoring oper- ates over the RBD (AA: 331–531) for all scores except VDS, which covers a larger region of Spike (AA: 14–913). Descriptions of the scoring system can be found in Supplementary Data S1. 2.3 Quality control Compared to Alpha and Delta, Omicron and it’s sublineages BA.1, BA.1.1 and BA.2 have signiﬁcantly higher mutational load in Spike RBD contributing to modiﬁed ACE2 binding and immune escape scores. The mutation driving increased class 2 escape in BA.1.1 (S: R346K) can be seen to be unique to this lineage. BA.2 can also be seen to be missing S: G446S mutation, leading to an observed reduced class 2 (and class 3) mAb escape score compared to BA.1/BA.1.1. (c) Heatmap of class 3 mAb escape. S: R346K stands out as being unique to BA.1.1 and drives an increase in escape in this lineage. (d) Heatmap of class 4 mAb escape. The mutation S: R408S drives an increase in class 4 mAb escape in BA.2 2.4 Spear annotation SnpEff (Cingolani et al., 2012) is leveraged to annotate basic vari- ant consequences. Compound HGVS.p format variants are then expanded by SPEAR to individual AA variants, e.g.: 2.3 Quality control SPEAR integrates several quality control (QC) checks for input sam- ples. All consensus and alignment inputs are checked for unknown base (N) content in the genome (default 50%), which is a sign of V C The Author(s) 2022. Published by Oxford University Press. V C The Author(s) 2022. Published by Oxford University Press. 1 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 unre- stricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ( ) y y 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 unre- stricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licens stricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. M.Crown et al. 2 2 Fig. 1. Scoring comparison of mutations in lineages of current and past global interest, taken from the SPEAR summary report. (a) Summary score table baselined to compare against BA.1. (b) Heatmap of residue mutation scores for class 2 mAb escape in each lineage. Compared to Alpha and Delta, Omicron and it’s sublineages BA.1, BA.1.1 and BA.2 have signiﬁcantly higher mutational load in Spike RBD contributing to modiﬁed ACE2 binding and immune escape scores. The mutation driving increased class 2 escape in BA.1.1 (S: R346K) can be seen to be unique to this lineage. BA.2 can also be seen to be missing S: G446S mutation, leading to an observed reduced class 2 (and class 3) mAb escape score compared to BA.1/BA.1.1. (c) Heatmap of class 3 mAb escape. S: R346K stands out as being unique to BA.1.1 and drives an increase in escape in this lineage. (d) Heatmap of class 4 mAb escape. The mutation S: R408S drives an increase in class 4 mAb escape in BA.2 Downloaded from https://academic.oup.com/bioinformatics/advance-article/doi/10.10 Fig. 1. Scoring comparison of mutations in lineages of current and past global interest, taken from the SPEAR summary report. (a) Summary score table baselined to compare against BA.1. (b) Heatmap of residue mutation scores for class 2 mAb escape in each lineage. 2.6 Baseline comparison d b d h SPEAR is distributed with a set of baseline scores for lineages and VoCs which can be selected to compare samples against. The default is to compare to BA.1 (Alpha, Delta, Omicron, BA.1, BA.1.1 and BA.2 can also be selected) or a user provided baseline can be used. Where scores exceed the baseline, these are highlighted in both the HTML and terminal sample summary tables. This enables samples scoring higher for any one metric than the current predominant lin- eage to be highlighted. S: p. G142_Y145delinsD to G142D, V143del, Y144del, Y145del 3 Application SPEAR can be used to identify new sets of mutations within samples of interest from genomic surveillance. The example report baselined to Delta (Supplementary Data S3) highlights this for historical and current lineages/VoCs. Both the heatmaps and scores summary table show that Omicron and its BA sublineages score higher for immune escape than both Alpha and Delta, immediately drawing attention to the emergence of new threats in a surveillance setting (Fig. 1a). Using a report baselined to BA.1 (predominant at time of writing in UK) (Supplementary Data S3), it is apparent that BA.1.1 (a recently designated sublineage) has increased immune escape specifically for epitope classes 2 and 3 (Fig. 1b and c), driven by S: R346K. BA.2 (a Variant Under Investigation) shows increased escape in classes 1 (driven by S: D405N) and 4 (driven by S: R408S, Fig. 1d), but reduced escape in classes 2 and 3 owing to the lack of S: G446S (Fig. 1b and c). SPEAR also annotates all structural, non-structural and acces- sory proteins for which structural information is present, including protein–protein interaction interfaces (such as replicase complex subunits and oligomerization), ligand binding and active sites resi- dues as well as domain boundaries, using a manually curated set of annotations derived from protein structures. Example per sample output for BA.1 is found in Supplementary Data S2. SPEAR also annotates all structural, non-structural and acces- sory proteins for which structural information is present, including protein–protein interaction interfaces (such as replicase complex subunits and oligomerization), ligand binding and active sites resi- dues as well as domain boundaries, using a manually curated set of annotations derived from protein structures. Example per sample output for BA.1 is found in Supplementary Data S2. SPEAR: Systematic ProtEin AnnotatoR 3 Funding Kraemer,M.U.G. et al. (2021) Spatiotemporal invasion dynamics of SARS-CoV-2 lineage B.1.1.7 emergence. Science, 373, 889–895. This work was supported by COG-UK; the Research England’s Expanding Excellence in England (E3) Fund to M.B. and M.C.; and the UK Health Security Agency to M.B. R.J.N. is a member of the Re´seau Que´be´cois de Recherche sur les Me´dicaments (RQRM) and the Quebec Network for Research on Protein Function, Engineering and Applications (PROTEO). Mo¨lder,F. et al. (2021) Sustainable data analysis with snakemake. F1000Res., 10, 33. ´ O’Toole,A´ . et al. (2021) Tracking the international spread of SARS-CoV-2 lin- eages B.1.1.7 and B.1.351/501Y-V2. Wellcome Open Res., 6, 121. g p Rambaut,A. et al. (2020) A dynamic nomenclature proposal for SARS-CoV-2 p Rambaut,A. et al. (2020) A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat. Microbiol., 5, 1403–1407. Conflict of Interest: none declared. Rambaut,A. et al. (2020) A dynamic nomenclature proposal for SARS CoV 2 lineages to assist genomic epidemiology. Nat. Microbiol., 5, 1403–1407. Starr,T.N. et al. (2020) Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding. Cell, 182, 1295–1310.e20. 4 Conclusion Graham,M.S. et al. (2021) Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an eco- logical study. Lancet Pub. Heal., 6, e335–e345. SPEAR provides rapid assessment of mutations in SARS-CoV-2 samples and can be run without reliance on external web servers. Together with its lightweight implementation, this allows for de- ployment both in the field and in pathogen surveillance labs worldwide. Greaney,A.J. et al. (2021a) Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe, 29, 463–476.e6. p , , Greaney,A.J. et al. (2021b) Mapping mutations to the SARS-CoV-2 RBD that bi di b diff l f ib di N C 12 4196 Greaney,A.J. et al. (2021b) Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies. Nat. Commun., 12, 4196. Greaney,A.J. et al. (2021b) Mapping mutations to the SARS-CoV- Hadﬁeld,J. et al. (2018) Nextstrain: real-time tracking of pathogen evolution. Bioinformatics, 34, 4121–4123. Downloaded from https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btac391/6607585 by guest on 27 June 2022 Conflict of Interest: none declared. References Starr,T.N. et al. (2021a) Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016. Cell Rep. Med., 2, 100255. Barnes,C.O. et al. (2020) SARS-CoV-2 neutralizing antibody structures in- form therapeutic strategies. Nature, 588, 682–687. Chen,R.E. et al. (2021) Resistance of SARS-CoV-2 variants to neutralization b l l d d d l l l b d d Chen,R.E. et al. (2021) Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies. Nat. Med., 27, 717–726. Starr,T.N. et al. (2021b) Prospective mapping of viral mutations that escape antibodies used to treat COVID-19. Science, 371, 850–854. Starr,T.N. et al. (2021c) SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature, 597, 97–102. Cingolani,P. et al. (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin), 6, 80–92. Teruel,N. et al. (2021) Modelling conformational state dynamics and its role on infection for SARS-CoV-2 spike protein variants. PLoS Comput. Biol., 17, e1009286. Davis,C. et al. (2021) Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination. PLoS Pathog., 17, e1010022. Tortorici,M.A. et al. (2021) Broad sarbecovirus neutralization by a human monoclonal antibody. Nature, 597, 103–108. Dong,J. et al. (2021) Genetic and structural basis for SARS-CoV-2 variant neu- tralization by a two-antibody cocktail. Nat. Microbiol., 6, 1233–1244. Twohig,K.A. et al. (2022) Hospital admission and emergency care attendance risk for SARS-CoV-2 Delta (B.1.617.2) compared with alpha (B.1.1.7) var- iants of concern: a cohort study. Lancet Infect. Dis., 22, 35–42. Edgar,R.C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 5, 113. iants of concern: a cohort study. Lancet Infect. Dis., 22, 35–42 Elliott,P. et al. (2021) Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant. Science, 374, eabl9551. Wang,P. et al. (2021) Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature, 593, 130–135.
https://openalex.org/W2267234772	https://www.scienceopen.com/document_file/e09c10e2-9be4-4d0a-9ee8-f631dba456a5/ScienceOpen/137_Gardiner.pdf	English	null	THE DIGITAL ATELIER: HOW SUBTRACTIVE TECHNOLOGIES CREATE NEW FORMS	Electronic workshops in computing	2,010	cc-by	3,942	CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner THE DIGITAL ATELIER: HOW SUBTRACTIVE TECHNOLOGIES CREATE NEW FORMS Professor Jeremy Gardiner Senior Research Fellow, Department of History of Art and Screen Media, Birkbeck, University of London, 43, Gordon Square, Bloomsbury, London WC1H OPD UK mail@jeremygardiner.co.uk www.technocultures.org Professor Jeremy Gardiner Senior Research Fellow, Department of History of Art and Screen Media, Birkbeck, University of London, 43, Gordon Square, Bloomsbury, London WC1H OPD UK mail@jeremygardiner.co.uk www.technocultures.org Professor Jeremy Gardiner Senior Research Fellow, Department of History of Art and Screen Media, Birkbeck, University of London, 43, Gordon Square, The Digital Atelier: For 50 years artists have been utilising the convergence and combination of different technologies to produce visually and intellectually challenging artworks. These artists create compelling artefacts that engage the pragmatics of technology and the free invention of art and bring them to a successful synthesis. A close examination of work from the past and present reveals how advanced digital design methods and subtractive fabrication processes have been used to make physical things from virtual data. INTRODUCTION The idea of a Digital Atelier comes from the French term for studio. Since the 1960s [1] the conceptual path of a small group of painters, printmakers and sculptors was considerably altered and redefined by the use of the computer. These artists began forging new forms utilising digital design systems and fabrication processes and have produced work that uses both subtractive and additive technologies. Their research encompasses the scientific exploration of materials, the development and use of new technologies, the cross-fertilisation of old and new technologies and the creation of new forms. TECHNIQUES AND TECHNOLOGIES The term solid freeform fabrication is applied to a range of techniques for manufacturing solid 3D objects directly from Computer Aided Design (CAD) data. To make sense of the many techniques and technologies used today we can divide them into either subtractive or additive processes. Subtractive fabrication is the term given to any fabrication process where material is taken away or reduced from a solid in order to reveal a new shape. This subtraction can take place using any combination of tooling techniques such as drills, lathes, and grinders, and more recently lasers and high-pressure water jets. With additive fabrication, the machine reads data from a CAD drawing and lays down successive layers of liquid or powder and in this way builds up the model from a series of cross sections. This paper will focus on artists using subtractive technologies. A second paper examining additive technologies will be delivered as part of Digital Continuities: From the History of Digital Art to Contemporary Transmedial Practices at the Association of Art Historians conference from 15th – 17th April 2010 at the University of Glasgow. 137 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner The following examples echo the forms of the past whilst utilising the latest technologies. Using the concept of ‘Ideas before their time – Connecting the past and present in Computer Art’ I am going to take a look at the work of two pioneering artists working with subtractive technologies: Robert Mallary and Richard Hamilton and how they have influenced the work of two specific contemporary artists; Bengtsson and Delvoye in the case of Mallary, and Grossman and Shafiei in the case of Hamilton. MALLARY, BENGTSSON AND DELVOYE Bengtsson and Delvoye laser cut a variety of sheet materials from plywood to corten steel to create their volumetric sculptural artworks. In 1968 the artist Robert Mallary began to experiment with computer sculpture, he manufactured Quad II and Quad III. To create these sculptures he developed his ideas on sequential contour projection and used them in the creation of sculptural computer forms [2]. The computer program he developed with his colleagues was called TRAN2, described in the following extract: “TRAN2 is a computer graphics program with twenty sub-routines to generate sculpture. The program presupposes a means of compiling form description data for use by the computer. This is done by breaking down the solid into a regular series of parallel cross sections, or contour “slices,” which are then graphed and digitized as X, Y and Z coordinates and transferred to punch cards. A sequence of mathematical transformation procedures is brought to bear on the contour sections whereby the computer, in effect, models and reshapes the contour sections into an original sculpture. The computer plotter reproduces a series of perspective views of the generated form together with a complete set of the transformed contour sections. These are used as patterns to complete the sculpture in some appropriate material.” Mallary considered the computer as an intelligence and information amplification device which could be linked synergistically with the unique, creative capacities of the human mind for creative activity, surpassing either human or machine capabilities functioning independently. 138 138 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner Figure 1. Quad II. 1968. Material: Plywood. Robert Mallary Figure 1. Quad II. 1968. Material: Plywood. Robert Mallary The information for the contour slices in Fig. 2 was transferred to computer punch cards as described before. The plotter produced a series of perspective views which Mallary called ‘Computer transformation templates’. These 2D slices were used as patterns for cutting the final sculpture from laminated wood veneer. Figure 2. Computer transformation templates. 1968. Robert Mallary. Figure 2. Computer transformation templates. 1968. Robert Mallary. Mallary began using rapid prototyping for his sculptures in the 1960s and since that time increasing numbers of visual artists have used rapid prototyping, as prices of hardware and software have dropped and performance, user interface and output technology have improved dramatically. The following contemporary artists employ lasers to cut the component parts of their work using paper, wood steel and plastics. A laser is an amplifier of light, focusing it into an intense beam that can burn, melt and evaporate the material it encounters and because the cutting tool is a beam of light it can move very quickly and makes cuts as fine as the focus of the beam. Mathius Bengtsson uses materials that are both natural and manufactured, each material seems to be carefully chosen to highlight the sinuous quality of the final design. ‘The Slice chair was constructed with the same adeptness an architect would employ to create a topological map of the landscape, evoking the illusion of a piece of furniture cut away from a cliff face and scaled to human proportions.’ Bradley Quinn, in Scandinavian Style [3]. First drawn by hand and later modelled in clay, the Slice chair combines organic shapes with cutting-edge technology. Slice is constructed as an assemblage of horizontal cross-sections that stack together into a uniquely lateral 139 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner profile. Laser-cut to a thickness of only 3mm, each individual layer resembles a two- dimensional abstraction more than it does a hi-tech element. Although the process was inspired by rapid prototyping methods, Bengtsson worked with more traditional materials. His starting point was to create a new form by using clay, which he then sliced in horizontal layers and manipulated digitally. The result was a surprising shape that blurred the distinctions between armrests, backrest, legs and frame. Figure 3. Slice Chair. Material: Plywood. 1999. Mathius Bengtsson Figure 3. Slice Chair. Material: Plywood. 1999. Figure 1. Quad II. 1968. Material: Plywood. Robert Mallary Mathius Bengtsson ‘It has such a strong aerodynamic feel that it could be an aircraft or a Formula One racing car.’ Corinne Julius, in the Evening Standard ‘It has such a strong aerodynamic feel that it could be an aircraft or a Formula One racing car.’ Corinne Julius, in the Evening Standard Conceived as a single, sweeping curve, the ‘Slice chaise longe’ comprises a contouring backrest that arcs forward to support the legs and feet. Crafted in ninety nine individual layers, regular spaces between each allows the eye to travel far beyond the chaise’s structure. As with the aluminium Slice chair, Bengtsson used transparency as a device to deconstruct the conventional parts of a chaise longe. Bengtsson's technique makes every aspect of the chaise visible in a single glance, and breaks down the density of its complex surface area. The ‘Slice chaise longe’ echoes the form of Mallary’s ‘Quad I’ and ‘Quad II’ but in the horizontal plane. 140 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner Figure 4. Slice Chaise. Material: Acrylic. 2000. Mathius Bengtsson Figure 4. Slice Chaise. Material: Acrylic. 2000. Mathius Bengtsson Wim Delvoye takes laser-cutting in steel to a new scale with his highly detailed sculptures in ornate patterns referencing the industrial revolution and gothic and Victorian architecture. 141 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner Figure 5. Wim Delvoye. Peggy Guggenheim Museum 2009. Figure 5. Wim Delvoye. Peggy Guggenheim Museum 2009. Wim Delvoye’s artistic practice draws on the notion of the attraction of binary opposites: the past and the present, the triumph of ornamentation over functionality [4]. The Peggy Guggenheim collection presented Wim Delvoye’s latest creation, 'Torre': a corten steel tower, with ogival windows, tracery and turrets in the international gothic style, on the terrace of palazzo Venier dei Leoni, overlooking the grand canal in Venice in 2009. Wim Delvoye’s artistic practice draws on the notion of the attraction of binary opposites: the past and the present, the triumph of ornamentation over functionality [4]. The Peggy Guggenheim collection presented Wim Delvoye’s latest creation, 'Torre': a corten steel tower, with ogival windows, tracery and turrets in the international gothic style, on the terrace of palazzo Venier dei Leoni, overlooking the grand canal in Venice in 2009. 142 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner _____________________________________________________________________ Figure 6. Torre. Corten steel. 2009. Wim Delvoye. Peggy Guggenheim Museum. Figure 6. Torre. Corten steel. 2009. Wim Delvoye. Peggy Guggenheim Museum. “I tried to integrate the tower with the building, but the architecture of the building is Neo-Classical. My project is another style completely. The way I designed it is also very unorthodox—from the top down rather than from the base to the peak. ‘It has such a strong aerodynamic feel that it could be an aircraft or a Formula One racing car.’ Corinne Julius, in the Evening Standard I want every detail to be perfect. My design team has incorporated the Gothic style and Gothic Revival into the tower. These are architects who have worked with me for years and totally understand what I want. I’ve constructed other towers, but they were mere exercises in relation to this one. Last year I exhibited a couple of 6-meter-tall [20-foot] maquettes in Moscow and Basel, but the tower for the Peggy Guggenheim Collection is a scale model for a tower that I hope someday to build. It’s one-to-four, a quarter of the size, which is still huge. I’ve designed towers 80 meters [262 feet] high. One is 325 meters [1,066 feet] high to match the Eiffel Tower. They remain maquettes, but with the Venice tower I’m very 143 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner motivated to push the design into reality because I’m satisfied with what we’ve done. It starts where Gothic stopped. We somehow ate it all, we assimilated it, and now we’ve done a Gothic style that has never been done. It’s like we’ve continued a long tradition, but we are not just copying other people—we’re inventing. The tower is Cor-Ten steel, which is laser-cut, folded and welded. It’s layered and very sculptural. The surface will be rusted, but we’ll varnish it to keep it from bleeding into the stone of the historical building. By making it in this steel we can do things that the Gothic builders couldn’t do. Some of those things are more beautiful in steel than in stone. The designs are computer-generated. Since 2000 I’ve created 3-D images so I can see how my works might look in real life. We have several different programs and we play with their limitations. I’m always reminding my staff about the great cathedrals of Strasbourg, Cologne, Canterbury and Paris, which weren’t built with computers, cameras and helicopters”. Wim Delvoye HAMILTON, GROSSMAN AND SHAFIEI Richard Hamilton’s interest in technology began with his reading Giedion's 'Mechanization takes Command'. The impact of technology was also the theme of Hamilton's exhibition 'Man, Machine and Motion', at the Hatton Gallery of Newcastle University and the ICA in 1955. From 1951 to 1963 Hamilton made perspective drawings leading to the 1964 print,’Five Tyres Abandoned’ In an issue of a magazine called Technique et Architecture published around 1951, there was an illustration of five tyres in a row. On the centre of each tread was an oval panel labelling it with a date – 1902, 1905 and so on to 1950. “In 1963, I began to make a perspective drawing of the subject. I proposed to make a print; an embossed relief, printed blind, so that the effect would be of the varied treads of the five tyres pressing up from the back of the paper – but in perspective. After working for a good many weeks it became clear that to continue in the rigorously accurate manner that alone made the task worthwhile would require such an abundance of time that I would have to consider whether the result could possibly merit such devotion. It was then I regretfully decided not to complete the drawing, and ‘Five tyres abandoned’ became the title of the 1964 print.” Richard Hamilton. The project had been revived in 1970 when an American art dealer, Carl Solway, publisher of EYE Editions, offered to find a US computer programmer interested in plotting the perspective drawing with a computer. Sherill F. Martin, manager of computer animation at Kaye Instruments, organised the computer formulation of the perspective drawing using a general FORTRAN programme called CAPER (Computer Aided Perspective). “The uses to which the computer has been put by artists most often develop out of properties peculiar to that device which enable it to use a set of instructions to effect transformations of a given image, or develop sequences of kinetic patterns. There is a tendency to ask it to perform what it most likes doing, or at least what it does most fluently, so we have to come to recognise a computer graphics style. HAMILTON, GROSSMAN AND SHAFIEI [5] The use of a 144 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner computer to make a conventional perspective projection puts no claim on its capabilities as an image creator – that is to say, the stylish qualities are not prompted by the tool. This kind of problem might have been posed by anyone since Piero della Francesca and its solution can be precisely forseen. What the computer provides is an inhuman speed which makes possible the formulation of a complex perspective image in its purest terms.” Richard Hamilton Figure 7. Five Tyres. 1972. Printed by Frank Kicherer, Stuttgart. The relief cast was made by Hartmut Freilinghaus of Hamburg. Richard Hamilton. V&A print room ‘3D for Print’ symposium March 2009. Figure 7. Five Tyres. 1972. Printed by Frank Kicherer, Stuttgart. The relief cast was made by Hartmut Freilinghaus of Hamburg. Richard Hamilton. V&A print room ‘3D for Print’ symposium March 2009. “Major computer programmes usually have wider applications than that of giving an answer to the specific problem in hand. CAPER (computer aided perspective), itself an extension of CALD (computer aided line drawing), by S E Anderson of Syracuse University, May 1967, is a general programme, written in FORTRAN. This offers the essential notions as to how instructions to the plotter may be stated, together with card – coded commands which provide the potential for the generation of any line- drawing. Sherill Martin received the data and proceeded to inform CAPER so that it would tell a plotter how to draw the required perspective. A series of encoded messages was then converted into a deck of punched cards. An IBM 36075 computer read the deck and generated signals on a magnetic tape to control the movements of a pen on a drafting machine. In this case a Calcomp 763 was used, which co-ordinates rotary movements of the paper with lateral movements of the pen along the axis of the cylinder to produce any figure. 145 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner At this stage the original idea of producing an embossed print on paper was modified to a proposal to cast the relief to the treads integrally with a sheet of cold-curing rubber. HAMILTON, GROSSMAN AND SHAFIEI I filled in the linear drawing by hand with the intention of etching a metal plate to serve as a mould. Etching proved unsatisfactory, so the mould was mechanically engraved in a brass plate. Machine cutting permitted a variation of relief. To take advantage of this, a further drawing designated depth of cut in tenths of a millimetre. The ‘print’ is ‘cast’ by spreading on the plate a silicone elastomer (manufactured as a flexible mould material), then reinforcing with a non-woven Terylene cotton fabric”. Richard Hamilton. Today Grossman, a sculptor and Shafiei, an architect use CAD in different ways to realise their three dimensional hybrid forms. The sculptor Bathsheba Grossman uses a laser to cut her CAD drawings into crystal. The points created by a focused laser beam are tiny (.1mm) fractures. The conical beam, with a focal length of about 3", shines into the glass without damaging it except at the focal point. At that one point, concentrated energy heats the glass to the cracking point, causing a microfracture. To draw more points, the laser is pulsed on and off. To make the beam move between points, it is reflected from a mirror that is repositioned between pulses. The mirror is moved by computer-controlled motors, so many points can be drawn with great speed and accuracy. A typical design might use several hundred thousand points, half a million is not unusual in a large block, each placed with .001" accuracy. Figure 8. Insulin. Material: jLaser cut crystal block. 2007. Bathsheba Grossman. The images are produced with different types of laser, and the results vary. Grossman uses a high-frequency laser, which draws points that are barely visible to the naked eye Figure 8. Insulin. Material: jLaser cut crystal block. 2007. Bathsheba Grossman. The images are produced with different types of laser, and the results vary. Grossman uses a high-frequency laser, which draws points that are barely visible to the naked eye 146 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner using a Nd:YAG laser, named after its active medium: a yttrium-aluminum-garnet crystal doped with neodymium. The glass itself must be clear optical crystal, since any ripples or bubbles would block or blur the laser. HAMILTON, GROSSMAN AND SHAFIEI The process requires drawing in layers, moving from the rear to the front of the glass, so that previous points do not block the laser from drawing new ones. The glass surface must be flat, or refraction will blur and redirect the beam; that is why it is difficult to work with spheres or other curved shapes. Refraction is also an issue in viewing curved glass: in a 60mm sphere only the central 20mm can be used, because optical magnification makes that area seem to fill the whole sphere. If anything is drawn closer to the surface than that, it will look very distorted. The architect Sara Shafiei in her project ‘Anamorphic Tectonics Theatre for Magicians’ plays with the art of illusion, bending laser-cut card. The project is based on a site in the National Botanical Gardens in Rome, and proposes a dispersed magical illusion, with its central spectacle being a theatre for magicians. The building sits at the peak of the site overlooking the gardens. The use of text and cone anamorphosis along with other perspectival illusions, aid in the creation of a landscape of the imagination, which surround the theatre. The project attempts to portray how the foundations of magic and illusion can become an inherent part of an architectural design. Figure 9. Anamorphic Tectonics: Theatre for Magicians. Laser cut paper, longitudinal section. 2007. Sara Shafiei. Figure 9. Anamorphic Tectonics: Theatre for Magicians. Laser cut paper, longitudin section. 2007. Sara Shafiei. Sara Shafiei explains: “Anamorphosis is a distorted projection or representation of an image on a plane or curved surface, which, when viewed from the correct vantage point, or as reflected space from a curved mirror or through a polyhedron, appears regular and in proportion. This technique takes the form of signs within my architectural proposal. It 147 CAT 2010 London Conference ~ 3rd February Jeremy Gardiner CAT 2010 London Conference ~ 3rd February Jeremy Gardiner is used in order to allow the visitor to engage with the landscape and architecture and navigate their way through the site. The use of this technique will also allow the visitor to experience a slow progression of a landscape of illusions, from the onset of entering the site.” THE FUTURE In 1985 Lisa Phillips, Associate Curator at the Whitney Museum in New York, made the following statement: “So many artists are exploring the computer that it cannot be ignored”. But it wasn’t until 2001, sixteen years later, that the Whitney Museum acknowledged this aesthetic by organising a major exhibition of Computer Art entitled ‘Bitstreams’. Eight years on in 2009 the exhibitions ‘Decode’ and ‘Digital Pioneers’ were mounted at the V&A museum in London. During the next decade digital craftsmanship will develop and new forms will emerge as solid freeform fabrication becomes more accessible and affordable and artists and designers investigate the many techniques that are available using subtractive and additive processes. This includes developmental research that explores new Rapid Prototyping techniques and processes, e.g. 3D scanning, CNC routing (3 and 5 axis), SLS (Selective Laser Sintering in Nylon), 3D printing and all the new and modified materials that are being developed. The problems confronting these artists and designers will be the same that faced Mallary and Hamilton in the 1960s. How to consider specific items of technology in terms of what they can do and determine whether or not they are useful and whether or not they allow scope for an idea to be developed or communicated. Another challenge will be whether they can make the technology perform in a certain way even if this contravenes the intentions of its inventors. References [1] BROWN, P and LAMBERT, N and MASON, C and GERE, C (eds.), White Heat and Cold Logic: Early British Computer Art: MIT Press, 2008. [2] REICHARDT, J. The computer in art. Studio Vista, 1971. [3] QUINN, B. Scandanavian style. Conran Octopus. 2003. [4] LASTER, P. Wim Delvoye: Towering ambition. Art in America. 2009. [5] HAMILTON, R. Collected words. Thames and Hudson. 1982. [6] GLYNN, R and SHAFIEI, S Digital Architecture, Hinterlands, 2009. [1] BROWN, P and LAMBERT, N and MASON, C and GERE, C (eds.), White Heat and Cold Logic: Early British Computer Art: MIT Press, 2008. [1] BROWN, P and LAMBERT, N and MASON, C and GERE, C (eds.), White Heat and Cold Logic: Early British Computer Art: MIT Press, 2008. [2] REICHARDT, J. The computer in art. Studio Vista, 1971. [3] QUINN, B. Scandanavian style. Conran Octopus. 2003. [4] LASTER, P. Wim Delvoye: Towering ambition. Art in America. 2009. 148
https://openalex.org/W2626028924	https://europepmc.org/articles/pmc5470192?pdf=render	English	null	Underutilisation of routinely collected data in the HIV programme in Zambia: a review of quantitatively analysed peer-reviewed articles	Health research policy and systems	2,017	cc-by	9,250	REVIEW Open Access * Correspondence: munthalitendai@gmail.com 1School of Public Health, University of Zambia, Lusaka, Zambia 2Ministry of Health, Lusaka, Zambia Full list of author information is available at the end of the article Underutilisation of routinely collected data in the HIV programme in Zambia: a review of quantitatively analysed peer-reviewed articles Tendai Munthali1,2* , Patrick Musonda1, Paul Mee5, Sehlulekile Gumede1,3, Ab Schaap3,4, Alwyn Mwinga4, Caroline Phiri2, Nathan Kapata2, Charles Michelo1 and Jim Todd3 © 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. Munthali et al. Health Research Policy and Systems (2017) 15:51 DOI 10.1186/s12961-017-0221-9 Munthali et al. Health Research Policy and Systems (2017) 15:51 DOI 10.1186/s12961-017-0221-9 Open Access Abstract Background: The extent to which routinely collected HIV data from Zambia has been used in peer-reviewed published articles remains unexplored. This paper is an analysis of peer-reviewed articles that utilised routinely collected HIV data from Zambia within six programme areas from 2004 to 2014. Methods: Articles on HIV, published in English, listed in the Directory of open access journals, African Journals Online, Google scholar, and PubMed were reviewed. Only articles from peer-reviewed journals, that utilised routinely collected data and included quantitative data analysis methods were included. Multi-country studies involving Zambia and another country, where the specific results for Zambia were not reported, as well as clinical trials and intervention studies that did not take place under routine care conditions were excluded, although community trials which referred patients to the routine clinics were included. Independent extraction was conducted using a predesigned data collection form. Pooled analysis was not possible due to diversity in topics reviewed. Results: A total of 69 articles were extracted for review. Of these, 7 were excluded. From the 62 articles reviewed, 39 focused on HIV treatment and retention in care, 15 addressed prevention of mother-to-child transmission, 4 assessed social behavioural change, and 4 reported on voluntary counselling and testing. In our search, no articles were found on condom programming or voluntary male medical circumcision. The most common outcome measures reported were CD4+ count, clinical failure or mortality. The population analysed was children in 13 articles, women in 16 articles, and both adult men and women in 33 articles. Conclusion: During the 10 year period of review, only 62 articles were published analysing routinely collected HIV data in Zambia. Serious consideration needs to be made to maximise the utility of routinely collected data, and to benefit from the funds and efforts to collect these data. This could be achieved with government support of operational research and publication of findings based on routinely collected Zambian HIV data. Keywords: Routinely collected data, HIV, Zambia © 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. Background This creates a need to monitor the HIV epidemic by focusing on indicators of effective prevention and on the quality of the HIV ser- vices in Zambia [8]. Six key service areas are prioritised in the country for prevention and treatment of HIV. These are (1) voluntary medical male circumcision (VMMC); (2) condom programming; (3) behavioural change; (4) HIV testing and counselling (HTC); (5) prevention of mother-to-child transmission (PMTCT); and (6) treatment and retention in care [7]. At national level, these programmes are monitored using routinely collected data and periodic country representative surveys. Routinely collected data can also be used to under- stand the effectiveness of services and to improve decision-making in the healthcare system. The benefits in using routinely collected data include wider coverage of recorded items from across the whole country and the longitudinal nature of the data allowing estimation of trends and changes in the use of services [9]. The use of these data in this way is cost effective, as it is already collected and readily available for analysis. Therefore, re- search can be conducted in a timely and cost-efficient manner [10]. It can be the basis of sampling for clinical trials, cohort studies and case-control studies, as matched case-control analyses can be performed repeat- edly over long time periods [11]. Routinely collected data are usually clinic based, but results from analysis of such data can be generalisable to the whole population if the services are widely used and serve all sections of society [10]. One of the main data collection systems for the routine collection of data from the HIV programme is SmartCare, which is one of the largest electronic patient monitoring systems (PMS) in Africa and is used in South Africa and Ethiopia [12]. In Zambia, the SmartCare database is used as a PMS for HIV services, and the data are used to monitor and plan improvements in the country’s HIV programme. SmartCare has been used as a pilot since 2004, and was officially rolled out in 2006 [13], with 528 clinics using SmartCare in 2012, and Sub-Saharan Africa is home to approximately 71% of the people living with HIV [3]. Zambia is a high HIV burden country within sub-Saharan Africa, having a na- tional HIV prevalence of 11.6% and almost 980,000 people living with HIV in 2016 [4–6]. Background implemented in more than 700 clinics that provide anti- retroviral therapy services by 2013 [14, 15]. SmartCare data, in facilities where it is available, is used to provide aggregate reports for DHIS2, and other health manage- ment information systems at the district level. In some health facilities, the SmartCare data can inform the drug ordering and the laboratory information systems, but this is not possible in most health facilities in Zambia. Worldwide, many countries routinely collect data on HIV care and services, which is then used to provide national and international indicators about the HIV epidemic. These indicators provide information and insight to aid policymakers and planners when mak- ing important decisions about HIV services, to re- quest for further research, and in advocacy for new initiatives and funding [1, 2]. Research has revealed that countries like Zambia, with one of the highest HIV/AIDS prevalence rates in Africa, are not the largest contributors to research on HIV/ AIDS. This was evident in a review of three journals fo- cusing on HIV/AIDS [16], which showed that the United States of America and Western Europe accounted for 85% of all published articles between 1986 and 2003. In sub-Saharan Africa, 50% of all publications on Africa indexed in PubMed between 1981 and 2009 were from South Africa, Uganda and Kenya. Zambia was ranked seventh, with 922 publications within that period, trans- lating to approximately 32 publications per year [17]. This paper is a review of published studies using rou- tinely collected HIV data from Zambia from 2004 to 2015, within the six areas of focus (VMMC, condom programming, behavioural change, HTC, PMTCT, and treatment and retention in care). We sought to examine the extent to which routinely collected HIV data has been analysed quantitatively for publication and identify gaps that exist across the six prioritised areas. It is hoped that findings from this review will potentially inform guidelines and strategies as well as stimulate policy dialogue in the use of routinely collected data. g [ ] Sub-Saharan Africa is home to approximately 71% of the people living with HIV [3]. Zambia is a high HIV burden country within sub-Saharan Africa, having a na- tional HIV prevalence of 11.6% and almost 980,000 people living with HIV in 2016 [4–6]. The HIV epidemic in Zambia is generalised and is mainly attributed to un- protected heterosexual activity [7]. Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Page 2 of 10 Munthali et al. Health Research Policy and Systems (2017) 15:51 Background The HIV epidemic in Zambia is generalised and is mainly attributed to un- protected heterosexual activity [7]. This creates a need to monitor the HIV epidemic by focusing on indicators of effective prevention and on the quality of the HIV ser- vices in Zambia [8]. Six key service areas are prioritised in the country for prevention and treatment of HIV. These are (1) voluntary medical male circumcision (VMMC); (2) condom programming; (3) behavioural change; (4) HIV testing and counselling (HTC); (5) prevention of mother-to-child transmission (PMTCT); and (6) treatment and retention in care [7]. At national level, these programmes are monitored using routinely collected data and periodic country representative surveys. p y p y Routinely collected data can also be used to under- stand the effectiveness of services and to improve decision-making in the healthcare system. The benefits in using routinely collected data include wider coverage of recorded items from across the whole country and the longitudinal nature of the data allowing estimation of trends and changes in the use of services [9]. The use of these data in this way is cost effective, as it is already collected and readily available for analysis. Therefore, re- search can be conducted in a timely and cost-efficient manner [10]. It can be the basis of sampling for clinical trials, cohort studies and case-control studies, as matched case-control analyses can be performed repeat- edly over long time periods [11]. Routinely collected data are usually clinic based, but results from analysis of such data can be generalisable to the whole population if the services are widely used and serve all sections of society [10]. One of the main data collection systems for the routine collection of data from the HIV programme is SmartCare, which is one of the largest electronic patient monitoring systems (PMS) in Africa and is used in South Africa and Ethiopia [12]. In Zambia, the SmartCare database is used as a PMS for HIV services, and the data are used to monitor and plan improvements in the country’s HIV programme. SmartCare has been used as a pilot since 2004, and was officially rolled out in 2006 [13], with 528 clinics using SmartCare in 2012, and Literature search strategy gy We conducted a literature review of studies that reported results from routinely collected HIV data in Zambia. We utilised a detailed search protocol and standard systematic review procedures (Additional file 1) for papers which utilised routinely collected HIV data from primary to tertiary healthcare settings, using SmartCare or other electronic or paper-based PMS data in Zambia. We included studies published between 2004 (when SmartCare started) and November 2015. The search was conducted between July and November 2015. We selected only original articles from peer-reviewed journals on HIV studies conducted in Zambia utilising routinely collected data and quantitative methods of data analysis. All reported studies relevant to our search topic were reviewed, regardless of sample size. Articles were excluded if they were not written in English or where the specific results for Zambia were not reported from regional or multi-country studies. Clinical trials and intervention studies that did not take place under routine care conditions were also excluded, although Munthali et al. Health Research Policy and Systems (2017) 15:51 Page 3 of 10 Page 3 of 10 community trials that referred patients to the routine clinics were included. of which seven were found to be multi-country studies that did not use routinely collected data, and the remaining 62 were considered for categorisation. The articles were then classified into the six HIV service areas. We searched the PubMed, Google Scholar, Directory of Open Access Journals, and African Journals Online databases for articles on HIV in Zambia that utilised routinely collected data (Table 1). We used a combin- ation of search words that included “HIV”, “SmartCare” and “routinely collected data”, among others (Table 1). One of the authors (TM) searched for articles and ex- tracted the data from included studies, while another au- thor (SG) reviewed the extracted data for discrepancies. All discrepancies were discussed and resolved. A stand- ard data extraction form was used to review and extract data such as sample size, study design, number of study sites, dates of data collection, year of publication and main outcomes. Overall, 15 articles addressed PMTCT, four focussed on HTC, four covered social and behavioural change (Table 2), and 39 covered treatment and retention in care. Our search did not reveal any articles that used routinely collected HIV data in Zambia reporting out- comes in the areas of condom programming or VMMC utilising quantitative methods. Literature search strategy The majority of the papers were mostly large samples, with thousands of subjects, covering many different health facilities. The articles on HIV treatment and retention in care covered topics such as enrolment and retention into antiretroviral therapy, effectiveness of different drug regimens, coinfections with laboratory confirmed pathogens, comorbidities using clinical signs and symptoms, and food supplementation (Table 3). The 15 PMTCT articles found addressed elimination of paediatric HIV infections, transmission of HIV to the babies, and improvement of survival in infected mothers and their exposed children. Data analysis h l d The selected papers could only be categorised by the six programme areas as the range of topics covered pre- vented aggregated statistical analysis of findings. All eli- gible articles were further grouped by the populations used in the papers, namely adult (males and females above 15 years of age), women and children (under the age of 15 years) to assess how effectively the priority areas cover the different age categories. The eligible arti- cles were also analysed based on institutions that collab- orated to publish the articles. p We found four articles on HTC covering couple coun- selling and provider-initiated testing and counselling. Ar- ticles on social and behaviour change looked at creating demand for adherence, prevention interventions, im- proved biomarkers and treatment uptake. Treatment and care had the largest number of articles with 39 arti- cles covering the topic (Table 3). Of the 62 articles, 33 full length papers utilised adult only routinely collected data and addressed retention in care, access to HIV treatment, mortality and clinical outcomes. A total of 16 full-length articles used data from only women, covering contraception, PMTCT and antenatal HIV prevalence rates. The articles using data on women only were pub- lished between 2010 and 2011, and had sample sizes ranging from 1435 to 138,884. There were 13 peer- reviewed articles that addressed paediatric HIV care and treatment. These were published between 2007 and 2013, with sample sizes ranging from 1120 to 4975. Our search did not reveal any articles utilising routinely col- lected HIV data specifically on adolescents aged 10–24 years old. Results A total of 1846 titles were reviewed and 1048 were excluded because they were not published in journals (n = 482), were published before 2004 (n = 335), or the topics were not relevant (n = 231). A total of 791 ab- stracts were then reviewed. Of these, some were ex- cluded because they were clinical trials (n = 39), qualitative studies (n = 110), or did not use routinely collected data (n = 470), or were multi-country studies that did not include specific data on Zambia (n = 103) (Fig. 1). From these, 69 full length articles were selected, Table 1 Search strategy Database Search terms Google Scholar (July 3, 2015) HIV + SmartCare + Zambia HIV + routine + data HIV/AIDS + “routinely collected data” + Zambia Condom + HIV + Zambia PubMed (July 15, 2015) HIV/AIDS + “routinely collected data” + Zambia + routine data Condom + HIV African Journals Online (November 6, 2015) HIV + routine + data + Zambia + condom + "routinely collected data" + SmartCare Directory of Open Access Journals (November 11, 2015) HIV + Zambia + condom + "routinely collected data" + SmartCare The 62 papers analysed were published in collabor- ation with partner institutions (Fig. 2). The Centre for Infectious Disease Research in Zambia and the Univer- sity of Alabama had the highest contribution, with col- laboration on 42 and 29 papers, respectively. The staff from national and district levels of the Ministry of Health participated in 40 of the published articles, while the lower collaboration was from institutions based in the United Kingdom, with collaboration on Munthali et al. Health Research Policy and Systems (2017) 15:51 Page 4 of 10 Fig. 1 Flow diagram of review of routinely collected HIV data in Zambia Fig. 1 Flow diagram of review of routinely collected HIV data in Zambia only 8 articles, four from LSHTM and four from other universities. This study was a collection of articles covering a di- verse range of topics, which meant that no meta-analysis of the studies was possible. One of the goals of this paper was to highlight the range and diversity of the topics available for analysis using routinely collected data, and to explore the gaps in the published literature so far. The main topics were grouped into treatment and retention in care, PMTCT, HTC, condom programming and VMMC. Results Our search on VMMC and condom programming revealed no quantitatively analysed papers and few qualitatively analysed papers. Discussion The review of published articles showed that consider- able strides are being made in utilisation of routinely collected HIV data in Zambia. A total of 62 articles were found and considered in this review. Treatment and retention in care and PMTCT had the highest contribu- tion, with counselling and social and behavioural change having four articles each. However, we could not find published papers that utilised quantitative data analysis methods in our search on VMMC and condom pro- gramming despite the importance of these programmes and the inclusion of data from these programmes in SmartCare. The broad focus of the literature search on HIV in Zambia should have identified many papers on condom programming or VMMC, but the only papers found were qualitative studies on these topics. It was also observable during the search process that quantita- tively analysed studies on HIV in adolescents in the country were limited and information for this age group has to be extracted from paediatric and adult studies. We did not include a large number of qualitatively analysed, clinical trial and survey-based articles, which have made important contributions to policy change in HIV care and treatment in Zambia. Further, risk of bias in individual study papers selected was not prioritised during the selection process since the rationale of the re- view was to assess the extent of utilisation of routinely collected data in the country. Only peer-reviewed articles where included because the assumption was that the peer-review process implies some form of quality control for biases in selected papers. In addition, only one of the authors reviewed the titles and abstracts, Munthali et al. Health Research Policy and Systems (2017) 15:51 Page 5 of 10 Table 2 Studies utilising routinely collected data in HIV testing and counselling, prevention of mother-to-child transmission (PMTCT) and social behavioural change programmes in Zambia Table 2 Studies utilising routinely collected data in HIV testing and counselling, prevention of mother to child transmission (PMTCT) and social behavioural change programmes in Zambia First author, year of publication Data collection period Sample size Sampled population Outcomes assessed HIV testing and counselling Topp et al. [31] 2008–2011 2239 Adults only CD4 count, haemoglobin level, BMI, education level, partner’s HIV status Topp et al. [32] 2008–2010 44,420 Adults only HIV testing, enrolment into care Czaicki et al. [33] 2011–2012 10,806 Adult couples Cohabitation length, prior HIV testing, current antiretroviral use Kankasa et al. Discussion [34] 2006–2007 15,670 Children HIV testing, testing coverage, HIV counselling, PMTCT Killam et al. [35] 2007–2008 13,917 Women initiating ART Women eligible for ART, women initiating ART Stringer et al. [36] 2001 17,263 Women only Women tested, mothers and babies receiving NVP Stringer et al. [37] 2003 8787 Mother baby pairs Gravidity, offered testing, tested, infant given NVP Chibwesha et al. [38] 2007–2010 1813 Mother baby pairs CD4 count, date of highly active ART initiation, infant HIV status Liu et al. [39] 2007– 2009 13,888 Women initiating ART CD4 count, haemoglobin level, syphilis, tuberculosis, HIV status Chintu et al. [40] 2004–2006 6740 Women on ART Mortality, NVP exposure, CD4 count, haemoglobin level Mandala et al. [41] 2007–2008 14,815 Women on ART CD4 count, initiated on ART Torpey et al. [20] 2005–2008 9723 Women on ART HIV testing, enrolled to care, received ART Chibwesha et al. [42] 2009–2010 18,407 Women initiating ART CD4 count, haemoglobin level, use of contraceptives Stringer et al. [43] 2002–2006 243,302 Women and baby pairs HIV status, number testing positive, attended antenatal care Mulindwa [44] Not stated 146 Women initiating ART NVP toxicity, hepatic toxicity, WHO grading of toxicity Ngoma et al. [45] 2008–2009 279 Women only HIV-free at 12 months, mortality rates, HIV transmission Torpey et al. [46] 2007–2010 28,320 Children only HIV testing, type of PMTCT regimen Torpey et al. [47] 2007–2009 8237 Children HIV testing, type of PMTCT regimen Albrecht et al. [48] 2001–2003 760 Women only PMTCT drug adherence, partner disclosure Social and behavioural change Kankasa et al. [34] 2004–2007 27,115 Adults on ART Adherence, mortality, loss to follow-up, CD4 count Goldman et al. [49] 2006–2007 913 Adults on ART Adherence, viral load Carlucci et al. [50] 2006 542 Adults on ART Drug adherence Birbeck et al. [51] 2005–2006 255 Adults on ART Drug adherence ART antiretroviral therapy, BMI body mass index, NVP nevirapine First author, year of publication Data collection period Sample size Sampled population Outcomes assessed information from the districts rarely used for decision- making at district levels. The reasons for these low numbers could be the limited data analysis skills, un- availability of data analysis software, disapproval or lack of support from supervisors, and lack of time and op- portunity [9–11, 18–20]. Discussion It could be further argued that the limited use of routinely collected data was due to lack of knowledge on the benefits of analyzing such data at facility and district levels and poor data man- agement, which could be alleviated by deliberate policy from government to support existing staff capacity building, operational research and publication of find- ings [18, 21]. which could be a source of bias. However, as far as we are aware, this is the first study to provide such a baseline of studies for future referral. The total number of published articles found in our literature search on the six HIV programmatic areas using routinely collected data meeting our criteria was quite low (an average of six articles per year) consider- ing that these have been published in the past 10 years. This finding is lower than the 32 articles per year re- ported by Uthman [17], but in line with findings by the Ministry of Health [18], where the use and analysis of routinely collected data were found to be inadequate in Zambia, with analysed data displayed in graphs and Munthali et al. Health Research Policy and Systems (2017) 15:51 Page 6 of 10 Table 3 Studies utilising routinely collected data on HIV treatment and retention in care in Zambia First author Data collection period Sample size Sampled population Outcomes assessed Cantrell et al. [52] 2004–2005 636 Adults on ART Adherence to medication, CD4, weight gain Koethe et al. [53] 2004–2008 27,915 Adults initiating ART Weight gain, death, treatment failure, BMI Tirivayi et al. [54] 2009 291 Adults on ART Adherence to medication, CD4, BMI Koethe et al. [55] 2004–2009 56,612 Adults on ART CD4, mortality, BMI Stringer et al. [56] 2004–2007 14,736 Adults on ART Single dose substitution, CD4 count, haemoglobin level, BMI, mortality Chi et al. [57] 2007–2010 18,866 Adults initiating ART CD4, clinical disease staging, BMI, serum creatinine adherence, mortality Chi et al. [58] 2007–2009 10,485 Adults on ART Drug substitution, mortality, loss to follow-up, withdrawal and death Chi et al. [59] 2004–2008 24,366 Adults on ART CD4 counts, age clinical staging, haemoglobin, tuberculosis co-infection, adherence Chi et al. [60] 2007 33,704 Adults on ART Cut-off points defining loss to follow-up, sensitivity, specificity, misclassification rate Giganti et al. [61] 2004–2010 40,410 Adults on ART Haemoglobin level, CD4, ART regimen Vinikoor et al. Discussion This is in line with global trends in HIV prevention strategies where treatment and retention in care have been identified as the most effective HIV prevention tool among the biomedical prevention tools analysed to date [22, 23]; more research is encouraged in these areas. However, considering the period under review, the number of studies found on retention and care were rather low. Similar trends of low levels of pub- lication in this area have been attributed to long follow- up periods required to monitor retention in care as well as to inconsistent information systems that make it difficult to track patients that seek care from multiple facilities [24]. from paediatric and adult studies. Similar findings have been reported in studies conducted in southern Africa in 2009 [26] and 2010 [27], where few data were reported on perinatally infected adolescents with most of the available data on adherence and outcomes emerging from the developed world. It is further argued that data for adolescents in southern Africa are disaggregated into 0–14, 15–19 and 15–24 year age groups, which makes it difficult to ascertain adolescent-specific data since, in most cases, the data includes very young children or adults [28]. The search on condom programming revealed mostly intervention studies in settings where prospective users could access them. Reasons for this could be the mode of distribution, which is restricted to public health facil- ities and to private health facilities only on request [29]. Similarly, Kane et al. [30] argued that use of aggregate data on condom sales does not provide information on utilisation of condoms after they are obtained, resulting in the need for in-depth analysis on factors associated with condom use. The same could be concluded on use- fulness of aggregate condom distribution data. Moreover, condom distribution data are difficult to document in routinely collected data and thus there is heavy reliance There was also a limited number of studies that looked at children born with HIV infection identified in our search. This is in line with findings from a systematic re- view of care and retention in HIV-infected children in low- and middle-income countries, where limited data were also found in Asia, Eastern Europe and Latin America [25], attributed to emphasis on studies on adult data. Discussion Health Research Policy and Systems (2017) 15:51 Page 7 of 10 Table 3 Studies utilising routinely collected data on HIV treatment and retention in care in Zambia (Continued) Van Dijk et al. [85] 2007–2008 192 Children on ART Years of receiving ART, distance from clinic, CD4 percentage, weight-for-age Z score Sinkala et al. [86] 2005–2006 5609 Adults only Colonoscopy, laparoscopy, culture results Sheyo [87] 2009–2010 452 Adults and children HIV status, burn history, burn outcome and management Brugha et al. [88] 2004–2007 39 Health facilities VCT, ART, PMTCT, childhood immunisation service and coverage trends Kancheya et al. [89] 2003–2006 203 Adults HIV status, VCT Kaile et al. [90] 2004 18 Adults on ART Blood pressure serum potassium, creatinine and sodium, Karnofsky score, WHO staging ART antiretroviral therapy, BMI body mass index, PMTCT prevention of mother-to-child transmission, VCT voluntary counselling and testing ble 3 Studies utilising routinely collected data on HIV treatment and retention in care in Zambia (Continued) Treatment and retention in care had the largest num- ber of studies. This is in line with global trends in HIV prevention strategies where treatment and retention in care have been identified as the most effective HIV prevention tool among the biomedical prevention tools analysed to date [22, 23]; more research is encouraged in these areas. However, considering the period under review, the number of studies found on retention and care were rather low. Similar trends of low levels of pub- lication in this area have been attributed to long follow- up periods required to monitor retention in care as well as to inconsistent information systems that make it difficult to track patients that seek care from multiple facilities [24]. There was also a limited number of studies that looked at children born with HIV infection identified in our search. This is in line with findings from a systematic re- view of care and retention in HIV-infected children in low- and middle-income countries, where limited data were also found in Asia, Eastern Europe and Latin America [25], attributed to emphasis on studies on adult data. It was also apparent that no quantitative peer- reviewed studies on treatment and retention among ado- lescents already in HIV care in Zambia were found in our search. Data on this age group has to be extracted Treatment and retention in care had the largest num- ber of studies. Discussion [62] 2015 20,308 Adults on ART HBsAg, CD4, BMI, WHO staging Mulenga et al. [63] 2004–2007 25,779 Adults on ART Mortality, creatinine clearance Stringer et al. [64] 2004–2005 21,755 Adults initiating ART Clinical staging, CD4, mortality, BMI, haemoglobin level, adherence Seu et al. [65] 2009–2012 68 Adults failing treatment CD4 count, adherence, HIV drug resistance mutations Krebs et al. [66] 2005 1343 Adults lost to follow-up CD4 count, BMI, mortality, home visit categories (traced, untraceable, died) Vinikoor et al. [67] 2004–2010 53,015 Adults missing pharmacy refills CD4 count, clinical staging, pharmacy refills, adherence, ART regimen Vinikoor et al. [68] 2004–2011 92,130 Adults on ART Adherence, CD4 count, mortality, long-term follow- up Harris et al. [69] 2005–2007 20,153 Tuberculosis/HIV co-infected adults Enrolment on ART, CD4 count, WHO staging Mweemba et al. [70] 2011–2013 91,130 Adults initiating ART Hepatitis B co-infection, WHO staging, CD4 count Deo et al. [71] 2007 13 Laboratories CD4 count, haemoglobin, liver function test Chi et al. [72] 2004–2006 6740 Women exposed to nevirapine WHO stage, CD4 cell count, status, BMI Bolton-Moore et al. [73] 2004–2007 4975 Children on ART CD4 percentage, weight-for-age Z scores, clinical staging, haemoglobin level, mortality Mubiana‐Mbewe et al. [74] 2004–2006 1705 Children enrolled into care CD4 percentage, clinical staging, haemoglobin level Scott et al. [75] 2006–2011 1334 Children on ART CD4 percentage, fixed and variable unit costs Kiage et al. [76] 2009–2011 822 Mother-infant pairs WHO staging, CD4/CD8 percentage, HIV, haemoglobin panel, maternal-CD4 count Sutcliffe et al. [77] 2004–2008 1278 Children on ART Enrolment in ART, loss to follow-up, mortality, clinical staging, CD4 percentage Iyer et al. [78] 2006–2011 1102 Children initiating ART Age, CD4 percentage, ART initiation, full blood count, blood chemistry Sutcliffe et al. [79] 2004–2008 863 Children on ART Mortality, CD4, HIV, haemoglobin level Sutcliffe [80] 2000–2002 492 Children on ART CD4 count, haemoglobin level, mortality Van Dijk et al. [81] 2007–2012 77 Children on ART Weight-for-age Z scores, CD4 percentage Van Dijk et al. [82] 2007–2010 198 Children on ART Treatment outcomes, viral load, CD4 percentage, retention in care, mortality Sutcliffe et al. [83] 2007–2009 193 Children initiating ART Weight-for-age and height-for-age Z scores Nkamba [84] Not stated 59 Children on ART T cell subsets CD4 and CD8 memory Table 3 Studies utilising routinely collected data on HIV treatment and retention in care in Zambia First author Data collection period Sample size Sampled population Outcomes assessed Munthali et al. Discussion It was also apparent that no quantitative peer- reviewed studies on treatment and retention among ado- lescents already in HIV care in Zambia were found in our search. Data on this age group has to be extracted Fig. 2 Graph showing number of articles published by each collaborating institution Munthali et al. Health Research Policy and Systems (2017) 15:51 Page 8 of 10 Page 8 of 10 on survey data [7]. There is an urgent need to under- stand the demographics of condom distribution in the country. Similar trends were revealed in the VMMC programme, which also yielded low numbers of peer- reviewed articles that utilised routinely collected quantitative data, despite the country not meeting its circumcision targets [7]. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding h This writing or this paper was supported by the Sustainable Evaluation through Analysis of Routinely Collected HIV data (SEARCH) project Zambia. 13. Ministry of Health. National Health Report, Directorate of Public Health and Research. Lusaka: MoH; 2012. 14. Ministry of Health. List of Health Facilities in Zambia, Preliminary Report. Lusaka: MoH; 2013. Abbreviations HTC: HIV testing and counselling; PMS: patient monitoring system; PMTCT: prevention of mother to child transmission; VMMC: voluntary male circumcision 10. Grzeskowiak LE, Gilbert AL, Morrison JL. Methodological challenges in using routinely collected health data to investigate long-term effects of medication use during pregnancy. Ther Adv Drug Saf. 2013;4(1):27–37. 11. Bain MR, Chalmers JW, Brewster DH. Routinely collected data in national and regional databases-an under-used resource. J Public Health. 1997;19(4):413–8. 10. Grzeskowiak LE, Gilbert AL, Morrison JL. Methodological challenges in using routinely collected health data to investigate long-term effects of medication use during pregnancy. Ther Adv Drug Saf. 2013;4(1):27–37. 10. Grzeskowiak LE, Gilbert AL, Morrison JL. Methodological challenges in using routinely collected health data to investigate long-term effects of medication use during pregnancy. Ther Adv Drug Saf. 2013;4(1):27–37. 11. Bain MR, Chalmers JW, Brewster DH. Routinely collected data in national and regional databases-an under-used resource. J Public Health. 1997;19(4):413–8. 11. Bain MR, Chalmers JW, Brewster DH. Routinely collected data in national and regional databases-an under-used resource. J Public Health. 1997;19(4):413–8. Availability of data and materials The dataset supporting the conclusions of this article is included with the article and its additional files. 15. PEPFAR. Zambia Operational Plan Report FY 2013. Washington, DC: PEPFAR; 2013. https://www.pepfar.gov/documents/organization/222188.pd . Accessed July 2015. Authors’ contributions 16. Uusküla A, Toompere KLK, et al. HIV research productivity and structural factors associated with HIV research output in European Union countries: a bibliometric analysis. BMJ Open. 2015;5(2):e006591. TM and PM participated in the conception of the study. SGM reviewed the analysed articles. CM, PM, JT, NK and CP reviewed all the drafts for intellectual content, participated in the interpretation of the findings. The whole team participated in the critical review and editing of all the manuscript drafts for scientific merit and depth. All authors read and approved the final manuscript. 17. Uthman OA. Pattern and determinants of HIV research productivity in sub- Saharan Africa: bibliometric analysis of 1981 to 2009 PubMed papers. BMC Infect Dis. 2010;10:47. 18. Ministry of Health. Assessment of the Health Information System in Zambia. Lusaka: Ministry of Health, Department of Planning and Development, Monitoring and Evaluation Unit; 2007. 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To improve the use of routinely collected data for use in publications the government could deliberately put in place policies to prioritise training of civil servants work- ing in various programmes in operational research and consequently fund publishing of findings. These published articles would aid in international resource mobilisation for most programmes in the country as programme level data can be easily accessed in peer-reviewed articles. 5. UNAIDS. Global Report: UNAIDS Report on the Global AIDS Epidemic 2013. Geneva: UNAIDS; 2013. 6. Ministry of Health. Zambia Population-Based HIV Impact Assessment (ZAMPHIA) 2015–2016, Summary Sheet: Preliminary Findings. Lusaka: Ministry of Health; 2016. 7. Ministry of Health. National AIDS Strategic Framework 2014-2016: A Nation Free from the Threat of HIV and AIDS. Lusaka: Ministry of Health; 2014. 7. Ministry of Health. National AIDS Strategic Framework 2014-2016: A Nation Free from the Threat of HIV and AIDS. 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Author details 1 1School of Public Health, University of Zambia, Lusaka, Zambia. 2Ministry of Health, Lusaka, Zambia. 3Department of Population Health, London School of Hygiene and Tropical Medicine, London, United Kingdom. 4Zambia AIDS Related Tuberculosis (ZAMBART) Project, Lusaka, Zambia. 5MeSH Consortium, Department of Social Economic and Health Research, Faculty of Public Health and Policy, London School of Hygiene and Tropical Medicine, London, United Kingdom. Conclusion There are positive advances being made in the HIV programme in the use of routinely collected data in Zambia. This progress must be nurtured and enhanced if Zambia is to reach elimination stages in HIV control. However, more efforts must be put into research and publishing results in critical areas, such as paediatric and adolescent care, VMMC and condom distribution, in order to build the skills and knowledge-base to deliver HIV services. Research on adolescent and childhood HIV morbidity and mortality outcomes as well as social behavioural change needs is important because HIV- infected adolescents and children are the key population in reducing HIV spread in their generation. Received: 9 June 2016 Accepted: 30 May 2017 Received: 9 June 2016 Accepted: 30 May 2017 Acknowledgements We acknowledge the SEARCH Project team in London for the additional training in HIV data analysis and support while in London. 12. Tassie J-M, Malateste K, Pujades-Rodríguez M, Poulet E, Bennett D, Harries A, Mahy M, Schechter M, Souteyrand Y, Dabis F. Evaluation of three sampling methods to monitor outcomes of antiretroviral treatment programmes in low-and middle-income countries. PLoS One. 2010;5(11):e13899. 14. Ministry of Health. List of Health Facilities in Zambia, Preliminary Report. Lusaka: MoH; 2013. Competing interests Results from an analysis of an urban cohort of HIV-positive patients in Lusaka, Zambia. 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Submit your next manuscript to BioMed Central and we will help you at every step: 74. Mubiana‐Mbewe M, Bolton‐Moore C, Banda Y, Chintu N, Nalubamba‐Phiri M, Giganti M, Guffey MB, Sambo P, Stringer EM, Stringer JS. Causes of morbidity among HIV‐infected children on antiretroviral therapy in primary care facilities in Lusaka, Zambia. Trop Med Int Health. 2009;14(10):1190–8. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: • We accept pre-submission inquiries 75. Scott CA, Iyer H, Bwalya DL, McCoy K, Meyer-Rath G, Moyo C, Bolton-Moore C, Larson B, Rosen S. 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https://openalex.org/W1985491326	http://cds.cern.ch/record/1490063/files/CR2012_253.pdf	English	null	Recent Results of the CMS Experiment	EPJ web of conferences	2,014	cc-by	7,632	Recent Results from The CMS Experiment Tommaso Dorigo for the CMS Collaboration Tommaso Dorigo for the CMS Collaboration The Compact Muon Solenoid Experiment Mailing address: CMS CERN, CH-1211 GENEVA 23, Switzerland Conference Report 04 October 2012 (v2, 15 October 2012) 04 October 2012 (v2, 15 October 2012) 04 October 2012 (v2, 15 October 2012) CMS CR -2012/253 CMS CR -2012/253 CMS CR -2012/253 Available on CMS information server Presented at ICFP2012: First International Conference on New Frontiers in Physics a e-mail: dorigo@pd.infn.it Tommaso Dorigo1,a for the CMS Collaboration INFN - Sezione di Padova Abstract. The CMS experiment obtained a large number of groundbreaking results from the analysis of 7- and 8-TeV proton-proton collisions produced so far by the Large Hadron Collider at CERN. In this brief summary only a sample of those results will be discussed. A new particle with mass mH = 125.3 ± 0.4(stat.) ± 0.5(syst.) GeV and char- acteristics compatible with those expected for a standard model Higgs boson has been observed in its decays to photon pairs, WW pairs, and ZZ pairs. Searches for the rare de- cays Bd →µµ and Bs →µµ have allowed to set limits on the branching fractions which are close to standard model predictions, strongly constraining new physics models. The top quark has been studied with great detail, obtaining among other results the world’s best measurement of its mass as mt = 173.49 ± 0.43(stat. + JES ) ± 0.98(syst.) GeV. New physics models have been strongly constrained with the available data. Abstract The CMS experiment obtained a large number of groundbreaking results from the analysis of 7- and 8-TeV proton-proton collisions produced so far by the Large Hadron Collider at CERN. In this brief summary only a sample of those results will be discussed. A new particle with mass mH = 125.3 ± 0.4(stat.) ± 0.5(syst.) GeV and characteristics compatible with those expected for a standard model Higgs boson has been observed in its decays to photon pairs, WW pairs, and ZZ pairs. Searches for the rare decays Bd →µµ and Bs →µµ have allowed to set limits on the branching fractions which are close to standard model predictions, strongly constraining new physics models. The top quark has been studied with great detail, obtaining among other results the world’s best measurement of its mass at mt = 173.49 ± 0.43(stat. + JES) ± 0.98(syst.) GeV. New physics models have been strongly constrained with the available data. Presented at ICFP2012: First International Conference on New Frontiers in Physics Recent Results of the CMS Experiment Tommaso Dorigo1,a for the CMS Collaboration INFN - Sezione di Padova 2 The CMS Detector CMS –an acronym for Compact Muon Solenoid– is a multi-purpose magnetic detector designed to study proton-proton collisions delivered by the CERN Large Hadron Collider. The detector is located in a underground cavern at a depth of 100m at the site of Cessy, near the border of France and Switzer- land. Particles emitted in hard collisions at the center of CMS cross in succession a silicon tracker, electromagnetic and hadron calorimeters, a solenoid magnet, and muon drift chambers embedded in the solenoid iron return yoke. A drawing of the CMS detector is shown in Fig. 1. Fig. 1. An exploded view of the CMS detector, showing the outer muon chambers (white) embedded in iron (red). Internally can be seen the calorimeter system (ECAL, in green, and HCAL, in orange). The tracker is located in the core of the central barrel. Fig. 1. An exploded view of the CMS detector, showing the outer muon chambers (white) embedded in iron (red). Internally can be seen the calorimeter system (ECAL, in green, and HCAL, in orange). The tracker is located in the core of the central barrel. 1 Introduction The observation of a particle with characteristics closely matching those predicted for a standard model Higgs boson, announced jointly by the ATLAS and CMS experiments on July 4th this year, constitutes a turning point for the experiments at the CERN Large Hadron Collider (LHC). The main purpose of building the LHC and its giant detectors was indeed that of discovering the Higgs boson and under- standing the origin of electroweak symmetry breaking: we have completed a very important step in that direction, but we are certainly only at the beginning of a long journey. Even assuming that the new 125 GeV resonance is indeed the standard model Higgs boson many questions remain unanswered; in the process of answering those questions we might however discover that the new-found particle is a Supersymmetric Higgs boson, or an even more exotic object. In other words, the study of the proper- ties of the new 125 GeV particle constitutes a whole new chapter at the frontier of particle physics, one which will take the next decade to write. In parallel with the new particle discovery, the CMS experiment has kept expanding our knowledge of frontier particle physics with several new precise measurements of electroweak physics observables, and with the search of new particles and phenomena predicted by physics models extending the stan- dard model. In the present document we can only supply a couple of examples of the wide-range searches and measurements that are being produced from the analysis of 7- and 8-TeV proton-proton collision data. Section 2 describes brieﬂy the experimental environment. Our summary of the recent CMS results starts in Sec. 3, where we highlight the precise new measurements of top quark mass and the search for rare B meson decays. In Sec. 4 we brieﬂy review the current measurements of Higgs boson mass, cross section, and properties. We brieﬂy mention searches for new physics in Sec. 5. We oﬀer some conclusions in Sec. 6. EPJ Web of Conferences 2.1 Overview of the CMS Detector The momenta of charged particles emitted in the collisions at the center of the CMS detector are measured using a 13-layer silicon pixel and strip tracker; 66 million silicon pixels of dimensions 100x150 µm are arranged in three barrel layers, and are surrounded by 9.6 million 180 µm-wide silicon strips arranged in additional concentric barrels in the central region and disks in the endcap region. In order to allow a precise measurement of charged particle momenta, the silicon tracker is immersed in the 3.8 T axial ﬁeld produced by a superconducting solenoid. The tracker covers the pseudorapidity range \|η\| < 2.5, where pseudorapidity is deﬁned as η = −ln tan θ/2 and θ is the polar angle of the trajectory of a particle with respect to the direction of the counter-clockwise proton beam. j y p p p Surrounding the tracker are an electromagnetic calorimeter (ECAL) composed of lead tungstate crystals, and a brass-scintillator hadron calorimeter (HCAL). These detectors are used to measure the energy of incident particles from the produced electromagnetic and hadronic cascades; they con- sist of a barrel assembly covering the central region, plus two endcaps covering the solid angle for particles emitted at lower angle with respect to the beams direction. The ECAL and HCAL extend to a pseudorapidity range of \|η\| < 3.0; at still smaller angles particles emitted in the collision en- counter a steel/quartz-ﬁber Cherenkov forward detector (HF) which extends the calorimetric coverage to \|η\| < 5.0. The outermost component of the CMS detector is the muon system, consisting of four layers of gas detectors placed within the steel return yoke. The CMS muon system performs a high-purity iden- tiﬁcation of muon candidates and a stand-alone measurement of their momentum, and in combination International Conference on Frontier Physics 2012 with the inner tracker information provides a high-resolution determination of muon kinematics. M detail on the CMS detector is provided elsewhere [1]. p [ ] CMS collects data with a two-level trigger system. Level 1 is a hardware trigger based on custom- made electronic processors that receive as input a coarse readout of the calorimeters and muon detec- tors and perform a preliminary selection of the most interesting events for data analysis, with an output rate of about 100 kHz. 3 Precision Measurements of Electroweak Observables At centre-of-mass energies of proton-proton collisions of 7 TeV and above, the LHC can be aptly described as a top quark factory. As an example, of the order of 800 thousand top quark pairs have been produced in the core of CMS in the 2011 run. Even larger is of course the number of produced W and Z bosons (respectively 500 millions and 150 millions). Electroweak interactions parameters can be determined with unprecedented accuracy by the analysis of CMS data, challenging theoretical predictions. In what follows we summarize only a few of the many new measurements produced by CMS with vector bosons and top quarks. 2.2 The LHC in 2011 and 2012 The 2011 proton-proton run of the LHC started on March 14th and terminated on October 30th. Proton- proton collisions were produced at the centre-of-mass energy of 7 TeV. In the course of seven months of data taking CMS acquired a total of 5.3 inverse femtobarns of integrated luminosity; 5.0 of these were collected with all the CMS subdetectors fully operational. During the 2011 run the instantaneous luminosity reached up to 3.5 × 1033cm−2s−1. At a bunch crossing rate of 50 ns, the average number of pp interactions per bunch crossing was approximately 10. In such conditions, the rare hard collision which produces the physics objects (electrons, muons, taus, photons, energetic jets, missing transverse energy) recognized by the trigger system and fulﬁlling the criteria for data acquisition is usually accompanied by several additional pp interactions overlapping with it in the same bunch crossing. These additional collisions, which are typically of low energy but may still produce signiﬁcant contributions to global event characteristics such as total visible energy or charged particle multiplicity, are denoted as pile-up events. The analysis of the hard collision properly includes the eﬀect of pile-up, which is also modeled in all the necessary Monte Carlo (MC) simulated samples. p In 2012 the Large Hadron Collider has been operating at the increased energy of 4 TeV per beam, for a centre-of-mass energy of 8 TeV. The run started on April 4th, and at the time of writing (late September) over 15 fb−1 of proton-proton collisions have been delivered to the CMS experiment. The LHC in 2012 has been running at higher instantaneous luminosities than in 2011. The peak instantaneous luminosity usually peaks at 7.5 × 1033cm−2s−1. This produces a higher pileup than in 2011; CMS has responded to the resulting reconstruction challenge with more sophisticated algorithms and calibration procedures, which have allowed to maintain the same physics output despite the harsher experimental conditions. The results discussed in the following sections are based on data collected in 2011 and until June 2012, for a total of up to 10 fb−1. 2.1 Overview of the CMS Detector Level 2, also called ”High-Level Trigger” (HLT), uses ﬁne-grained information from all sub-detectors in the regions of interest identiﬁed by Level 1 to produce a ﬁnal decision, se- lecting events at a rate of about 300 Hz by means of speed-optimized software algorithms running on commercial computers. EPJ Web of Conferences as well as the production rate of WW, WZ, and ZZ pairs [6–9]. In all these measurements, W boson candidates have been selected by searching for their decay to eνe and µνµ ﬁnal states, and Z bosons by searching for their ee and µµ ﬁnal states. In the case of ZZ production the second boson has also been identiﬁed in its decay to τ-lepton pairs. as well as the production rate of WW, WZ, and ZZ pairs [6–9]. In all these measurements, W boson candidates have been selected by searching for their decay to eνe and µνµ ﬁnal states, and Z bosons by searching for their ee and µµ ﬁnal states. In the case of ZZ production the second boson has also been identiﬁed in its decay to τ-lepton pairs. Fig. 2. The total production cross section of ﬁnal states including W and Z bosons, in picobarns (red markers) are compared to theoretical predictions (blue lines). For single boson production are also reported the cross sections of processes including at least one to at least four hadronic jets with transverse energy ET > 30 GeV and pseudorapidity \|η\| < 2.4. Fig. 2. The total production cross section of ﬁnal states including W and Z bosons, in picobarns (red markers) are compared to theoretical predictions (blue lines). For single boson production are also reported the cross sections of processes including at least one to at least four hadronic jets with transverse energy ET > 30 GeV and pseudorapidity \|η\| < 2.4. The general picture that can be drawn is one of excellent agreement with theoretical calcula- tions, which are available at next-to-leading order (NLO) [10–12] and next-to-next-to-leading order (NNLO) [13–17] in perturbative QCD. Figure 2 provides a nice summary of the CMS measurements of these processes. 3.1 Vector Boson Production Cross Sections The production cross section of W and Z bosons at a centre-of-mass energy of 7 and 8 TeV has been studied both inclusively [2,3] and as a function of the number of hadronic jets accompanying the bosons [4]. Additional measurements have determined the cross section of Wγ and Zγ production [5], 3.3 Search for Bd →µµ and Bs →µµ decays In an era when heavy ﬂavor physics was to be dominated by B factories -machines providing electron- positron collisions at the Y(4S) centre-of-mass energy- experiments studying heavy ﬂavor properties in hadronic collisions have gone far beyond being complementary to the dedicated electron-positron experiments. In particular, the CDF experiment could match and in some cases surpass the precision of several BaBar and Belle results in the latter’s own ﬁeld of excellence, mostly thanks to its ca- pability of triggering on the impact parameter of tracks, thus selecting large samples of hadronic B and D meson decays. The triggering on track impact parameter, however, is not currently possible in CMS and ATLAS, mainly because of two otherwise strong points in the design of these experiments: the one-order-of-magnitude higher bunch-crossing rate of the LHC, together with the two-orders-of- magnitude larger number of readout channels of the silicon trackers of new generation of which the LHC experiments are endowed. The huge cross section of processes yielding bottom quarks in the ﬁnal state of 7-TeV proton- proton collisions again comes to the rescue. CMS can collect semileptonic B-hadron decays of medium to large transverse momentum thanks to inclusive electron and muon triggers, as well as exploit the signiﬁcant branching ratio of B-hadron decays to J/ψ and ψ(2S ) mesons by triggering on the resulting pairs of low transverse momentum muons. Indeed, a simple graph showing the invariant mass of muon pairs collected by muon triggers speaks volumes about the heavy ﬂavor potential of the experiment (see Fig. 3). In the light of the above discussion, it is maybe not surprising that CMS has placed competitive limits on the rate of rare B →µµ decays, nor that many other world-class results are being produced in this area of research. In the following we only make a brief mention of the search for the rare decays of neutral Bd and Bs mesons to muon pairs, which has been a hot topic in the last few years. These decays are heavily suppressed in the standard model due to the absence of ﬂavor-changing neutral-current diagrams at tree level. International Conference on Frontier Physics 2012 The top quark mass remains a parameter of great interest even after the precise measurements produced by the Tevatron experiments. The most recent CMS result [24], which employs the full statistics of the 2011 run, is the most precise top quark mass determination in the world at the time of writing. The analysis uses top pair decays in the “lepton plus jets” topology, which arises when one of the two W bosons emitted in the t¯t →W+bW−¯b reaction decays via W →lν, while the other W boson creates a pair of quarks. By selecting events from lepton-triggered data which contain a clean and isolated electron or muon candidate of pT > 30 GeV and four or more hadronic jets of ET > 30 GeV, two of them with identiﬁed secondary vertices from b-quark decay, a sample of 17,985 candidates is isolated. A kinematic ﬁt using the world average value of the W boson mass as a constraint is used to determine an estimate of the top quark mass for each possible combination of jet assignments to the ﬁnal state partons. The estimated masses of all combinations in 5174 events passing a selection based on the probability of the best ﬁt are ﬁnally used in a global likelihood which combines information on the top quark mass and the jet energy scale factor, the latter obtained by comparing the pre-constraint mass of the jet pair assigned to the W decay with the world average W mass. The top quark mass is measured to be mt = 173.49 ± 0.43 ± 0.98 GeV, where the ﬁrst uncertainty quoted is the combination of statistical with jet-energy-scale-related systematic uncertainty, and the second is the quadrature sum of all other systematic uncertainties. 3.2 Top Quark Mass and Cross Section Measurements The large samples of top quark events produced in the 2011 run of the LHC have allowed the CMS experiment to measure with great accuracy the top pair production cross section in 7- and 8-TeV proton-proton collision using several diﬀerent ﬁnal states. The most precise CMS determinations come from the analysis of the clean dilepton ﬁnal state of top pair decay [19,20]. These have reached an equal or smaller total uncertainty than existing theoretical estimates at NLO [21] and NNLO [22,23]: with data corresponding to 2.3 inverse femtobarns the 7-TeV production cross section is measured at σ7 TeV t¯t = 162 ± 2 ± 5 ± 4pb, and with data corresponding to 2.4 inverse femtobarns of 2012 integrated luminosity the 8-TeV cross section is measured at σ8 TeV t¯t = 227 ± 3 ± 11 ± 10pb. In both cases the ﬁrst two quoted uncertainties are statistical and systematic, while the latter comes from the uncertainty in the total integrated luminosity. International Conference on Frontier Physics 2012 3.3 Search for Bd →µµ and Bs →µµ decays Two additional factors further reduce their rate: the ratio m2/m2 B between the squared masses of muons and B mesons implied by the helicity conﬁguration of zero-total-momentum energetic fermion-antifermion ﬁnal states, and the ratio f 2 B/m2 B (where fB is the B decay constant) due to the inner annihilation of quarks in the decaying meson. The smallness of the total predicted branching fractions, B(Bs →µµ) = (3.2±0.2)10−9 and B(Bd →µµ) = (1.0±0.1)10−10 [25], constitute an opportunity to search for indirect evidence of new physics, which could intervene in the form of the exchange of new virtual particles, with signiﬁcant increases in the rate of these decays for speciﬁc values of the new physics parameters. The search method is a counting experiment of events in the signal region of the dimuon mass distribution for both B species. Because of the dependence of mass resolution and background levels EPJ Web of Conferences Fig. 3. Invariant mass distribution of pairs of muon candidates of opposite sign collected by muon triggers in 1.1 inverse femtobarns of 7-TeV proton-proton collision in 2011. One immediately recognizes the signals due to muon pair decays of the φ, the ω, the J/ψ and the ψ(2S ), as well as the three lowest bottomonium states and the Z boson peak. Fig. 3. Invariant mass distribution of pairs of muon candidates of opposite sign collected by muon triggers in 1.1 inverse femtobarns of 7-TeV proton-proton collision in 2011. One immediately recognizes the signals due to muon pair decays of the φ, the ω, the J/ψ and the ψ(2S ), as well as the three lowest bottomonium states and the Z boson peak. on the pseudorapidity of detected muons, two separate samples are analyzed independently and then combined: “barrel” candidates have both muons with \|η\| < 1.4, and “endcap” candidates include all remaining events. MC simulations are used to estimate backgrounds from other B decays, while combinatorial backgrounds are evaluated from the data in suitable mass sidebands. A normalization sample of B+ →J/ψK+ decays, with the subsequent J/ψ →µµ decay, is collected by a similar trigger to the one used for the rare decay search, and is used to remove the uncertainties of B hadron production cross section and integrated luminosity of the data sample. Muon candidates are selected using a variety of criteria, including the quality of their track ﬁts. 4.1 Production and Decay The standard model Higgs boson has a non-zero coupling to all massive particles, and can therefore be produced by several diﬀerent mechanisms in proton-proton collisions. The reactions studied so far at the LHC include gluon-fusion diagrams, where a Higgs boson is emitted most frequently by a virtual top-quark loop; vector-boson-fusion processes, where the Higgs is produced together with two characteristic high-rapidity hadronic jets resulting from the emission of two virtual W or Z bosons oﬀ the initial state quarks; and Higgs-strahlung diagrams where the particle is radiated by a highly-oﬀ- shell W or Z boson. At a mass of mH = 125 GeV, the Higgs boson exhibits also a very rich decay phenomenology. The decays to weak boson pairs (H →WW∗, H →ZZ∗) are possible when one of the two ﬁnal-state objects is oﬀ-mass-shell, but their branching ratios are comparatively small, allowing other processes to contribute signiﬁcantly. The LHC experiments therefore search for ﬁve diﬀerent decay modes of the Higgs boson: the two mentioned above as well as decays to b-quark pairs, τ-lepton pairs, and photon pairs. The latter, although quite rare (with a predicted branching fraction of 2.3 × 10−3), was in fact crucial for the ﬁrst observation of the Higgs boson. Other still rarer decay modes (e.g. H →Zγ) will also become accessible to investigation and measurement in the future. 4 Higgs Boson Physics On July 4th, 2012 the ATLAS and CMS collaborations released preliminary results of their Higgs boson searches. Both experiments claimed the observation of a new particle decaying into photon pairs or Z boson pairs, with a mass of approximately mH = 125 GeV; and both excluded basically all the remaining part of the searched mass spectrum from the LEP II lower limit of 114.4 GeV up to 600 GeV. The observed rates of decays of the new particle into the searched ﬁnal states, as well as the production mechanisms inferred by the event topology, are in agreement with expectations for a standard model Higgs boson [28–33]. After a short review of the predicted phenomenology of Higgs production and decay, we summarize below the status of the CMS measurements of Higgs boson mass, cross section, and properties. We refer the reader to the bibliography for preliminary papers describing the recent status of individual searches [34–41]. 3.3 Search for Bd →µµ and Bs →µµ decays Muon pairs with invariant mass in the 4.9 < Mµµ < 5.9 GeV range are then preselected, and candidates in the signal regions (5.2 < Mµµ < 5.3 GeV for the Bd and 5.3 < Mµµ < 5.45 GeV for the Bs) are blinded to avoid potential bias in the selection procedure. A random-grid search of 1.6 million possible selection strategies is performed on a set of variables capable of discriminating the rare B decay signals from backgrounds, optimizing the sensitivity to the expected upper limit; among the used variables are the χ2 of the ﬁt of muon trajectories to a common vertex, the transverse momentum of the higher-pT and the lower-pT muon, the B candidate transverse momentum, the isolation of the muons from other tracks in the event, the number of nearby tracks, and the smallest impact parameter of these tracks with respect to the common vertex of the muon pair. In the “barrel” sample two candidates are found for each B meson species, while in the “endcap” four Bs candidates and zero Bd candidates are observed. Upper limits on the branching ratios are determined at 95% C.L. using the CLs criterion [26,27]: for the Bs meson the limit is B(Bs →µµ) < 7.7 × 10−9, and for the Bd meson the limit is B(Bd →µµ) < 1.8 × 10−9. These limits can be used to constrain several proposed extensions of the standard model. International Conference on Frontier Physics 2012 4.2 Determinations of Higgs boson cross section, mass, and coupling strengths CMS has recently published [42] results of searches for the Higgs boson which employ the full 2011 statistics of proton-proton collisions and all data collected until June 2012. The following experimen- tally signiﬁcant production modes of Higgs particles have been exploited: gluon-gluon fusion, vector- boson fusion, and Higgs-strahlung oﬀvector bosons. Five decay modes have been considered: photon pairs [34], Z boson pairs [35,36], W boson pairs [37,38], bottom quark pairs [39,40], and τ-lepton pairs [41]. The results of these searches are combined by CMS by taking into account all statistical and sys- tematic uncertainties and their correlations [43,44]. The combination is performed by constructing a global likelihood function, with each systematic source assigned to a nuisance parameter; each of these has a corresponding probability density function. Most of the systematic uncertainties are constrained by subsidiary measurements. The left panel in Fig. 4 shows the local p-value of observed excesses in the data, as a function of mH. For mH = 125 GeV an excess corresponding to a 5σ signiﬁcance is observed, using the γγ and ZZ ﬁnal states. The diﬀerent analyses provide rate measurements of Higgs decays in the various considered ﬁnal states. The individual measurements are shown in the right panel of Fig. 4, where the cross section is measured in units of the standard model expectation. The combined measurement of CMS is µ = 0.87 ± 0.23, in agremeent with the prediction. g From the analysis of the diphoton and four-lepton ﬁnal states, which are the ones with the largest signal-to-noise ratio and the best mass resolution, the Higgs mass has been measured to be mH = EPJ Web of Conferences Fig. 4. Left: Local p-value of the background-only hypothesis as a function of mH. Diﬀerent search channels are indicated by diﬀerent colored curves; the p-value predicted in the presence of a real Higgs boson is indicated with a dashed curve. Right: Measured Higgs production cross sections for the studied decay modes, in units of the standard model prediction (a mass mH = 125.5 GeV is assumed). The shaded band shows the ﬁtted average of experimental measurements. Fig. 4. Left: Local p-value of the background-only hypothesis as a function of mH. Diﬀerent search channels are indicated by diﬀerent colored curves; the p-value predicted in the presence of a real Higgs boson is indicated with a dashed curve. 5.1 Searches for Supersymmetry Signatures Among all hypothesized extensions of the standard model, Supersymmetry (SUSY) is one of the most studied alternatives. The symmetry between standard model fermions and bosons with supersymmetric counterparts of bosonic and fermionic nature automatically cancels the large quantum contributions to the Higgs boson mass due to virtual loops of standard model particles [45–48], solving the naturalness puzzle in a very elegant way; the added bonus is a uniﬁcation-ready merging of coupling constants below the Planck scale. Supersymmetric particle searches have been carried out in the past thirty years without success, pushing the allowed mass of the hypothetical SUSY particles to higher and higher values. Despite those early results, the much larger centre-of-mass energy of the LHC led many to trust that SUSY particles would suddenly pop up soon after the start of data taking, with unmistakable and striking signatures. But Nature has chosen otherwise. The CMS experiment has searched the 2011 data for supersymmetric particle signatures in a num- ber of ﬁnal states and with a variety of advanced methods [49–65]. Here, for the sake of brevity, only a summary of those searches is provided. In general, SUSY particles can be copiously produced in LHC proton-proton collisions in the form of pairs of squarks or gluinos, which carry color quantum num- bers and are thus subject to strong interactions. Depending on the mass spectrum of SUSY particles, the decay of squarks and gluinos may give rise to several lighter supersymmetric states in succession, with a typical “cascade” signature and characteristic kinematic features. At the end of the decay chain, a quite general signature of R-parity-conserving SUSY theories is the production of neutral weakly- interacting particles called neutralinos, which are the lightest in the supersymmetric spectrum and are thus stable. Their escape from the detector with large transverse momentum can be ﬂagged by the same experimental observable used to detect neutrinos, i.e. a large energy imbalance in the plane transverse to the beams. Experimental searches often require large values of missing transverse energy, in some cases along with hadronic jets, in others accompanied with charged leptons or more complex ﬁnal states. Figure 7 summarizes the status of CMS searches for SUSY particles in 2011 datasets correspond- ing to up to 5 inverse femtobarns of integrated luminosity. Upper limits in production cross sections are turned into exclusion regions in the plane described by the universal scalar and gaugino masses. International Conference on Frontier Physics 2012 Fig. 6. Left: 1-sigma contours of mass and cross section measurements in the three bosonic ﬁnal states, and their combination (black curve). Right: Fit results for the bosonic and fermionic coupling strength modiﬁers. The light-coloured marker at (1,1) indicates the standard model value. Fig. 6. Left: 1-sigma contours of mass and cross section measurements in the three bosonic ﬁnal states, and their combination (black curve). Right: Fit results for the bosonic and fermionic coupling strength modiﬁers. The light-coloured marker at (1,1) indicates the standard model value. 4.2 Determinations of Higgs boson cross section, mass, and coupling strengths Right: Measured Higgs production cross sections for the studied decay modes, in units of the standard model prediction (a mass mH = 125.5 GeV is assumed). The shaded band shows the ﬁtted average of experimental measurements. Fig. 5. Left: Reconstructed mass of photon pairs in 10.4f b−1 of 2011+2012 data. Entries in the histogram in the main panel are weighted by signal-to-noise ratio. Right: Reconstructed mass of Z pairs. The tall peak on the left is due to Z →llγ →llll events; the inset on the upper right shows the result of a tighter selection based on a kinematic multivariate discriminant. Fig. 5. Left: Reconstructed mass of photon pairs in 10.4f b−1 of 2011+2012 data. Entries in the histogram in the main panel are weighted by signal-to-noise ratio. Right: Reconstructed mass of Z pairs. The tall peak on the left is due to Z →llγ →llll events; the inset on the upper right shows the result of a tighter selection based on a kinematic multivariate discriminant. 125.3±0.4±0.5 GeV, where the ﬁrst uncertainty is statistical and the second one is systematic. Figure 5 shows the signals in the reconstructed invariant mass distribution of diphoton and ZZ candidates. The left panel of Fig. 6 shows the individual measurements in the mass-cross section plane, and their combination. 125.3±0.4±0.5 GeV, where the ﬁrst uncertainty is statistical and the second one is systematic. Figure 5 shows the signals in the reconstructed invariant mass distribution of diphoton and ZZ candidates. The left panel of Fig. 6 shows the individual measurements in the mass-cross section plane, and their combination. Finally, the observed signals can be used to ﬁt for coupling strength modiﬁers, to allow for diﬀerent coupling of the Higgs boson to fermions and bosons from the predictions of the standard model. The result is shown in the right panel of Fig. 6. Although the larger-than-expected rate of decays to photon pairs could be suggestive of anomalous couplings, the CMS measurement supports the standard model interpretation. International Conference on Frontier Physics 2012 International Conference on Frontier Physics 2012 5.1 Searches for Supersymmetry Signatures The most sensitive searches are the one for jets and missing energy and the ”razor” analysis, which exploits the kinematic conﬁgurations of the jets in the reconstructed reference frame of super-particle decay. Those searches are expected to produce much tighter limits on superparticle masses when per- EPJ Web of Conferences Fig. 7. Summary of results of SUSY searches by CMS using 2011 data, here shown for a representative choice of SUSY parameters (see legend in the upper right corner). Grey curves show iso-contours in the value of squark and gluino masses. Fig. 7. Summary of results of SUSY searches by CMS using 2011 data, here shown for a representative choice of SUSY parameters (see legend in the upper right corner). Grey curves show iso-contours in the value of squark and gluino masses. formed on the data from the full 2012 run, because of the larger statistics as well as the signiﬁcant increase of the production cross section for very massive objects. 5.2 Other Exotica Searches A number of exotic extensions of the standard model have been tested by CMS; in no case a departure from standard model predictions has been observed. For lack of space here we may only refer the interested reader to the public pages of the CMS experiment [66] for a comprehensive list of all results of exotica searches. 7 Acknowledgements We wish to congratulate our colleagues in the CERN accelerator departments for the excellent per- formance of the LHC machine. We thank the technical and administrative staﬀat CERN and other CMS institutes, and acknowledge support from: FMSR (Austria); FNRS and FWO (Belgium); Cap, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, Most, and NSFC (China); COLCIENCIAS (Colombia); MSES (Croatia); RPF (Cyprus); More, SF0690030s09 and ERDF (Es- tonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NKTH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); NRF and WCU (Korea); LAS (Lithuania); CINVESTAV, CONACYT, SEP, and UASLPFAI (Mexico); MSI (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Armenia, Belarus, Georgia, Ukraine, Uzbekistan); MON, Rosa tom, RAS and RFBR (Russia); MSTD (Serbia); MICINN and CPAN (Spain); Swiss Funding Agencies (Switzerland); NSC (Taipei); TUBITAK and TAEK (Turkey); STFC (United Kingdom); DOE and NSF (USA). References 1. 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https://openalex.org/W3203277111	https://www.banglajol.info/index.php/AAJFSS/article/download/55760/39080	English	null	Evaluation of pathogenicity of motile Aeromonas species in air-breathing catfish Magur (Clarias batrachus)	Asian-Australasian journal of food safety and security	2,017	cc-by	5,082	Asian Australas. J. Food Saf. Secur. 2017, 1 (1), 45-50 Asian Australas. J. Food Saf. Secur. 2017, 1 (1), 45-50 Asian-Australasian Journal of Food Safety and Security ISSN 2523-1073 (Print) 2523-2983 (Online) www.ebupress.com/journal/aajfss Received: 07 November 2017/Accepted: 20 November 2017/ Published: 21 November 2017 Received: 07 November 2017/Accepted: 20 November 2017/ Published: 21 November 2017 Abstract: The present study was carried out to evaluate the comparative capability of producing infections and causing mortality of the experimental Magur (Clarias batrachus) with motile Aeromonas species. A total of 200 apparently healthy C. batrachus were acclimatized to the laboratory conditions for experimental study. Nine different groups (each group consisting of 20 fish) of healthy C. batrachus was injected with nine motile Aeromonas isolates (A. hydrophila-3, A. sobria-3 and A. caviae-3). Experimental C. batrachus were infected with motile A. hydrophila, A. sobria and A. caviae to groups 1-3, 4-6 and 7-9, respectively while group 10 was injected with sterile physiological saline (0.85% NaCl) and served as the control. The selected motile bacterial species via intramuscularly were injected at the rate of 4.5 × 105 cfu/fish for pathogenicity study on C. batrachus and monitored up to two weeks. The highest clinical infections were noticed 90% in group-3 whereas only 35% in group-8 within the experimental period. After two weeks of the experiment, the cumulative mortality rate was also found highest (60%) in group-3 but lowest (15%) in group-9 while no infection or mortality showed in group-10 (control group). The development of infection and mortality to the injected C. batrachus was associated more severely by Aeromonas hydrophila than A. sobria and A. caviae used in this study. However, the isolates motile Aeromonas species could serve as the primary cause of skin lesions as well as mortality in cultured C. batrachus. Keywords: motile Aeromonas species; Magur (Clarias batrachus); pathogenicity; artificial infected Md. Shirajum Monir1, Nazneen Bagum1, S. M. Lutful Kabir2, Shuvho Chakra Borty2 and Mohammad Ashaf- Ud-Doulah3 Md. Shirajum Monir1, Nazneen Bagum1, S. M. Lutful Kabir2, Shuvho Chakra Borty2 and Mohammad Ashaf- Ud-Doulah3 1Bangladesh Fisheries Research Institute, Freshwater Station, Mymensingh-2201, Bangladesh 2Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh 3Bangladesh Fisheries Research Institute, Head Quarter, Mymensingh-2201, Bangladesh Corresponding author: Md. Shirajum Monir, Senior Scientific Officer, Bangladesh Fisheries Research Institute, Freshwater Station, Mymensingh-2201, Bangladesh. Phone: +0880 1721624623; E-mail: monir_bau22@yahoo.com Corresponding author: Md. Shirajum Monir, Senior Scientific Officer, Bangladesh Fisheries Research Institute, Freshwater Station, Mymensingh-2201, Bangladesh. Phone: +0880 1721624623; E-mail: monir_bau22@yahoo.com Asian Australas. J. Food Saf. Secur. 2017, 1 (1) Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 46 Catfish like Shing (H. fossilis), Magur (Clarias batrachus) and Pangasius (Pangasianodon hypophthalmus) are teleosts having entire body surfaces, fins and barbells those covered with skin composed with non-keratinized stratified squamous epithelial cells (Zhao et al., 2008; Esteban, 2012; Monir et al. 2016). Actually fish skin plays an important role with the environment to maintain homeostatic conditions including protection from external environment and antimicrobial activity (Bordas, 1996; Esteban, 2012). It provides the first attachment site for a wide range of microorganisms in aquatic environment (Bordas, 1996; Esteban, 2012). As a result, the attachment of microorganisms in skin frequently cause lesions and rupture which make possible for the pathogens to invade and multiply into the body. Thus, skin lesions and rupture causes harmful effect on normal growth and reproduction of the fishes as well as mass mortality (Monir et al., 2015). Intensification of aquaculture has increased the various disease outbreaks in different cultured fish in the past few decades. The motile Aeromonad species by far the most common among the bacterial diseases of freshwater fish that has been associated with diffrent species of the Aeromonas such as A. hydrophila, A. caviae, A. veronii, A. schuberti, A. salmonicida and A. sobria etc. However, A. hydrophila were documented as causative agents for large scale mortality in fish (Wahli et al., 2005). Additionally, the pathogenesis of fish diseases or disease outbreak can be medium to high in fishes (Yardimci and Aydin, 2011; Kumar et al., 2016) but all types of disease results in economic losses. The acute form of the disease may result in fatal sepsis without any symptoms (Yardimci and Aydin, 2011) while chronic infections may show the symptoms like hemorrhagic septicaemia with ulceration, inflammation, and dermal lesions (Cipriano et al., 2001). Despite the knowledge of severity and symptoms of the Aeromonad septicaemia in other species, factors such as host- pathogen interaction, temperature requirement of the bacteria, course of pathogenesis, and immunity to pathogenesis may vary for fish species (Wu et al., 2007). So far a very few experimental works were conducted on bacterial diseases especially on pathogenicity of Aeromonas species in Magur (C. batrachus). Therefore, the present study was carried out to evaluate the comparative pathogenicity of motile Aeromonas hydrophila, A. sobria and A. caviae in air-breathing catfish Magur (C. batrachus) of Bangladesh. Catfish like Shing (H. . Bacterial culture and preparation of suspension p p p The collected stocks of A. hydrophila, A. sobria and A. caviae were grown on Typtic Soya Agar (TSA) at 30 °C for 24 hours and identity confirmed using different biochemical characteristics prior to the experiment. The bacterial suspensions were prepared with 0.85% NaCl (physiological saline) that resulted in a concentration of 4.5 to 5.6 x 105 cfu/ml. Asian Australas. J. Food Saf. Secur. 2017, 1 (1) fossilis), Magur (Clarias batrachus) and Pangasius (Pangasianodon hypophthalmus) are teleosts having entire body surfaces, fins and barbells those covered with skin composed with non-keratinized stratified squamous epithelial cells (Zhao et al., 2008; Esteban, 2012; Monir et al. 2016). Actually fish skin plays an important role with the environment to maintain homeostatic conditions including protection from external environment and antimicrobial activity (Bordas, 1996; Esteban, 2012). It provides the first attachment site for a wide range of microorganisms in aquatic environment (Bordas, 1996; Esteban, 2012). As a result, the attachment of microorganisms in skin frequently cause lesions and rupture which make possible for the pathogens to invade and multiply into the body. Thus, skin lesions and rupture causes harmful effect on normal growth and reproduction of the fishes as well as mass mortality (Monir et al., 2015). g p y ( , ) Intensification of aquaculture has increased the various disease outbreaks in different cultured fish in the past few decades. The motile Aeromonad species by far the most common among the bacterial diseases of freshwater fish that has been associated with diffrent species of the Aeromonas such as A. hydrophila, A. caviae, A. veronii, A. schuberti, A. salmonicida and A. sobria etc. However, A. hydrophila were documented as causative agents for large scale mortality in fish (Wahli et al., 2005). Additionally, the pathogenesis of fish diseases or disease outbreak can be medium to high in fishes (Yardimci and Aydin, 2011; Kumar et al., 2016) but all types of disease results in economic losses. The acute form of the disease may result in fatal sepsis without any symptoms (Yardimci and Aydin, 2011) while chronic infections may show the symptoms like hemorrhagic septicaemia with ulceration, inflammation, and dermal lesions (Cipriano et al., 2001). Despite the knowledge of severity and symptoms of the Aeromonad septicaemia in other species, factors such as host- pathogen interaction, temperature requirement of the bacteria, course of pathogenesis, and immunity to pathogenesis may vary for fish species (Wu et al., 2007). So far a very few experimental works were conducted on bacterial diseases especially on pathogenicity of Aeromonas species in Magur (C. batrachus). Therefore, the present study was carried out to evaluate the comparative pathogenicity of motile Aeromonas hydrophila, A. sobria and A. caviae in air-breathing catfish Magur (C. batrachus) of Bangladesh. 2.1. Experimental fish and set up 2.1. Experimental fish and set up 2.1. Experimental fish and set up p p A total of 200 healthy Magur (C. batrachus) weighting 80-90 g were collected from local commercial fish farms of Mymensingh district located in 24o383N 90o164E of Bangladesh during this experiment. Prior to the artificial infection by selected motile Aeromonas spp., the collected fish were kept in aquariums to acclimatize in laboratory conditions from 28 to 30°C for at least 7 days providing adequate feed and better aeration by circulating water. The pathogenicity test was conducted at the Fish Disease and Health Management Laboratory of Bangladesh Fisheries Research Institute, Mymensingh. 2.2. Collection of motile Aeromonas species 2.2. Collection of motile Aeromonas species Motile Aeromonas hydrophila, A. sobria and A. caviae were isolated from infected Shing (Heteropneustes fossilis) showing severe disease symptom of erosions at the bases of fins and tail, hemorrhages and skin lesions on body surface (Monir et al., 2015; Monir et al., 2016). 1. Introduction 1. Introduction Among the different air-breathing catfishes, Magur (Clarias batrachus) is very popular and highly valuable fish species in Bangladesh. It is not only recognized for its delicious taste and market value but it is also considered as a medicinal fish and traditionally remained a strike among the pregnant & lactating mothers, the elderly and children. It is prescribed prophylactically to the anemic & malnourished individuals as well as for the convalescent of the patients due to the nutritional superiority (Debnath, 2011). It is a very hardy fish that can survive for quite a few hours outside the water due to presence of accessory respiratory organs. C. batrachus was abundantly available in open water of Bangladesh but presently, it is threatened due to over exploitation and various ecological changes in its natural habitat. Although, the appropriate breeding, nursing and rearing technology of fry and fingerlings of C. batrachus had been developed by Bangladesh Fisheries Research Institute (BFRI) in few years ago but various diseases of this fish causes huge economic losses because of their high mortality under farming conditions. Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 2.4. Experimental infection For the purpose of this study, intramuscular injection method was used for the experimental infection to know the efficacy of the selected motile Aeromonas spp. in initiating the infection as well as observe mortality. After acclimatization for 7 days, 80-90 g, apparently healthy Magur (C. batrachus) were randomly assigned to 10 groups as 20 fish per group. For the intramuscular (IM) injection, one ml insulin syringes (sterile and disposable) were used to inject intramuscularly with 0.1 ml of pre-selected (Ahmed, 2009) bacterial dose (4.5 × 105 cfu/fish) as follows: groups 1 to 3 - A. hydrophila, groups 4 to 6 - A. sobria, while groups 7 to 9 were infected with A. caviae. A negative control group-10 of 20 fish were injected with sterile physiological saline as above Monir et al., 2015). Prior to the experiment, one day before, feeding was stopped. During experiment fish were fed with commercial feed once in a day. Water was exchanged thrice in a week and residual feed was removed every two days by siphoning. The temperature, pH and dissolved oxygen, ammonia concentration of Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 47 the water was kept at acceptable limit during the experiment (Sofiq et al., 2013). The injected fishes were then observed clinical signs, symptoms and mortalities daily up to 14 days. the water was kept at acceptable limit during the experiment (Sofiq et al., 2013). The injected fishes were then observed clinical signs, symptoms and mortalities daily up to 14 days. 2.5. Re-isolation of challenged pathogens g p g Re-isolation of inoculated bacteria were carried out by collecting samples from skin lesions, kidney, liver of moribund and freshly dead or sacrificed experimental infected Magur (C. batrachus) fish and grown on TSA plates to check the presence and absence of bacterium. Positive bacterial culture was confirmed by the morphological and biochemical characteristics of the re-isolated bacteria were identical with those of the isolates used in the experimental infection. 2.6. Statistical analysis The data collected for rates of developing infection and mortality were subjected to descriptive statistics and expressed in percentages. 3.1. Clinical and gross pathology of experimental Magur (C. batrachus) However, the highest average infection was found 82% among the groups-1, 2 and 3 where Aeromonas hydrophila was used but the lowest average infection was noticed 42% in group-7, 8 and 9 during the experimental period where Aeromonas caviae was used (Table 1). Table 1. Cumulative progression of infection in experimental Magur (C. batarchus) infected with Aeromonas species. ulative progression of infection in experimental Magur (C. batarchus) infected with Table 1. Cumulative progression of infection in experimental Magur (C. batarchus) infected with Aeromonas species. Group Infected bacteria Number of healthy Magur used per group Development of infections at days after injection Percentage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 A. hydrophila 20 0 0 3 7 9 13 15 16 16 16 16 16 16 16 80 2 20 0 0 3 8 10 14 14 15 15 15 15 15 15 15 75 3 20 0 0 4 9 12 15 16 18 18 18 18 18 18 18 90 Average 0 0 3 8 10 14 15 16 16 16 16 16 16 16 82 4 A. sobria 20 0 0 2 5 7 9 12 14 14 14 14 14 14 14 70 5 20 0 0 1 3 5 8 10 11 11 11 11 11 11 11 55 6 20 0 0 1 3 3 6 8 12 12 12 12 12 12 12 60 Average 0 0 1 4 5 7 10 12 12 12 12 12 12 12 62 7 A. caviae 20 0 0 0 2 3 5 6 9 10 10 10 10 10 10 50 8 20 0 0 0 0 2 4 5 7 7 7 7 7 7 7 35 9 20 0 0 0 1 3 4 4 6 8 8 8 8 8 8 40 Average 0 0 0 1 2 4 5 7 8 8 8 8 8 8 42 10 Control 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 2. Cumulative progression of mortality rate in experimental Magur (C. batrachus) infected with Aeromonas species. Group Infected bacteria Number of healthy Magur used per group Mortality rate at days after injection Percentage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 A. 3.1. Clinical and gross pathology of experimental Magur (C. batrachus) g p gy p g ( ) It was observed that after one day post-infection of intramuscularly injected all Magur (C. batrachus) groups expressed abnormal movement, loss of balance and constant rubbing of body with the aquaria glass except control groups. By day 4 post-infection, most of the fishes were noticed to develop hemorrhages at the base and tips of the fins. The liver, spleen and kidney of the infected freshly dead fish were observed to be hemorrhage, enlarged, unsmooth, and turned slide blackish. After day 6 post-infection, severally diffused hemorrhage was observed on fin bases, edge of head and body surface in groups 1, 2, 3 (Figure 1). In some fishes, hyperaemic patches of the fins were also observed in these groups. The hemorrhagic ulcerative lesions, body, fins and tail erosions were noticed in groups 4, 5, 6, 7, 8, 9 (Figure 2) after day 6 post-infection. But corrosion of the barbells and severe hemorrhagic ulcerative lesions on the caudal area were also observed in most of the fishes especially in groups 7, 8, 9 (Figure 3). However, bacteria showed clinical signs in natural infection were found to be more or less similar in the injected experimental fish. But, no clinical signs and mortality were observed in the control group 10. Figure 1. Hemorrhages in fins bases, edge of head and skin lesions in Magur (C. batrachus) experimentally-infected with Aeromonas species. Figure 1. Hemorrhages in fins bases, edge of head and skin lesions in Magur (C. batrachus) experimentally-infected with Aeromonas species. Figure 2. Hemorrhagic ulcerative lesions on body in Magur (C. batrachus) experimentally-infected with Aeromonas species. Figure 2. Hemorrhagic ulcerative lesions on body in Magur (C. batrachus) experimentally-infected with Aeromonas species. Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 48 Figure 3. Corrosion skin lesions on caudal region in Magur (C. batrachus) experimentally-infected with Aeromonas species. Figure 3. Corrosion skin lesions on caudal region in Magur (C. batrachus) experimentally-infected with Aeromonas species. 3.2. Effect of the selected Aeromonas species to develop infections in experimental Magur (C. batrachus) After 14 days of the experiment, the highest clinical infection in the challenged fishes showed up to 90% in groups-3 but lowest 35% in group-8. Additionally, seventy percent of the challenged fish in group-4 had developed infection and only 40% in groups-9. 3.1. Clinical and gross pathology of experimental Magur (C. batrachus) hydrophila 20 0 0 0 2 4 6 7 8 9 9 9 9 9 9 45 2 20 0 0 0 1 3 5 6 7 7 7 7 7 7 7 35 3 20 0 0 2 3 4 7 8 10 12 12 12 12 12 12 60 Average 0 0 1 2 3 4 6 7 8 9 9 9 9 9 47 4 A. sobria 20 0 0 0 1 3 5 6 7 7 7 7 7 7 7 35 5 20 0 0 0 1 2 4 4 5 6 6 6 6 6 6 30 6 20 0 0 0 0 2 3 3 4 4 4 4 4 4 4 20 Average 0 0 0 1 2 4 4 5 5 5 5 5 5 5 28 7 A. caviae 20 0 0 0 0 1 2 2 3 3 4 4 4 4 4 20 8 20 0 0 0 0 1 2 2 2 4 4 4 4 4 4 20 9 20 0 0 0 0 0 1 1 2 2 3 3 3 3 3 15 Average 0 0 0 0 1 2 2 2 3 3 3 3 3 3 18 10 Control 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 1. Cumulative progression of infection in experimental Magur (C. batarchus) infected with Aeromonas species. Group Infected bacteria Number of healthy Magur used per group Development of infections at days after injection Percentage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 A. hydrophila 20 0 0 3 7 9 13 15 16 16 16 16 16 16 16 80 2 20 0 0 3 8 10 14 14 15 15 15 15 15 15 15 75 3 20 0 0 4 9 12 15 16 18 18 18 18 18 18 18 90 Average 0 0 3 8 10 14 15 16 16 16 16 16 16 16 82 4 A. 3.1. Clinical and gross pathology of experimental Magur (C. batrachus) sobria 20 0 0 2 5 7 9 12 14 14 14 14 14 14 14 70 5 20 0 0 1 3 5 8 10 11 11 11 11 11 11 11 55 6 20 0 0 1 3 3 6 8 12 12 12 12 12 12 12 60 Average 0 0 1 4 5 7 10 12 12 12 12 12 12 12 62 7 A. caviae 20 0 0 0 2 3 5 6 9 10 10 10 10 10 10 50 8 20 0 0 0 0 2 4 5 7 7 7 7 7 7 7 35 9 20 0 0 0 1 3 4 4 6 8 8 8 8 8 8 40 Average 0 0 0 1 2 4 5 7 8 8 8 8 8 8 42 10 Control 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 2. Cumulative progression of mortality rate in experimental Magur (C. batrachus) infected with Aeromonas species. Group Infected Number of Mortality rate at days after injection Table 2. Cumulative progression of mortality rate in experimental Magur (C. batrachus) infected with Aeromonas species. Group Infected bacteria Number of healthy Magur used per group Mortality rate at days after injection Percentage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 A. hydrophila 20 0 0 0 2 4 6 7 8 9 9 9 9 9 9 45 2 20 0 0 0 1 3 5 6 7 7 7 7 7 7 7 35 3 20 0 0 2 3 4 7 8 10 12 12 12 12 12 12 60 Average 0 0 1 2 3 4 6 7 8 9 9 9 9 9 47 4 A. sobria 20 0 0 0 1 3 5 6 7 7 7 7 7 7 7 35 5 20 0 0 0 1 2 4 4 5 6 6 6 6 6 6 30 6 20 0 0 0 0 2 3 3 4 4 4 4 4 4 4 20 Average 0 0 0 1 2 4 4 5 5 5 5 5 5 5 28 7 A. 4. Discussion In the present study, intramuscular injected all Magur (C. batrachus) groups expressed abnormal movement, loss of balance and constant rubbing of body with the aquaria glass and develop hemorrhages at the base and tips of the fins. The internal organs such as liver, spleen and kidney of the infected freshly dead fish were observed to be hemorrhage, enlarged, unsmooth, and turned slide blackish. The intramuscular inoculation of motile Aeromonas spp. produced similar signs and symptoms, reported during the disease progression in other fish species (Rashid et al., 2008; Kumar et al., 2016). Khalil and Mansour (1997) reported that clinical signs including weakness, slower movement, swimming closer to the surface, fin hemorrhages and red patches at the gut region were observed in the challenge fish with Aeromonas spp. Yarmidici and Aydin (2011) noticed body rubbing against tank walls in Nile tilapia infected with A. hydrophila isolate after 8 hours of infection. However, similar observations were also documented by Borty et al. (2016); Monir et al. (2015). y y ( ) ( ) The average highest percentage (82%) of infection in challenge fish were found among the groups-1, 2 and 3 indicates that A. hydrophila species used to infect the groups might be more pathogenic than the other used Aeromonas species in this experiment. Paniagua (1990), Janda (2010); Daood (2012) were observed that A. hydrophila is more pathogenic and the most frequently isolated species from naturally-infected fish than other Aeromonas species. The average percentage of infection in A sobria groups (28%) was higher than the A. caviae groups (15%) that suggests A sobria was more pathogenic compared to A. caviae in this experiment. However, these findings are in agreement with those reported by Daood (2012) and Monir et al. (2015). g g p y Pathogenicity of A. hydrophila to Magur (C. batrachus) by intramuscular injection was ranged from 35-60% mortality among the groups-1, 2 and 3. This mortality rate was higher than that recorded in the groups of A. sobria-injected (20-35%) and A. salmonicida-injected (15-20%) groups. This result indicates that the A. hydrophila strains used in this experiment were more pathogenic than the other Aeromonas species. Kumer et al. (2016) reported that the LD50 value of 1.74 × 105 cfu per 100 g of body weight was standardized for A. hydrophila in Golden Mahseer. Sarkar and Rashid (2002) noticed that 100% and 60-80% mortality in Shing (H. fossilis) and Magur (C. 5. Conclusions Motile Aeromonas species used in this study those were capable to develop infections as well as cause mortality in challenge Magur (C. batrachus). In the experiment, challenge through motile Aeromonas species will serve as a baseline data for further studies on Magur (C. batrachus). Also, the knowledge of various symptoms during pathogenesis help to understand the course of pathogenesis and overall immune response of fishes which can be very beneficial to control and prevent the disease outbreaks in farmed fishes. Further researches are necessary to prepare antibody against these bacteria strains to prepare vaccines and to try vaccination in susceptible to save catfishes against these pathogenic bacteria. 4. Discussion batrachus), Carps and Thai koi (Anabas testudineus) injected with 6.7x107 and 6.7x106 cfu/ml of A. hydrophila, successively. Mostafa et al. (2008) carried out an experimental infection of Shing (H. fossilis) with A. hydrophila by two different methods viz. intraperitoneal and intramuscular injection at a dose of 9.6 × 107 cfu/fish that caused in 100% mortality of the tested fish within 1-9 days. Additionally, Monir et al. (2015) found 100% mortality by 14 days of injection when Shing (H. fossilis) was challenged with 6.7 x 105 cfu/fish of A. hydrophila isolate, successively. However, the infection and mortality rates were found variation in this study than other studies might be due to different species of fish used in experiment, immunity of the fish, various strains of Aeromonas species, environmental factors, used different doses of the infective pathogens, route of administration as well as experimental duration. 3.1. Clinical and gross pathology of experimental Magur (C. batrachus) caviae 20 0 0 0 0 1 2 2 3 3 4 4 4 4 4 20 8 20 0 0 0 0 1 2 2 2 4 4 4 4 4 4 20 9 20 0 0 0 0 0 1 1 2 2 3 3 3 3 3 15 Average 0 0 0 0 1 2 2 2 3 3 3 3 3 3 18 10 Control 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 2. Cumulative progression of mortality rate in experimental Magur (C. batrachus) infec species. Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 49 3.3. Effect of the selected Aeromonas species on mortality of experimental Magur (C. batrachus) 3.3. Effect of the selected Aeromonas species on mortality of experimental Magur (C. batrachus) At the end of the experiment (after 14 days), a total of 2 severely infected fish were died in group-3 after 3 days of post infection whereas no fish was died in other groups. The cumulative mortality rate was recorded highest (60%) in group-3 but the lowest (15%) in group-9. However, the highest average mortality rate was found 47% among the groups-1, 2 and 3 where Aeromonas hydrophila was used but the lowest average mortality rate was noticed 18% in group-7, 8 and 9 during the experimental period where Aeromonas caviae was used (Table 2). Conflict of interest Conflict of interest None to declare. Acknowledgements The authors would like to cordial thanks and extend gratitude to all the staffs of the Fish Diseases and Fish Health Manamgemt Labortory, Bangladesh Fisheries Research Institute, Mymensingh, Bangladesh and the Department of Microbiology and Hygiene, Bangladesh Agricultural University to fulfill the experimental work successfully and also for providing continuous support and sincere cooperation. Asian Australas. J. Food Saf. Secur. 2017, 1 (1) 50 References Bordas MA, MC Balebona, I Zorrilla, JJ Borrego and MA Morinio, 1996. Kinetics of adhesion of selected fish- pathogenic Vibrio strains to skin mucus of gilt-head sea bream (Sparus aurata L.). Appl. Environ. Microbiol., 62: 3650-3654. Borty SC, F Rahman, AKM Ali Reza, S Khatun, ML Kabir, MH Rahman and MS Monir, 2016. Isolation, molecular identification and antibiotic susceptibility profile of Aeromonas hydrophila from cultured indigenous Koi (Anabus testudineus) of Bangladesh. Asian J. Med. Biol. 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https://openalex.org/W3014147413	https://lipidworld.biomedcentral.com/track/pdf/10.1186/s12944-020-01252-4	English	null	The benign c.344G > A: p.(Arg115His) variant in the LDLR gene interpreted from a pedigree-based genetic analysis of familial hypercholesterolemia	Lipids in health and disease	2,020	cc-by	3,945	SHORT REPORT Open Access Hori et al. Lipids in Health and Disease (2020) 19:62 https://doi.org/10.1186/s12944-020-01252-4 Hori et al. Lipids in Health and Disease (2020) 19:62 https://doi.org/10.1186/s12944-020-01252-4 The benign c.344G > A: p.(Arg115His) variant in the LDLR gene interpreted from a pedigree-based genetic analysis of familial hypercholesterolemia Mika Hori1, Atsushi Takahashi2, Cheol Son3, Masatsune Ogura1 and Mariko Harada-Shiba1 Abstract Background: We previously identified the c.344G > A: p.(Arg115His) variant in the low-density lipoprotein receptor (LDLR) gene, which was interpreted as “conflicting interpretations of pathogenicity” in ClinVar, based on a genetic analysis of patients with familial hypercholesterolemia (FH). However, whether this variant affects the pathophysiology of FH remains unclear. Therefore, our aim was to annotate the c.344G > A: p.(Arg115His) variant in the LDLR gene in FH. We present 2 families harboring the c.344G > A: p.(Arg115His) variant in the LDLR gene. Methods: Genetic analyses were performed for the coding regions and the exon-intron boundary sequence of the LDLR and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes in 2 FH families. Next, the family without pathogenic variants in the LDLR and PCSK9 genes was screened by whole-exome sequencing. Detailed clinical and biochemical data were gathered from family members. Results: In one family, the index case had biallelic c.1567G > A: p.(Val523Met) and c.344G > A: p.(Arg115His) variants in the LDLR gene, while the sibling had only the c.1567G > A: p.(Val523Met) variant in the LDLR gene. There was no difference in the FH phenotype between the siblings. In another family, the index case and the sibling had no pathogenic variants in the LDLR, PCSK9, and apolipoprotein B (APOB) genes, but the sibling’s wife with nonFH had the c.344G > A: p.(Arg115His) variant in the LDLR gene. The sibling and his wife had 4 children, including an unaffected child and an affected child who had the c.344G > A: p.(Arg115His) variant in the LDLR gene. In addition, the allele frequency of the c.344G > A: p.(Arg115His) variant (0.0023–0.0043) in Japanese and East Asian populations is relatively high compared with that of the other LDLR pathogenic variants (0.0001–0.0008). Conclusions: The c.344G > A: p.(Arg115His) variant in the LDLR gene is interpreted as benign in individuals with FH. Keywords: LDL receptor Familial hypercholesterolemia Variant Benign Annotation Conclusions: The c.344G > A: p.(Arg115His) variant in the LDLR gene is interpreted as benign in individuals with FH. K d LDL t F ili l h h l t l i V i t B i A t ti rds: LDL receptor, Familial hypercholesterolemia, Variant, Benign, Annotation * Correspondence: mihori@ncvc.go.jp; mshiba@ncvc.go.jp 1Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-Shimmachi, Suita, Osaka 564-8565, Japan Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: mihori@ncvc.go.jp; mshiba@ncvc.go.jp 1Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-Shimmachi, Suita, Osaka 564-8565, Japan Full list of author information is available at the end of the article DNA analysis Genomic DNA was extracted from whole blood from the sibling of Family 1 and the index case of Family 2 using an automated DNA extraction machine (QIA- symphony; QIAGEN, Valencia, CA) and from the other members of Family 2 using the Genome Extraction Kit (GENOMIX; Biologica, Nagoya, JAPAN) by SRL Inc. (Tokyo, JAPAN). The coding regions and exon-intron boundary sequences of the LDLR and PCSK9 genes were examined by Sanger sequencing as described previously [4]. For the index case of Family 2, multiplex ligation- dependent probe amplification (MLPA) was performed to detect large rearrangements of the LDLR gene using the P062B LDLR MLPA Kit (MRC Holland, Amsterdam, the Netherlands). Next, whole-exome sequencing was performed for Family 2. Exome libraries were prepared using the SureSelect Human All Exon V7 Kit (Agilent Technologies, Santa Clara, CA). Sequencing was per- formed by NovaSeq 6000 (Illumina, San Diego, CA) with 150 bp paired-end reads at RIKEN GENESIS CO., LTD. Clinical and laboratory data Serum total cholesterol (TC), triglycerides (TG), and high-density lipoprotein-cholesterol (HDL-C) levels were measured using enzymatic methods. LDL-C levels were calculated by the Friedewald formula or were measured using enzymatic methods. Achilles tendon thickness (ATT) was measured by X-ray. CAD was evaluated by the presence of myocardial infarction, angina pectoris, or coronary arteries with ≥75% stenosis by coronary angiography or electrocardiogram. In our FH cohort, patients were found to harbor the c.344G > A: p.(Arg115His) variant in the LDLR gene, which was interpreted as “conflicting interpretations of pathogenicity” in ClinVar. However, whether this variant affects the pathophysiology of FH remains to be eluci- dated. Herein, we present 2 families harboring the c.344G > A: p.(Arg115His) variant in the LDLR gene, in- cluding an index case with biallelic LDLR variants that comprised the c.344G > A: p.(Arg115His) and a patho- genic variant (Family 1) and nonFH patients with the c.344G > A: p.(Arg115His) variant (Family 2). © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hori et al. Lipids in Health and Disease (2020) 19:62 Page 2 of 5 Page 2 of 5 Hori et al. Lipids in Health and Disease (2020) 19:62 Methods The protocol of this study was approved by the Ethics Re- view Committee of the National Cerebral and Cardiovas- cular Center (M17–56 or M24–80). Each patient provided written informed consent to participate in the study. Case presentation Family 1 y The index case was a 19-year-old woman who had bial- lelic LDLR c.1567G > A: p.(Val523Met) and c.344G > A: p.(Arg115His) variants (Fig. 1, Table 1). She was referred to our lipid clinic with her brother at the age of 19 years for dyslipidemia, and her untreated LDL-C level was 208 mg/dL. She did not have ATT. Her medications in- cluded 2.5 mg of rosuvastatin, and her LDL-C level was 100 mg/dL on this medication regimen. Her sibling (II- 1) was a 21-year-old male who had the LDLR c.1567G > A: p.(Val523Met) variant. His untreated LDL-C level was 204 mg/dL at 21 years old. His ATT values were 6.7 and 9.1 mm. His medication included 2.5 mg of rosuvastatin, and his LDL-C level was 99 mg/dL under this medica- tion regimen. Their father was diagnosed with FH, but his lipid profile was unknown. Background Sequence reads were aligned to the human reference genome (hg19) using BWA-MEM. Single nucleotide var- iants and small indels were called with HaplotypeCaller of the Genome Analysis Tool Kit. The presence/absence of the c.344G > A:p.(Arg115His) variant in the LDLR gene was confirmed by Sanger sequencing. g Familial hypercholesterolemia (FH) is a disease that leads to a high risk of coronary artery disease (CAD) be- cause FH patients are exposed to high serum LDL- cholesterol (LDL-C) levels from birth. The prevalence of FH is 1 per 200–500 individuals in the general popula- tion [1]. FH is caused by mutations in the low-density lipoprotein receptor (LDLR) and in the related genes apolipoprotein B (APOB) and proprotein convertase sub- tilisin/kexin type 9 (PCSK9). More than 4970 variants in the LDLR gene, 580 variants in the APOB gene, and 350 variants in the PCSK9 gene are shown in ClinVar [2]. In Japan, pathogenic variants in the APOB gene have not been reported [3]. Whether some variants of the LDLR and PCSK9 genes affect the pathophysiology of FH re- mains unclear. Family 2 The index case was a 52-year-old man who had no patho- genic variants in the LDLR and PCSK9 genes (Fig. 1). He was referred to the lipid clinic at the age of 52 years for dyslipidemia (II-1). His untreated LDL-C level was 237 mg/dL, and his ATT values were 7.1 mm and 9.2 mm. He was diagnosed with hypertension at the age of 45 years. His medication regimen included 5 mg of rosuvastatin and 10 mg of ezetimibe, and his LDL-C level was 111 mg/ dL under this regimen. The lipid profiles of his parents were unknown. His father died of liver cancer at 65 years old, and his mother died at 64 years old due to unknown causes. The sibling (II-2) of the index case was a 49-year- old man who was diagnosed with FH. His medication regi- men included 2 mg of rosuvastatin, 10 mg of ezetimibe, and 2 g of omega-3 fatty acid ethyl, and his LDL-C level was 106 mg/dL under this regimen. The index case and Hori et al. Lipids in Health and Disease (2020) 19:62 Page 3 of 5 Hori et al. Lipids in Health and Disease (2020) 19:62 (2020) 19:62 Hori et al. Lipids in Health and Disease Page 3 of 5 Fig. 1 Two family pedigrees with the c.344G > A: p.(Arg115His) variant in the LDLR gene. Arrows show the index cases Fig. 1 Two family pedigrees with the c.344G > A: p.(Arg115His) variant in the LDLR gene. Family 2 The sibling and the wife had 4 children, of whom 3 sib- lings (III-1, III-2, and III-3) were diagnosed with nonFH and 1 sibling (III-4) was diagnosed with FH. The eldest daughter (III-1) and the youngest son (III-4) were hetero- zygous for the c.344G > A: p.(Arg115His) variant in the LDLR gene. The presence/absence of the c.344G > A: p.(Arg115His) variant in the LDLR gene was confirmed by Sanger sequencing. g p g The c.344G > A: p.(Arg115His) variant in the LDLR gene has been reported in several individuals of Asian eth- nicity [3, 8]. The allele frequency of the c.344G > A: p.(Arg115His) (0.029/0.0043) variant in the LDLR gene among the Japanese population was similar to that ex- pected based on the prevalence of heterozygous FH among the Japanese population (0.002–0.005). The allele frequency of the c.344G > A: p.(Arg115His) variant in the LDLR gene is relatively high compared with that of other LDLR pathogenic variants (0.0001–0.0008) in Japanese da- tabases. The LDLR gene is located on chromosome 19p13.1–13.3 and contains 18 exons, encoding a mature protein of 839 amino acids with a 21 amino acid signal peptide. The c.344G > A: p.(Arg115His) variant is located in the cysteine-rich, 40 amino acid repeat region of the binding domain of the LDLR, but the 115th Arg is not conserved among this region [9]. In addition, the Arg (pI = 10.76) to His (pI = 7.59) substitution does not change the positive charge of this sequence. For the c.344G > A: p.(Arg115His) variant, only one study exists; functional assay results revealed that receptor activities were 64% of normal in COS7 cells transfected with LDLR cDNA con- taining the c.344G > A: p.(Arg115His) variant [10]. How- ever, the genotype-phenotype correlation of the c.344G > A:p.(Arg115His) in the LDLR gene in pedigrees has not been demonstrated. Thus, based on 2 pedigree-based gen- etic analyses, the c.344G > A: p.(Arg115His) variant in the LDLR gene was classified as benign according to the guidelines issued by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Thus, examining the genotype-phenotype cor- relation in a pedigree is important for providing a genetic diagnosis with high accuracy. Allele frequency of the c.344G > A: p.(Arg115His) vari- ant in the LDLR gene in Japanese and East Asian populations. Family 2 Arrows show the index cases Table 1 Clinical and genetic characteristcs of the family with the c.344G > A: p.(Arg115His) variant in the LDLR gene Family 1 2 Case II-1 II-2 II-1 II-2 II-3 III-1 III-2 III-3 III-4 Clinical diagnosis FH FH FH FH nonFH nonFH nonFH nonFH FH Age (y) 22 19 52 49 48 21 19 16 9 Sex M F M M F F F M M TC (mg/dL) 280 286 350 – 198 167 181 157 214 TG (mg/dL) 42 58 239 137 262 77 53 71 153 HDL-C (mg/dL) 68 66 – 49 45 70 58 68 44 LDL-C (mg/dL) 204 208 237 236 101 82 112 75 139 Achilless tendon thickness Yes No Yes Yes No No No No No Xanthoma No No No No No No No No No Corneal Acrus No No Yes Yes No No No No No CAD No No No No No No No No No LDLR pathogenic variant c.1567G > A: p.(Val523Met) c.1567G > A: p.(Val523Met) None None None None None None None LDLR variant None c.344G > A: p.(Arg115His) None None c.344G > A: p.(Arg115His) c.344G > A: p.(Arg115His) None None c.344G > A: p.(Arg115His) PCSK9 pathogenic variants None None None None None None None None None CAD coronary artery disease Untreated serum lipid levels are shown linical and genetic characteristcs of the family with the c.344G > A: p.(Arg115His) variant in the LDLR gene 1 2 Page 4 of 5 Page 4 of 5 Hori et al. Lipids in Health and Disease (2020) 19:62 Hori et al. Lipids in Health and Disease c.344G > A: p.(Arg115His) variant in the LDLR gene was detected in 1 unaffected sibling and 1 affected sibling. The FH phenotype of the youngest son is suggested to be derived from his father harboring unknown pathogenic variants. the sibling’s family were screened by whole-exome se- quencing. We examined the coding regions and exon- intron boundary sequences in the LDLR, PCSK9, and APOB genes. The index case and the sibling had no patho- genic variants in the LDLR, PCSK9, and APOB genes, but the wife of the sibling (II-3) with nonFH had the c.344G > A: p.(Arg115His) variant in the LDLR gene. They had no loss-of-function mutations in the PCSK9 and APOB genes. Discussion We present 2 families harboring the c.344G > A: p.(Arg115His) variant in the LDLR gene. In the first fam- ily (Family 1), we identified an index case of biallelic LDLR variants that included c.1567G > A: p.(Val523Met) and c.344G > A: p.(Arg115His), and in the second family (Family 2), we identified that the wife of the index case’s sibling with FH, who was normolipidemic, had the LDLR c.344G > A: p.(Arg115His) variant. In Family 1, the index case had biallelic LDLR variants that included c.1567G > A: p.(Val523Met) and c.344G > A: p.(Arg115His), while the sibling had only the heterozygous c.1567G > A: p.(Val523Met) variant in the LDLR gene. The c.1567G > A: p.(Val523Met) variant is defined as pathogenic/likely pathogenic in ClinVar. FH patients harboring biallelic LDLR pathogenic variants generally show a much more severe phenotype than those harboring heterozygous LDLR pathogenic variants. However, the phenotype of the index case in Family 1 was identical to that of het- erozygous FH. In Family 2, the index case’s sibling with FH had 4 children, namely, 1 affected and 3 unaffected siblings. His wife with nonFH had the c.344G > A: p.(Arg115His) variant in the LDLR gene. A heterozygous In our cohort, we showed that unrelated patients har- boring no pathogenic variants in the LDLR and PCSK9 genes comprised approximately 40% of FH patients [4]. FH may be partly explained by the accumulation of com- mon SNPs [11]. A number of factors, including somatic genetic changes, environmental factors, and other genetic factors, might contribute to the pathogenesis and pheno- typic variations observed in FH. We are currently search- ing for new candidate FH genes that may be responsible for FH in Family 2. Finally, in vitro functional analyses are needed to quantify the actual effect of the c.344G > A: p.(Arg115His) variant in the LDLR gene. Family 2 The allele frequency of the c.344G > A: p.(Arg115His) variant in the LDLR gene in Japanese and East Asian pop- ulations was 0.0029, 0.0043, and 0.0023 according to the Japanese Human Genetic Variation Database (n = 1201; HGVD: http://www.genome.med.kyoto-u.ac.jp/SnpDB) [5], the Tohoku Medical Megabank Organization database (n = 3381; tommo_3.5KJPN) [6], and ExAC (n = 66,000, [7]), respectively. The allele frequency of the c.344G > A: p.(Arg115His) variant in the LDLR gene was relatively high compared with that of the other LDLR pathogenic variants (0.0001–0.0008) detected in these databases. Funding 11. Talmud PJ, Shah S, Whittall R, Futema M, Howard P, Cooper JA, et al. Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study. Lancet. 2013;381:1293–301. This work was partly supported by grants from the Ministry of Health, Labor and Welfare of Japan for Clinical Research on Intractable Diseases (H26-nanji- ippan-056, H30-nanji-ippan-003), JSPS KAKENHI Grant Number JP17K08681, the Japan Agency for Medical Research and Development (16ek0210075h0001), the Intramural Research Fund (28–2-2; 29–6-11) for Car- diovascular Diseases of the National Cerebral and Cardiovascular Center, the Japan Heart Foundation & Astellas Grant for Research on Atherosclerosis Up- date, the Takeda Science Foundation, the Kanae Foundation for the Promo- tion of Medical Science, Novartis Research Grants, and The Japanese Circulation Society. This work was partly supported by grants from the Ministry of Health, Labor and Welfare of Japan for Clinical Research on Intractable Diseases (H26-nanji- ippan-056, H30-nanji-ippan-003), JSPS KAKENHI Grant Number JP17K08681, the Japan Agency for Medical Research and Development (16ek0210075h0001), the Intramural Research Fund (28–2-2; 29–6-11) for Car- diovascular Diseases of the National Cerebral and Cardiovascular Center, the Japan Heart Foundation & Astellas Grant for Research on Atherosclerosis Up- date, the Takeda Science Foundation, the Kanae Foundation for the Promo- tion of Medical Science, Novartis Research Grants, and The Japanese Circulation Society. Ethics approval and consent to participate The protocol of this study was approved by the Ethics Review Committee of the National Cerebral and Cardiovascular Center (M17–56 or M24–80). Each patient provided written informed consent to participate in the study. Acknowledgments 6. Nagasaki M, Yasuda J, Katsuoka F, Nariai N, Kojima K, Kawai Y, et al. Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat Commun. 2015;6:8018. We thank Dr. Yoshihiro Miyamoto, Mr. Suguru Yamamoto, Mr. Naotaka Ohta, Mr. Hiroaki Masuda, and Ms. Rieko Isoda for DNA analysis and Ms. Rie Oishi for clerical support. We thank Dr. Yoshihiro Miyamoto, Mr. Suguru Yamamoto, Mr. Naotaka Ohta, Mr. Hiroaki Masuda, and Ms. Rieko Isoda for DNA analysis and Ms. Rie Oishi for clerical support. 7. Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016; 536:285–91. Abbreviations APOB A li Abbreviations APOB: Apolipoprotein B; ATT: Achilles tendon thickness; CAD: Coronary artery disease; FH: Familial hypercholesterolemia; HDL: High-density lipoprotein; HDL-C: HDL-cholesterol; LDL: Low-density lipoprotein; LDL-C: LDL-cholesterol; LDLR: LDL receptor; MLPA: Multiplex ligation-dependent probe amplification; PCSK9: Proprotein convertase subtilisin/kexin type 9; TC: Total cholesterol; TG: Triglycerides 4. Hori M, Ohta N, Takahashi A, Masuda H, Isoda R, Yamamoto S, et al. Impact of LDLR and PCSK9 pathogenic variants in Japanese heterozygous familial hypercholesterolemia patients. Atherosclerosis. 2019;289:101–8. 5. Higasa K, Miyake N, Yoshimura J, Okamura K, Niihori T, Saitsu H, et al. Human genetic variation database, a reference database of genetic variations in the Japanese population. J Hum Genet. 2016;61:547–53. 6. Nagasaki M, Yasuda J, Katsuoka F, Nariai N, Kojima K, Kawai Y, et al. Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat Commun. 2015;6:8018. Author details 1 1Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-Shimmachi, Suita, Osaka 564-8565, Japan. 2Department of Genomic Medicine, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-Shimmachi, Suita, Osaka 564-8565, Japan. 3Laboratory of Clinical Genetics, National Cerebral and Cardiovascular Center, 6-1 Kishibe-Shimmachi, Suita, Osaka 564-8565, Japan. Received: 18 November 2019 Accepted: 31 March 2020 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors’ contributions 8. Kim JH, Choi HK, Lee H, Park HY, Kim JH, Kim JW, et al. Novel and recurrent mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia. Mol Cells. 2004;18:63–70. M.H. collected the clinical data, and M.H., M.O. and M.H-S. contributed to the interpretation of the data. A.T. performed bioinformatic analysis in whole- exome sequencing. M.H. and C. S performed the genetic analysis by Sanger se- quencing. M.H. drafted the manuscript and contributed to the conception and design of the study. M.H-S. contributed to the study’s supervision. All authors gave final approval of the submitted version. 9. Sudhof TC, Goldstein JL, Brown MS, Russell DW. The LDL receptor gene: a mosaic of exons shared with different proteins. Science. 1985;228:815–22. 10. Chang JH, Pan JP, Tai DY, Huang AC, Li PH, Ho HL, et al. Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese. J Lipid Res. 2003;44:1850–8. Conclusions The c.344G > A: p.(Arg115His) variant in the LDLR gene was not shared by the sibling with FH in Family 1, and patients with nonFH also had the variant in Family 2. In Page 5 of 5 Page 5 of 5 Page 5 of 5 Hori et al. Lipids in Health and Disease (2020) 19:62 Hori et al. Lipids in Health and Disease conclusion, the c.344G > A: p.(Arg115His) variant in the LDLR gene is interpreted as benign based on pedigree- based genetic analysis. 2. Iacocca MA, Chora JR, Carrie A, Freiberger T, Leigh SE, Defesche JC, et al. ClinVar database of global familial hypercholesterolemia-associated DNA variants. Hum Mutat. 2018;39:1631–40. 3. Yu W, Nohara A, Higashikata T, Lu H, Inazu A, Mabuchi H. Molecular genetic analysis of familial hypercholesterolemia: spectrum and regional difference of LDL receptor gene mutations in Japanese population. Atherosclerosis. 2002;165:335–42. Availability of data and materials The data are not available because some data are being used by another study. Consent for publication All the participants provided written informed consent for the publication of the results of this study. Competing interests Competing interests The authors declare that they have no competing interests. 1. Mabuchi H, Nohara A, Noguchi T, Kobayashi J, Kawashiri MA, Tada H, et al. Molecular genetic epidemiology of homozygous familial hypercholesterolemia in the Hokuriku district of Japan. Atherosclerosis. 2011; 214:404–7. References 1. Mabuchi H, Nohara A, Noguchi T, Kobayashi J, Kawashiri MA, Tada H, et al. Molecular genetic epidemiology of homozygous familial hypercholesterolemia in the Hokuriku district of Japan. Atherosclerosis. 2011; 214:404–7.
https://openalex.org/W3126452548	https://www.e3s-conferences.org/10.1051/e3sconf/202123604043/pdf	English	null	Research on Modeling of Vocal State Duration Based on Spectrogram Analysis	E3S web of conferences	2,021	cc-by	3,458	Research on Modeling of Vocal State Duration Based on Spectrogram Analysis Xiaoyan Zhang1 1School of Preschool and Elementary Education of CWNU CWNU (China West Normal University), Nanchong, Sichuan, China Xiaoyan Zhang1 1School of Preschool and Elementary Education of CWNU CWNU (China West Normal University), Nanchong, Sichuan, China Abstract-In the early stage of vocal music education, students generally do not understand the structure of the human body, and have doubts about how to pronounce their voices scientifically. However, with the continuous development of computers, computer technology has become more and more developed, and computer processing speed has been greatly increased, which provides favorable conditions for the development of the application of vocal spectrum analysis technology in vocal music teaching. In this paper, we first study the GMM-SVM and DBN, and combine them to extract the deep Gaussian super vector DGS, and further construct the feature DGCS on the basis of DGS; then we study the convolutional neural network (CNN), which has achieved great success in the image recognition task in recent years, and design a CNN model to extract the deep fusion features of vocal music. The experimental simulations show that the CNN fusion-based speaker recognition system achieves very good results in terms of recognition rate. initiative in learning. The use of advanced spectral analysis technology, the usual abstract concepts of sound into computer graphics, so that students can be more vivid and concrete understanding of their own voice, breaking through the traditional "one-to-one" teaching mode, to achieve the "mouth - ear - nose! " a hybrid teaching model [6]. Sound visualization focuses on the use of graphics to plot the different frequencies and amplitudes of collected sounds, representing the interaction between the frequency component and the overall sound. The use of advanced computer technology allows the use of text, video, images, and sound to add a variety of fun and excitement to the classroom [7]. As a result, advanced computerized spectral analysis techniques are widely used in vocal music classrooms, and they have achieved a very good result. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202123604043 https://doi.org/10.1051/e3sconf/202123604043 E3S Web of Conferences 236, 04043 (2021) ICERSD 2020 * Corresponding author: 275809262@qq.com © 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/) y EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). 2.1 Associative Hyper-Vector-based Speaker Recognition System 1 1 ( , ) I J i i j j i j E v h a v b h       (2) (2) The traditional GMM-SVM speaker recognition system is shown in Figure 1. In the training phase, the feature parameters are extracted from the training speech material after the preemphasis and endpoint detection processes. Then, the feature parameters are input into the GMM to extract the super vector, and the super vector is used to train the classifier SVM; for the test phase, the test feature parameters are extracted after the same pre-processing process, and then they are used to extract the super vector. Finally, the test super-vector is used for pattern recognition with the trained SVM to obtain the result. where ω denotes the weight matrix, 𝑎 and b denote the thresholds for the visible and hidden layer units, respectively, and I and J are the number of visible and hidden layer neural units, respectively. From E(v) in the above equation, a joint probability distribution can be obtained. ( , ) E v h e q Z   (3) (3) where h is the normalization factor, which represents the sum of the energy values between the hidden layer and the visible layer cells. Further an independent distribution of ν can be obtained, which can also be called a likelihood function. In this chapter, MFCC is selected as the input eigenparameter of GMM because it better fits the auditory characteristics of human ear. The Gaussian mixture model is estimated by parameter λ (mixture weight, mean vector, and covariance), and then the appropriate M is chosen to characterize the phonetic personality. The vector m can be expressed by the following formula. Then the hypervector m can be expressed by: ( , ) ( , ) , ( ) E v h E v h v h e q v e     (4) (4) 1 2 . . i m m m m                  (1) The units in the same visible or hidden layer of the RBM are not connected to each other, so that the units in the hidden layer are independent of each other given the parameter ν for the visible layer. Given random h, one can obtain the probability of ν. 2.2 In-depth e-learning recognition system. It is shown that the number of layers in the CNN network has an important impact on the system performance. In order to make better use of the features between different layers, the CNN features in two different layers with better recognition rate are fused in this paper. The experimental simulation shows that the CNN fusion-based speaker recognition system achieves a good recognition rate. RBMs are energy-based and can learn an unknown distribution of data. Each RBM is a Markov random field with a two-layer structure, i.e., a visible layer and a hidden layer. While the traditional Boltzmann machine layers are fully connected to each other, the RBMs have no connections between the visible layer and the nodes in the visible layer and between the hidden layer and the nodes in the hidden layer; only the hidden layer is connected to the nodes in the visible layer, and this connection is bidirectional. Given the statistics of the visible layer, then the RBM energy function for the hidden and visible unit states can be expressed by the following equation. 1 INTRODUCTION Through the use of computers in the classroom, the organic unification of science and technology with school education can be fully reflected, which can effectively mobilize students' E3S Web of Conferences 236, 04043 (2021) ICERSD 2020 https://doi.org/10.1051/e3sconf/202123604043 1 INTRODUCTION The development of integrated technology has driven the development of semiconductor chips, each chip is able to store tens of thousands of transistors, allowing calculators and controllers to be concentrated on a single chip, which led to the emergence of the microprocessor, combined with large-scale, very large-scale integrated circuits to form the microcomputer, which is now a notebook and desktop computer [1]. In general, PPT is used in vocal music teaching, which not only ensures the quality of the classroom, but also enhances the entertainment of the classroom and achieves a good teaching effect [2]. In vocal music teaching, spectral analysis technology is widely used in vocal music teaching through computers [3]. g This paper provides a comprehensive introduction to the basics of speaker recognition, including the basic principles of speaker recognition, the feature extraction process, and the main recognition models. First, the extraction process of the speech feature parameters MFCC and LPCC is outlined in this paper. Then, the classical speaker recognition models, such as GMM, GMM-UBM general background model, support vector machine SVM, and deep neural network, are introduced in detail. According to the previous studies, these models have good performance in speaker recognition systems, and this thesis is also based on these models. Based on the superior performance of the fusion feature, this paper constructs a CNN fusion feature using a convolutional neural network. In this paper, the speech material is first converted into a speech map, and then the speech map is used as the input of the CNN to construct a speaker [ ] The rapid conversion of analog signals to digital signals using computers is a way for students to visualize their voices on a computer screen. The computer displays the time, frequency, and intensity of sound occurring from the computer screen primarily through three-dimensional images; the y-axis represents the frequency spectrum, and the darker the grayscale, the stronger the intensity, and the lighter the grayscale, the weaker the intensity [4]. The spectrum is a two-dimensional image, usually with curves and data to represent the frequency and intensity of the sound. The use of computer technology in the vocal music teaching process has significantly improved the teaching quality of vocal music education [5]. 2.1 Associative Hyper-Vector-based Speaker Recognition System (1) p y ( , ) ( , ) , ( / ) E v h E v h v h e q v h e      (5) (5) In a GMM-SVM-based speaker recognition system, the super vector can be seen as a refinement of the input feature parameters, which can not only effectively erase the text information but also highlight the speaker's identity and personality characteristics; the super vector is also a process of data downscaling, thus reducing the complexity of SVM processing to some extent. Because of the lack of connectivity between units in the same layer of the RBM, the probability of activation is also conditionally independent in two cases. 1 ( 1/ ) ( ) J i j q vi h a hw      (6) 1 ( 1/ ) ( ) J i j q hi v b vw      (7) (6) Figure 1. Vocal recognition based on traditional CMM-SVM. Figure 1. Vocal recognition based on traditional CMM-SVM. (7) where σ(𝑥) = 11 + e-𝑥 is the sigmoid activation function. Combining the two equations above, that is, given v, first the hidden layer state p can be calculated by v=1/h, and then by v can be computed for the visual layer state reconstructions. The error between the visual layer units and the reconstructed visual layer units is minimized by Figure 1. Vocal recognition based on traditional CMM-SVM. 2 2 E3S Web of Conferences 236, 04043 (2021) ICERSD 2020 https://doi.org/10.1051/e3sconf/202123604043 Figure 3 Experimental simulation results based on correlation super vector. 0 100 200 0.0 0.2 0.4 0.6 Rate (%) Correlation super vector this quasi-side, and the hidden layer can be seen as an alternative representation of the visual layer. To conclude, the hidden layer units are the result of feature extraction from the visible layer input units, and thus the structure of the RBM can achieve the goal of feature extraction, as shown in Figure 2. Figure 2. The relationship between hidden and visible layers. Emission frequency Voice mapping MD=（λ1+ λ2 + λ3 ）/3 RD=（λ2+ λ3 ）/2 λ1+ λ2 + λ3 V λ2+ λ3 Note: λ1>> λ2 >> λ3 Signal sensor Detector Mapping Figure 2. The relationship between hidden and visible layers. Figure 2. The relationship between hidden and visible layers. Figure 3 Experimental simulation results based on correlation super vector. 2.1 Associative Hyper-Vector-based Speaker Recognition System As with M, the Gaussian correlation number q takes the power of 2. It can be seen that the radial basis kernel function performs best under the four kernel function conditions and the polynomial kernel function performs worst. Based on the line graph of the radial basis kernel function, it can be seen that the lowest recognition rate is obtained when q=1, i.e., the original hypervector is the worst case. M is inversely related to p. As q increases, the number of samples in the mean vector decreases, but the number of dimensions per sample increases significantly. The increase in the number of dimensions leads to redundancy and introduces noise interference, so that q is too large and the recognition rate decreases. It can be concluded from the line graph that the recognition rate of systems with i > 1 is higher than i = 1, i.e., the performance of the associated super-vector is better than the original super-vector for any Gaussian number of correlations. 3 EXPERIMENTAL SIMULATION This thesis uses speech material recorded by the team under anechoic chamber conditions with 210 speakers, where the number of statements per speaker is 180 and the average duration of each statement is 3 seconds. In addition, the signal is sampled at 16k Hz and quantized at 16bit, and the frame length N is set to 256 before extracting the parameters. To analyze the results of the comparative experiments, 10 speakers are selected in all experiments and 80 statements are randomly selected from each speaker's speech material. 60 statements are used for training and 20 statements are used for testing. In this thesis, the initial input features of the speaker recognition system are all MFCCs, and it is shown that the original MFCC contains only static information about the speech, but in order to make the features more dynamic, the first order reciprocal of the MFCC can be obtained and spliced with the original MFCC. 3.1 Experimental Simulation of Associated Supervectors We can use the spectral software on the computer to display the sound graphically, as shown in Figure 5. (9) Next, standardize using the following formula. Next, standardize using the following formula. m m o o      (10) (10) Figure 5. Sound picturization changes For DBN models, increasing the number of hidden layers can reduce the network error, but the training time of the network increases and is accompanied by a tendency to overfit. So in order to find the most appropriate number of hidden layers, fix the number of nodes for all hidden layers (including bottlenecks) and change the number of hidden layers. In the case of two hidden layers, the first hidden layer is set as the bottleneck layer; in the case of 3, 4, and 5 hidden layers, the second layer is set as the bottleneck layer. The experimental results are shown in Figure 4. Figure 4. Number of nodes in the bottleneck layer. 0 20 40 60 80 100 3rd algorithm Signal amplitude (a.u.) Pixel value Face tracking Body tracking bottleneck laye 2nd 1st 0 20 40 60 80 100 3rd algorithm Signal amplitude (a.u.) Pixel value Face tracking Body tracking bottleneck laye 2nd 1st Figure 5. Sound picturization changes The use of computerized noise acoustics detection systems is the main embodiment of the use of sound spectral analysis technology in computers. The system is able to identify the spectral differences between artistic and non-artistic vocalizations, where the bandwidth of the artistic vocalization increases with the increase of the resonant peak energy and vice versa, and can effectively identify the differences between professional and amateur vocalists using resonant vocalizations. The detection system analyzed the vowel signals of 100 professional vocalists and analyzed the spectral characteristics of the spectra, which were mainly influenced by harmonics, resonance peaks, and noise components. Figure 4. Number of nodes in the bottleneck layer. 3.1 Experimental Simulation of Associated Supervectors In a deep learning model, if the problem to be solved is uncorrelated with the variation of the sample mean, the mean of the features should be zeroed to reduce the effect of the features on the DBN network model and reduce the correlation. In speech processing, the use of inverted spectral mean normalization can reduce the undesirable effects of channel distortion by subtracting the mean of the MFCC parameters from the statement. Global feature normalization scales the feature data in each dimension so that the feature vectors lie within a similar dynamic range. In speakers, global transformations are typically used to normalize the feature parameters to zero mean and unit variance. Given a training data set, one can compute the mean and standard for each dimensional feature i of it. In order to verify the validity of the correlation concept proposed in this chapter, an experimental simulation of a GMM-SVM system based on the correlation super vector is performed. The Gaussian mixed number M is a power of 2 (1, 2, 4, 8, 16,...). The choice of M has a great impact on the performance of the GMM. If M is too small, the GMM fits the training data poorly and the recognition rate is unsatisfactory; if M is too large, the model is difficult to converge and the parameter errors are large. The type of kernel function greatly affects the performance of the SVM classifier, however, there is no specific theory guiding which kernel function should be chosen in which case. Therefore, the experiments in this section simulate all four basic types of kernel functions. The effect of the Gaussian correlation number on the speaker recognition rate is shown in Figure 3. 1 M i m O M    (8) 1 M i m O M    (8) 1 M i m O M    (8) M M 3 3 https://doi.org/10.1051/e3sconf/202123604043 E3S Web of Conferences 236, 04043 (2021) ICERSD 2020 E3S Web of Conferences 236, 04043 (2021) 2 1 ( ) M i m o M      (9) remains multiplicative with the fundamental tone. The frequency of the second harmonic is twice the frequency of a harmonic, and the third harmonic is three times the frequency of a harmonic, and the frequency of the harmonic maintains a multiple relationship with the fundamental tone. 4 CONCLUSION By configuring the computer with a corresponding sound frequency analysis software, it is possible to systematically evaluate and test the singer's voice through this software. By analyzing each frequency component and intensity, the sound frequency analysis software shows the singer in the form of an image of the sound spectrum. In a two-dimensional image of a sound spectrum, the x-axis represents the frequency and the y- axis represents the amplitude, and the spectral lines vary in length in the graph. The longer the line length, the greater the amplitude of the corresponding frequency, and the shorter the line length, the smaller the amplitude of the corresponding frequency. The frequency of the fundamental tone is called the fundamental frequency represented by the first spectral line, also called the first harmonic, and so on after the first harmonic is called the second harmonic, the third harmonic... The frequency of the second harmonic is twice the frequency of the first harmonic, the third harmonic is three times the frequency of the first harmonic, and the frequency of the harmonic This paper provides a comprehensive introduction to the basics of speaker recognition, including the basic principles of speaker recognition, the feature extraction process, and the main recognition models. First, the extraction process of the speech feature parameters MFCC and LPCC is outlined in this paper. Then, the classical speaker recognition models, such as GMM, GMM-UBM general background model, support vector machine SVM, and deep neural network, are introduced in detail. According to the previous studies, these models have good performance in speaker recognition systems, and this thesis is also based on these models. Based on the superior performance of the fusion feature, this paper constructs a CNN fusion feature using a convolutional neural network. In this paper, the speaker speech material is converted into a speech spectrogram, and then the spectrogram is used as the input of CNN to construct a speaker recognition system. 4 https://doi.org/10.1051/e3sconf/202123604043 E3S Web of Conferences 236, 04043 (2021) ICERSD 2020 REFERENCES 1. Alzamendi, Gabriel A., and Gastón Schlotthauer. "Modeling and Joint Estimation of Glottal Source and Vocal Tract Filter by State-Space Methods." Biomedical Signal Processing and Control, vol. 37, April 2017, pp. 5-15. 2. Galindo, G. E., Peterson, S. D., Erath, B. D., Castro, C., Hillman, R. E., & Zañartu, M. "Modeling the Pathophysiology of Phonotraumatic Vocal Hyperfunction with a Triangular Glottal Model of the Vocal Folds." Journal of Speech Language and Hearing Research, vol. 60, no. 9, March 2017, pp. 2452-2471. 3. Yoshinaga, T., Hirtum, A. V., & Wada, S. "Multimodal Modeling and Validation of Simplified Vocal Tract Acoustics for Sibilant /S/." Journal of Sound and Vibration, vol. 411, June 2017, pp. 247- 259. 4. Gamba, M., Favaro, L., Araldi, A., Matteucci, V., Giacoma, C., & Friard, O. "Modeling Individual Vocal Differences in Group-Living Lemurs Using Vocal Tract Morphology." Current Zoology, vol. 63, no. 4, July 2017, pp. 467-475. 5. Zhang, J., Honda, K., & Wei, J. "Tooth Visualization in Vowel Production MR Images for Three- Dimensional Vocal Tract Modeling." Speech Communication, vol. 96, July 2018, pp. 37-48. 6. Speed, M., Murphy, D., & Howard, D. "Modeling the Vocal Tract Transfer Function Using a 3D Digital Waveguide Mesh." IEEE Transactions on Audio, Speech, and Language Processing, vol. 22, no. 2, March 2014, pp. 453-464. 7. Nasir, Baucom, B., Bryan, C., Narayanan, S., & Georgiou, P. "Modeling Vocal Entrainment in Conversational Speech Using Deep Unsupervised Learning." IEEE Transactions on Affective Computing, no. 1, May 2020, pp. 11-21. 5
https://openalex.org/W2726205870	https://jle.hse.ru/article/download/6815/7437	English	null	Assessment and Evaluation Techniques	Journal of language and education	2,017	cc-by	6,390	Valentina Shaforostova Correspondence concerning this article should be addressed to Valentina Shaforostova, National Research University Higher School of Economics, Malaya Pionerskaya, 12, Moscow, Russian Federation, 115054. E-mail: shafo@hse.ru Assessment and evaluation have always been important; they are linked to language teaching methodology, program outcomes, language teacher competencies, language standards and second language acquisition training. They can serve many different policies and can come in different forms. Assessment and evaluation have always been seen as the responsibility of the specialists, but they have rarely been included as a component in English language teacher (ELT) training. However, the ELT field has been experiencing a major shift in assessment and evaluation with effects on teachers, and learners around the world. It has also been influenced by a major questioning of traditional forms of testing and the underlying psychometric principles of measurement in ELT. Recent studies reveal that the reconceptualization of English language assessment and evaluation provides systematic information about student learning in relation to their performance and contributes to better understanding of their strengths and weaknesses. In many ways ELT has lagged behind the rest of education in the exploration of new theories and assessment and evaluation tools, including self-assessment. This research gap was generated partly because of the lack of integration with mainstream educational theory and practice in many areas of ELT, and partly because of powerful positions of traditional English language tests. The attempt to bridge this gap has lead to the research carried out. The aim of this article is to elaborate different assessment techniques that may better address student learning needs, improve student learning and engage students in self-assessment, including the sequence of steps that could lead to self-assessment. The study shows that the techniques implemented to develop self-assessment enable students to perform well. Keywords: evaluation, assessment, important, English language This article examines the importance of assessment and evaluation A major concern of English language teaching has been assessing and evaluating students’ progress during the course of study as well as their achievements at the end of it. The methodology of this paper is a qualitative approach using classroom activities and library sources as well as other related research in an attempt to improve students’ knowledge and learning. Assessment and evaluation also give teachers useful information on how to improve their teaching methods. Assessment and evaluation are very important parts of the constructive alignment process. Baranovskaya, T., & Shaforostova, V. (2017). Assessment and Evaluation Techniques. Journal of Language and Education, 3(2), 30-38. doi:10.17323/2411- 7390-2017-3-2-30-38 Baranovskaya, T., & Shaforostova, V. (2017). Assessment and Evaluation Techniques. Journal of Language and Education, 3(2), 30-38. doi:10.17323/2411- 7390-2017-3-2-30-38 National Research University Higher School of Economics Journal of Language & Education Volume 3, Issue 2, 2017 Correspondence concerning this article should be addressed to Tatiana Baranovskaya, National Research University Higher School of Economics, Malaya Pionerskaya, 12, Moscow, Russian Federation, 115054. E-mail: tbaranovskaya@hse.ru Correspondence concerning this article should be addressed to Tatiana Baranovskaya, National Research University Higher School of Economics, Malaya Pionerskaya, 12, Moscow, Russian Federation, 115054. E-mail: tbaranovskaya@hse.ru Keywords: evaluation, assessment, important, English language This article is published under the Creative Commons Attribution 4.0 International License. Tatiana Baranovskaya Higher School of Economics Research University Tatiana Baranovskaya Higher School of Economics Research University making. can give the lecturer a clearer sense of how the task is assessing mastery and what aspects are being assessed. Evaluation of the course or module, by students and lecturers should feed back into the whole process of curriculum alignment, as well as reflect critically and constructively on the outcomes, the teaching and learning activities, the assessment and the experience of the course or module. Reflexivity, continuous learning and development are key aims of successful evaluation. In classroom assessment, since teachers themselves develop, administer and analyse the questions, they are more likely to apply the results of the assessment to their own teaching. Therefore, it provides feedback on the effectiveness of instruction and gives students a measure of their progress. As Biggs (1999) maintains, two major functions can be pointed out for classroom assessment: one is to show whether or not learning has been successful, and the other one is to clarify the expectations teachers have of the students (Dunn et al., 2004). Through the use of appropriate classroom assessment strategies and techniques, teachers can increase their students’ motivation and show them how well they have learned the language. Evaluation goes beyond learners’ achievements and language assessment to consider all aspects of teaching and learning. Although the terms ‘assessment’ and ‘evaluation’ are often used interchangeably, they can be considered two parts of the same process. Assessment is the process of gathering evidence of what the child can do. Evaluation is the process that follows this collection of data, including analysis and reflection, as well as decisions based on the data. Assessment plays a number of roles in the life of a student, some of which they may be more aware of than others. It is widely accepted that students’ learning patterns, educational focus, and allocation of time will be directly influenced by assessment. It does more than allocate a grade or degree classification to students – assessment plays an important role in focusing their attention and, as Sainsbury & Walker (2007) observe, actually drives their learning. Gibbs (2003) states that assessment has 6 main functions: 1. Capturing student time and attention; 2. Generating appropriate student learning activity; This paper will present some useful assessment and evaluation techniques that can assist language teachers to create a dynamic classroom situation for evaluation. Materials and Methods He states that, with the exception of the last two points, these functions should occur as frequently as possible to support effective learning. making. It will show that the quality of the assessment and evaluation in the educational process has a profound link to students’ performance and can engage them in self-assessment which is most important in English language teaching. 3. Providing timely feedback which students pay attention to; 4. Helping students to internalise the discipline’s standards and notions of equality; 5. Generating marks or grades which distinguish among students or enable pass/fail decisions to be made; 6. Providing evidence enables them to judge the appropriateness of course standards. Valentina Shaforostova Well- designed assessments can allow students to use the knowledge and skills they have learnt and indicate their level of mastery. The feedback on the assessment will also provide students with clear information on the criteria they need to succeed at assigned tasks, 30 TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA It, therefore, gives lecturers valuable insights into how they teach and how effectively instruction has been taken up by the students. Assessment is perhaps one of the most important elements of curriculum design and alignment, because this is where it is possible to see if students can demonstrate mastery in terms of the knowledge and skills they need to have learnt. Assessment, in a constructively aligned curriculum, must speak to the outcomes listed for the course. It must draw in both the knowledge and the practical and intellectual skills and competencies that students have been taught and that they have practiced in lectures and tutorials. Assessment activities must test what has been learnt and taught, and should not be constructed so as to be ambiguous or inexplicit. One of the most important issues in evaluation is timing. Teachers can use quick exercises to check in with students during the course, at the end of a topic or after an assignment has been completed. Longer and more detailed evaluation for the end of a course can also be created. Students who did the course last year can be asked to complete a retrospective evaluation. The important thing to consider when thinking about the timing is the purpose of the evaluation (what do students need to know and why), and what teachers plan to do with the information students give them. Assessment tasks can be formative and summative. The former give students opportunities to make errors and get constructive, guiding feedback used to develop competency and understanding in further assessments and teaching and learning. Formative quizzes, essays that can be drafted and revised, and short written or verbal tasks that receive detailed feedback are examples of formative assessments. They are opportunities for the students to demonstrate mastery or competence in a particular area or across several areas that have been studied. The feedback is usually less detailed and aimed more at providing a summary of what they have and have not yet mastered. Examinations, some kinds p g Gensee and Upshur (1996) state that classroom assessment and evaluation is concerned primarily with improving instruction in order to help enhance students’ learning. Teachers in any educational system are actively and continuously involved in assessment and evaluation. Students can also be active participants in assessing their own achievements and in planning how they will study and learn a second language, i.e. TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA of tests and theses or dissertations are examples of summative assessments. student achievement and language assessment to consider all aspects of teaching and learning and to look at how educational decisions can be made on the basis of alternative forms of assessment. Gensee (cited in Carter & Nunan, 2001) believes that another purpose of evaluation is to guide classroom instruction and enhance student learning on a day-to-day basis. Cl d l i Feedback is a very important part of the assessment process, both formative and summative. Through receiving focused, relevant and guiding feedback, students are able to understand where their strengths and weaknesses are, and where they still need to concentrate their efforts in terms of their own learning. Through giving feedback, lecturers and tutors are better able to make similar assessments of strengths and weaknesses for students. This can enable more responsive teaching and tutoring to address gaps and weaknesses where necessary. It can also provide a better understanding of how students are responding to the methods and styles of teaching and tutoring. It can further show how deeply and accurately the students grasp and understand the relevant knowledge and employ the related skills and practices to explore and demonstrate their knowledge. Classroom assessment and evaluation concerns: • Suitability of general instructional goals and objectives associated with an individual lesson or unit plans; • Effectiveness of instructional methods, materials and activities used to attain instructional objectives; • Adequacy of professional resources required to deliver instruction. Classroom assessment and evaluation under the active management of teachers can also serve important professional development purposes since the information resulting from such evaluations provides teachers with valuable feedback about their instructional effectiveness that they can use to develop and improve their professional skills. As part of reflective teaching movement, teachers are encouraged to conduct research in their own classrooms (Nunan, 1989b; Allwright & Baily, 1991; Richards & Lockhart, 1994); classroom assessment and evaluation is an important part of such research. Evaluation is an important part of an aligned curriculum and an overall teaching and learning strategy because it is a part of the feedback and development cycle. It should be a part of any responsive and up-to-date teaching and learning strategy or plan. Evaluation gives students opportunities to speak to the lecturer about their experiences and impressions of the course content and the pedagogical approaches that have been used. Theoretical Background The present study focuses upon the qualitative approach of English language learning assessment and evaluation process in the educational system. Evaluation in teaching the English language is a process of collecting, analysing and interpreting information about teaching and learning in order to make informed decisions that enhance student achievement and the success of educational programs (Rea-Dickens & Germanie, 1993; Genesee & Upshur, 1996; O’Mally & Valdez-Pierce, 1996). Evaluation is a process that includes five basic components: The purpose of classroom assessment and evaluation is to give students the opportunity to show what they have learned rather than catching them out or to show what they have not learned. Needless to say, evaluation and assessment can focus on different aspects of teaching and learning: respectively, textbooks and instructional materials, student achievement, and whole programs of instruction. It is important to clarify the distinction between evaluation and assessment. These terms are often used interchangeably and are, in fact, related, but they are technically different. Assessment of an individual student’s progress or achievement is an important component of evaluation: it is that part of evaluation that includes the measurement and analysis of information about student learning. The primary focus of assessment in English Language Teaching has been language assessment and the role of tests in assessing students’ language skills. Evaluation goes beyond • Articulating the purpose of the educational system; • Identifying and collecting relevant information; • Having ideas that are valuable and useful to learners in their lives and professions; • Analysing and interpreting information for learners; • Classroom management or classroom decision 31 ASSESSMENT AND EVALUATION TECHNIQUES ASSESSMENT AND EVALUATION TECHNIQUES Figure 1. The context of classroom assessment and evaluation. 1 Input Factors ↓ 2 Student needs and abilities →→→ 3 Instructional purposes 10 Assessment Evaluation ↓ ↓ ↑ 4 Time, Attitudes, Resources, Facilities, Support →→→ 5 Instructional Plans 9 Redesigned Restaffed ↓ ↓ ↑ 6 Teacher Abilities →→→ 7 Instructional practices →→→ 8 Output Result learners’ attitudes or social behaviour that result from classroom instruction (e.g. changes in attitudes toward the target language, the target language group, or the learner’s first language group). However, in most cases these objectives are secondary to language learning objectives. Gensee (1996) deals with philosophical objectives as changes in attitudes, values, or beliefs of a more general nature than those associated with socio- affective objectives. And, finally, method or process objectives refer to methods, processes, experiences, materials, activities, or other aspects of instruction. Nevertheless, Gensee and Upshur (1996) state that the influence of these objectives is not equally useful for classroom instruction. They believe philosophical objectives, for example, are minimally useful. Strategic objectives help in understanding students’ performances in class, thus, play an important role in instructional planning. They are, however, secondary to language acquisition; in other words, the effective deployment of certain strategies should lead to enhanced second language attainment and usage. Clearly, language objectives are fundamental to second language evaluation. 3 Instructional purposes Figure 1. The context of classroom assessment and evaluation. Any instruction consists of three components: first, the purposes identify the objectives of instruction – the “WHY”; second, the plans describe the means of attaining those objectives – the “HOW”; third, practice reveals what actually takes place in the classroom – the “WHAT”. Gensee and Upshur (1996) also discuss other factors, which are not part of classroom instruction itself, but can have a significant effect on second language teaching and learning. They refer to these additional factors as “input factors.” Thus, it can be said that classroom assessment and instruction have four aspects, namely: purposes, plans, practices, and input factors. Gensee and Upshur (1996) argue that evaluation and assessment involve comparison. More specifically, decisions that result from assessment are arrived at by making comparisons. They claim that in order to evaluate and assess, it is necessary to understand the factors that influence student performance in class. This means going beyond the assessment of just achievement. TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA they can be engaged in the early stages of the process of self-assessment. The context of classroom assessment and evaluation is summarized by Gensee and Upshur (1996) in the following figure: 32 TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA Table 1 Assessment results of the first group (43 students) Number of students Score (60) 4 55-58 5 53 4 50 9 45-49 9 41-44 5 37-40 3 32-35 2 28-30 2 27 Table 2 Assessment results of the second group (47 students) Number of students Score (60) 5 55-57 6 52-54 5 51 10 47-49 8 41-45 4 38-40 3 34-36 4 29-32 2 26-27 Table 1 Assessment results of the first group (43 students) Number of students Score (60) 4 55-58 5 53 4 50 9 45-49 9 41-44 5 37-40 3 32-35 2 28-30 2 27 Table 1 Assessment results of the first group (43 students) This article includes analyses of the evaluation and assessment tools carried out by teachers at the National Research University Higher School of Economics with fourth-year students in the Department of Public Administration. The main idea of the experiment was to develop students’ ability to assess their own speaking skills. The expected skills of fourth-year students included: specialized knowledge and experience in conveying ideas and information clearly and in a well-organized manner; ability to give presentations; effective communication skills. To acquire these necessary skills in order to confidently and effectively interact in speaking situations, students should learn how to plan, organise and present information on a variety of topics. They should be able to give formal presentations at conferences as well as talk to experts, consultants, visiting researchers, etc. Table 2 Table 2 Assessment results of the second group (47 students) Number of students Score (60) 5 55-57 6 52-54 5 51 10 47-49 8 41-45 4 38-40 3 34-36 4 29-32 2 26-27 Focusing on this primary task, the authors conducted an experiment on developing, improving, mastering and assessing oral presentation skills among the fourth-year students. At the end of the course the students were supposed to give presentations to accompany the formal written paper, i.e. a project proposal. The 90 students who took part in the experiment were split into six smaller groups: three of these groups were organised as Group 1; the other three groups comprised Group 2. Both groups were given instruction on oral presentation skills. TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA But while the first group were given specific instruction about how they would be assessed and were shown the evaluation criteria for oral presentations, the second group received no explicit information regarding evaluation; the input they received was based solely on the fourth-year teaching materials (Kuzmenkova, 2011). ASSESSMENT AND EVALUATION TECHNIQUES Chastain (1988) believes that teachers need to constantly evaluate their teaching on the basis of student reaction, interest, motivation, preparation, participation, perseverance, and achievement. The conclusions drawn from such ongoing evaluation constitute their main source for measuring the effectiveness of selected learning activities. Instructional objectives are identified as the goals that a teacher sets while teaching. On the one hand, they provide direction for planning appropriate instruction and, on the other hand, they provide a basis for determining whether a student has achieved what a teacher has set out to accomplish. They provide criteria for assessing the outcomes of students’ learning and monitoring their performance. Different kinds of objectives can guide classroom instruction: 1) language, 2) strategic, 3) socio-affective, 4) philosophical, and 5) method or process. Evaluation of achievement is the feedback that makes improvement possible. By means of evaluation, strengths and weaknesses are identified. Evaluation, in this sense, is another aspect of learning. It enables learners to grasp what they missed previously and helps the teacher to comprehend what can be done in subsequent lessons to improve learning. To do so, alternative methods (e.g. dialogue journals, portfolio conferences, interviews and questionnaires, observation, etc.) are available for collecting useful information about language learning and about student-related factors that influence the processes of language teaching and learning. It is widely accepted that the assessment/evaluation process involves the use of multiple sources of information collected in a variety of contexts. At the primary level, many teachers use observation, work samples, and questionnaires as tools in the process of assessment and evaluation. Language objectives refer to language skills that learners are expected to acquire in the classroom. Objectives that are concerned with strategies for communicating, learning, and critical thinking are referred to as “strategic objectives”. Learning process refers to a “conscious processes and techniques that facilitate the comprehension, acquisition, and retention of new skills and concepts” (Chamot & O’Malley, 1989). According to Chamot and O’Malley, they may include metacognitive strategies (such as selective attention), cognitive strategies (such as summarizing and elaboration), or socio-affective strategies (such as questioning for clarification). Methodology Socio-affective objectives refer to changes in Socio-affective objectives refer to changes in Methodology 33 Concluding a Presentation • What are its current and future applications or uses? • What needs does the particular development meet? To make a strong impression on listeners, the conclusion of the presentation should be brief and to the point of the talk. For example, when presenting conclusions, students are instructed that it is not the time and place to introduce new ideas, but to remind the audience of what has already been presented by reviewing the main points and emphasising the major issues. The listeners are prepared for the end of the talk through some signalling strategies for concluding a presentation; for example, “And now let me quickly review the main points (advantages, reasons, effects, types) of _________.” • What, if any, are the problems associated with it? B. Discuss a research project that you have carried out: • the purpose of the research; • what you did; • when and where you conducted the research; • significant results/conclusions/ recommendations; • when and where you conducted the research; • significant results/conclusions/ recommendations; C. Imagine that you are speaking to some students who are interested in majoring in your particular field of study. Conduct a discussion on different job opportunities in this field. Having identified the main features of conclusion, the teacher assigned the tasks for writing conclusions using the same topics that were given as the examples for writing an introduction. Students worked individually to prepare a one- or two-minute conclusion to a presentation they selected. When everyone finished, they started to work in small groups, taking turns presenting conclusions to the group. Within the small groups, strengths and weaknesses were discussed and then results were reported to the whole group. Introducing a Presentation To assess students’ level of English language competence at the beginning of the course, both groups were given Objective Placement Test, Variant 1 CUP & FLTRP, 2010 (consisting of 60 multiple-choice questions divided into three sections Language Use (40 items), Reading (10 items), Listening (10 items)). In Group 1, information shared on how to introduce a presentation and make it effective was provided by the teacher’s input and the students were asked to select a problem that they felt deserved the special attention of the class. After that, they worked individually to prepare a one- to two-minute introduction for a presentation on the topic. When the task was completed, the students worked in small groups and they then took turns presenting their introductions to the group. Once all the introductions were presented, the strengths and weaknesses of each were discussed. Before the discussion, the instructor assigned different students to fill out the evaluation form and consider the following questions in analysing both positive and negative sides of each introduction. ( ) g ( ) g ( )) The results achieved of the first group of 43 students are presented in Table 1. The second group of 47 students (whose results were rather close to the first group) results are presented in Table 2. The pre-test showed that the level of the English language competence was practically equal in both groups. To accomplish the objective of the study, the authors attempted to verify the role of continuous evaluation of different stages on their ability to master the special skills associated with giving presentations. With this task in mind, the authors conducted research with the first group of 43 students. The essence of the study was to evaluate each part of the presentation, which was to be introduced during the academic course. 1. How did the speaker attract listeners’ interest and focus their attention on the topic? 2. What was the central idea of the presentation? Was it clearly stated? y 3. What preview did the speaker give of the 34 ASSESSMENT AND EVALUATION TECHNIQUES 5. What do your peers want or need to know about this subject? presentation organisation? 5. What do your peers want or need to know about this subject? 4. How did the speaker plan to handle questions from the audience? After that, the teacher wanted the students to write down two topics from their field of the research that they thought would be suitable for a five- to ten-minute presentation to be given to students in the class. The instructor collected these topics, listed them on the board and asked students to work in small groups. The students in groups analysed each topic, considered whether it was too limited, too general, too technical or too well-known for the audience. Moreover, if the students found the topic unsatisfactory, they revised it to make it adequate for delivering a precise message. 5. Can you offer any suggestions for improving the introduction? 5. Can you offer any suggestions for improving the introduction? After that the listeners carefully studied the assessment criteria and justified the grade allocated. Eventually the students were given a list of suggested topics (in alphabetical order) and optional guidelines: A. Discuss a recent development or innovation in your field A. Discuss a recent development or innovation in your field y f Guidelines: Guidelines: Guidelines: • How was it developed? Organising Information For example, the chosen subject ‘International Cooperation’, could be developed in a variety of ways: a) the history of creation; b) the importance of international cooperation to avoid dangers, solve problems; c) working together with the UN and other organisations to deal with international problems; d) priorities of international cooperation: a. the environment b. economic cooperation c. regional infrastructure d. the indigenous population e. social aspects f. cross-border cooperation, etc. To build up several different topics with a clear central idea, the teacher asked the students to work in small groups. Each group was given the list of general subjects: Further, the teacher asked the students to work in small groups to determine which pattern of organisation would work best with the general subjects suggested earlier. When the students finished the task, they discussed the results in their groups. After giving some time for comparing the patterns, the teacher invited one person from each group to present the outline of their topics on the board. The audience made comments, pointed to positive and negative sides and improved the imperfect ones. • Public Administration • Civil Service • Procurement • Bureaucracy • Corruption • Budgeting • E-Government • Knowledge Management • Public-Service Motivation • Public Administration • Civil Service • Procurement • Bureaucracy • Corruption • Budgeting • E-Government • Knowledge Management • Public-Service Motivation • Public Administration • Civil Service • Procurement • E-Government • Knowledge Management Determining Content Inevitably, at some point in preparing for a presentation, students began to be concerned about determining the content, that is, what specific information to include. Students were introduced to the structure and teachers emphasised that the focus of any informative presentation should be to communicate useful information in an explicit way. For the fourth- year students, the topic chosen for presentation was related to their studies or research projects. Once the subject issue was chosen, the topic would be limited in order to cover the information adequately within the time available for the presentation. When students selected the topic for their presentations they also considered the following points: After studying the information on determining the content and preparing the conclusion of the presentation, students prepared a four- or five-minute talk to give to a group or to the entire class, taking into account the following guidelines: • making an outline of the points to be presented (avoiding writing out every word of the presentation) • making sure the points were put in a logical order • planning the introduction and conclusion 1. Do you have enough time and resources to conduct the necessary research? • making up a short list of any specialised or technical terms, etc. 2. Have you narrowed the topic enough to cover it adequately within the time limits? The teacher assigned some students the task of evaluating the presentations. These listeners considered the following questions in analysing strengths and weaknesses of the content presented: 3. Is the topic of potential interest to your listeners? 1. What kind of details, examples or facts related to the topic did the speaker include? 4. Is the topic too easy/too difficult or too technical for the audience? 35 Using Transitions • Public-Service Motivation TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA 2. Did the speaker use the appropriate vocabulary? way to understand and remember. Some of the most commonly used patterns of organisation are: (a) topical, (b) chronological, (c) spatial, (d) problem-solution, (e) cause and effect, (f) comparison/contrast (Matthews & Marino, 1990). For example, in comparison/contrast pattern, there are two basic ways to follow when two things are compared or contrasted: A-B and point-by- point. In the first type, the two things to be compared are discussed in turn to give a general picture of the comparison by focusing first on A then on B. While in the second type, the point-by-point approach, specific details are emphasised, alternating between A and B. The plan for this pattern is given in a Table 3. 3. Was it the right level for these particular listeners in terms of understanding? 4. Was the information too simple or too complex for the audience? 5. Did the presentation meet the time requirements? They also examined the positive and negative sides of the conclusion focusing on the following questions: 1. Did the speaker use a fixed phrase to lead into the conclusion? What was it? 2. Was there a summary of the main points of the presentation? 3. Did the presenter highlight the major issues? Table 3 Two solutions to a problem 4. How did the speaker elicit questions from the audience? 5. If the conclusion did not meet the format how could it be improved? To compare two solutions to a problem To compare two solutions to a problem A-B type Point-by-point type I. Solution 1 I. Cost A. Cost A. Solution 1 B. Practicality B. Solution 2 C. Side effects II. Practicality D. Disadvantages A. Solution 1 E. Advantages B. Solution 2 II. Solution 2 III. Side effects A. Cost A. Solution 1 B. Practicality B. Solution 2 C. Side effects IY. Disadvantages D. Disadvantages A. Solution 1 E. Advantages B. Solution 1 Y. Advantages A. Solution 1 B. Solution 1 According to the teacher’s instructions, the listeners then reported the results to the speakers and the rest of the class, and finally discussed them. In terms of organising information, determining the central idea explains exactly what aspect of the topic is to be covered. Thus, the central idea controls what is included in the presentation and also determines the arrangement of the main points. Organising Information Table 4 Table 4 Table 4 Post-test scores of the first group Number of students (43) Score (60) 7 60 6 57-58 5 53-55 7 48-50 7 45-47 5 41-43 3 38-40 3 33-36 Post-test scores of the first group ASSESSMENT AND EVALUATION TECHNIQUES Table 5 Post-test scores of the second group Number of students (47) Score (60) 5 56-58 6 53-55 5 49-50 8 48-49 8 42-46 6 39-41 4 35-37 5 30-33 Table 5 Post-test scores of the second group transitions effectively, moving from point to point and connecting different parts of the talk. When individual work came to an end, the teacher instructed the students to work in groups, taking turns giving their presentations. After each speaker had finished, the discussion of strengths and weaknesses was initiated. Having practiced in groups, some of the students were asked by the teacher to give presentations to the entire class. Meanwhile the teacher also assigned some students to do the following listening task: 1. What was the central idea of the presentation? 2. What pattern of organisation did the speaker use? 3. What were the main points presented by the speaker? • Crowdfunding • Crowdfunding In order for the listeners to understand the relationship of the ideas and to show them how the pieces of information fit together into a logical pattern, transitions need to be used. The students were given the task to work individually, planning their presentations with the focus on outlining and using Working in a group, the students compared their topics, reported their results and finally selected those that sounded most relevant to the subject. The teacher emphasised that the central idea is the main body of the presentation. It consists of key points that need to be arranged for the audience in an easy 36 Discussion 4. Were the main points presented in a logical way? 5. How well-connected were the different parts and ideas of the presentation? 5. How well-connected were the different parts and ideas of the presentation? The present study provides an overview of assessment techniques pertinent to English language training in the field of oral speech. This exploration can help to understand the extent to which these learning- oriented techniques of assessment affect competence in English language learning and lead to self-assessment, which plays an active role in English Learning Teaching. This issue has not been studied so far and the main aim of the present study was to observe and characterise these effects. The research consistently showed that only assessment “for learning” and not “of learning” lead to self-assessment. A sequence of steps to develop self-assessment was worked out: setting goals for students; the assessment of each language component; guidance of the teacher in discussions; clear references if students needed further review; teacher insights into student motivation. These patterns were repeated several times. They proved to demonstrate permanent progress. The development of the assessment techniques encourages the active involvement of students in the process of their own learning and assessment. These results indicate that the techniques implemented to develop assessment and self-assessment are intended to shift the focus more to students, enabling them to become more effective learners and to succeed in English language learning. Turning to the conditions of the experiment the first group under research was aware of the examination speaking criteria while the students of the second group were not presented the criteria and were not evaluated according to these criteria as the first group was. To see the effect of the experiment and to assess students’ level of English language competences in terms of general English, the students were given Objective Placement Test, Variant 2, CUP & FLTRP, 2010 before the exam of the fourth-year academic course. This post-test revealed a definite progress in the first group: Table 4 Post-test scores of the first group Conclusion While the results of the second group did not improve much (see Table 5). Therefore, the authors were persuaded that continuous evaluation and awareness of the assessment criteria had a positive impact on the process of students’ further development in the field of English language training. Moreover, these methods led to remarkable results at the final examination. The research conducted by the authors shows that the framework used in assessment produces good results. It outlines the relationship between assessment and self-assessment. The authors argued that self- assessment plays a major role in ELT and explored 37 TATIANA BARANOVSKAYA, VALENTINA SHAFOROSTOVA the main stream. TESOL Quarterly, 21, 227-249. the main stream. TESOL Quarterly, 21, 227-249. the assessment activities which they embedded in the curriculum to develop self-assessment. The research shows that the impact on learning outcomes was great. The degree of English learning competence improvement helped students to develop and perfect their language skills. Chastain, K. (1988). Developing second language skills. New York, NY: Harcourt Brace Jovanovich. Davison, C., & Cummins, J. (2007). Assessment and evaluation in ELT: Shifting paradigms and practices. In International Handbook of English Language Teaching (Vol. 15, pp. 415-420). Boston, MA: Springer Science Business Media, LLC. The research into assessment and evaluation in ELT highlights several broad themes for further research into teachers’ professional development. More detailed classroom studies of assessment practices and their effects on students’ learning are needed. By investigating the effect of using some self-assessment techniques on language competence, this study hypothesized that the development of self- assessment can foster language skills. In order to verify this hypothesis different techniques were adapted and injected while teaching “Oral Presentation Skills”. It was found that the development of self-assessment helped improve and foster language skills and contributed to the development of reliable monitoring and evaluation, thereby influencing students’ progress and attainment. However, an important variable not investigated in this study was the effect of assessment on students’ motivation. This might be a focus for a further experimental study and calls for a wider range of assessment strategies. Dunn, B., et al. (2004). Genetic footprinting: A functional analysis of the S. cerevisiae genome. Stanford, CA: Stanford University Press. Genesee, F., Upshur, J. (1996). Classroom-based evaluation in second language education. Cambridge, UK: Cambridge University Press. Gibbs, G. (2003). Using assessment to support student learning at University of East Anglia. Leeds, UK: Leeds Metropolitan University. Jabbarifar, T. (2009). The importance of classroom assessment and evaluation in educational system. In Proceedings of the 2nd International Conference of Teaching and Learning (ICTL 2009). INTI University College, Kuching, Malaysia. Matthes, C., & Marino, J. (1990). Professional interactions: Oral communication skills in science, technology, and medicine. London, UK: Prentice Hall. Nunan, D. (1989). Understanding language classrooms. London, UK: Prentice Hall. O’Malley, J. M., & Valdez-Pierce, L. (1996). Authentic assessment for English language learners: Practical approaches for teachers. Reading, MA: Addison- Wesley. References Allwright, D., & Bailey, K. (1991). Focus on the language classroom: An introduction to classroom research or language teachers. New York, NY: Cambridge University Press. Rea-Dickins, P. (1994). Evaluation and English language teaching. Language Teaching, 27, 71-91. Richards, J. C., & Lockhart, C. (1994). Reflective teaching in second language classrooms. New York, NY: Cambridge University Press. Biggs, J. (1999). What the student does: Teaching for enhanced learning. Hager Education Research and Development, 18(1), 57-75. Sainsbury, E., & Walker, R. (2007). Assessment as a vehicle for learning: Extending collaboration into testing. Assessment and Evaluation in Higher Education, 33(2), 1-18. Carter, R., & Nunan, D. (2001). The Cambridge guide to teaching English to speakers of other languages. Cambridge, UK: Cambridge University Press. Kuzmenkova, Iu. (2011). Academic project presentations. Мoscow, Russia: MSU. Chamot, A. U., & O’Malley, J. M. (1989). The cognitive academic language learning approach: A bridge to 38
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https://openalex.org/W3118814887	https://hal.science/hal-02986272/document	English	null	SAGA GIS for Information Extraction on Presence and Conditions of Vegetation of Northern Coast of Iceland Based on the Landsat TM	Acta Biologica Marisiensis	2,020	cc-by	4,804	SAGA GIS for information extraction on presence and conditions of vegetation of northern coast of Iceland based on the Landsat TM Polina Lemenkova Polina Lemenkova To cite this version: Polina Lemenkova. SAGA GIS for information extraction on presence and conditions of vegetation of northern coast of Iceland based on the Landsat TM. Acta Biologica Marisiensis, 2020, 3 (2), pp.10 - 21. 10.2478/abmj-2020-0007. hal-02986272 Distributed under a Creative Commons Attribution 4.0 International License SAGA GIS FOR INFORMATION EXTRACTION ON PRESENCE AND CONDITIONS OF VEGETATION OF NORTHERN COAST OF ICELAND BASED ON THE LANDSAT TM Polina LEMENKOVA1* 1Analytical Center, Moscow, 115035, Russian Federation Correspondence: Polina LEMENKOVA pauline.lemenkova@gmail.com eived: 17 September 2020; Accepted: 20 October 2020; Published: 30 December 2020 Abstract: The paper aims to evaluate the presence and condition of vegetation by SAGA GIS. The study area covers northern coasts of Iceland including two fjords, the Eyjafjörður and the Skagafjörður, prosperous agricultural regions. The vegetation coverage in Iceland experience the impact of harsh climate, land use, livestock grazing, glacial ablation and volcanism. The data include the Landsat TM image. The methodology is based on computing raster bands for simulating Tassel Cap Transformation (wetness, greenness and brightness) and Enhanced Vegetation Index (EVI) sensitive to high biomass. The results include modelled three bands of brightness, greenness and wetness. Greenness variation shows the least values in ice-covered areas (-56.98 to -18.69). High values (-23.48 to 9.12) are in the valleys with dense vegetation, correlating with the geomorphology of the river network, the vegetation-free areas and ocean which corresponds to the peak of 30.87 to 41.19. The bell-shaped data distribution shows frequency 43.19–141.74 for vegetation indicating healthy state and canopy density. Maximal values are in ice-covered regions and glaciers (64°N- 65°N). Very low values (0 to -20) show desertification and mountainous rocks. Moderate values (20-40) indicate healthy vegetation. The most frequent data: -28,17 to 11,8. The EVI shows data variations (-0.14 to 0.04). The study contributes both to the regional studies of Arctic Iceland and methodological approach of remote sensing data processing by SAGA GIS. Keywords: Iceland, Landsat TM, SAGA GIS, cartography, vegetation index, machine learning, automatization, mapping. Keywords: Iceland, Landsat TM, SAGA GIS, cartography, vegetation index, machine learning, automatization, mapping. HAL Id: hal-02986272 https://hal.science/hal-02986272v1 Submitted on 2 Nov 2020 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 ABMJ 2020, 3(2): 10-21 DOI: 10.2478/abmj-2020-0007 Acta Biologica Marisiensis 1. Introduction Because the land is a natural complex and comprehensive system of geomorphic landforms, geologic factors (mineral rocks), climate settings, hydrologic conditions, and ecology (vegetation and fauna), land degradation and desertification can have worrying consequences for the fragile Arctic environment. Benefits of the presented study include a contribution to the environmental monitoring of the selected regions of Iceland, which includes the two fjords, Skagafjörður and Eyjafjörður which can be used as information on the appropriate level (ecologists, environmentalists, authorities). Because the study is fully based on the open source software (SAGA GIS and occasional GMT) for mapping and data analysis, the benefits for broad public, scolars and students consists in the repeatability of the described methods, algorithms and advices for data capture and resources. Thus, this study presents a broad spectrum of remote sensing data processing and visualization by SAGA GIS, and the use of the standard suite of high-quality raster Landsat TM datasets, as demonstrated and described in this work. Generic Mapping Tools (GMT) was also used for topographic mapping (Fig. 1) aimed at the advanced cartographic visualization of Iceland using available mapping techniques (Lemenkova, 2020a, 2020b, 2020c). Therefore, this study can be effectively reused for analysis of similar landscapes in Arctic regions using presented workflow of the SAGA GIS and calculation of the Landsat TM raster bands for analysis of vegetation. Analytical studies in the remote sensing of satellite images have unveiled the presence of indicators in plants that may be used to detect the healthiness of the canopy. For instance, chlorophyll as a health indicator of leaves absorbs visible light, and the leaf cell strongly reflects near-infrared light. Parameters of spectral reflectance of leaves are discussed (e.g. Kauth and Thomas, 1976; Broge and Leblanc, 2001; Kim et al., 2010; Lemenkova, 2011; Gao et al., 2020) and widely used for detecting vegetation coverage stage and monitoring their ecological stage. The bands constituting the Landsat TM image scene play an essential role in detecting vegetation health and quality assessed by such indicators as greenness, brightness or wetness. In this context, the use of cartographic methods applied for the satellite image processing, raster bands calculation and visualization have shown promising results in experiments carried out on Landsat TM imagery (Lemenkova, 2015a, 2015b, 2015c; Taufik et al., 2016; Ahmet and Akter, 2017; Zaitunah et al., 2018). 1. Introduction breeding. Today the vegetation coverage in Iceland has gained more attention in landscape studies due to changes under the impact of various factors including impacts of harsh climate (Brombacher et al., 2020), specifics of land use, intensive livestock grazing, glacial ablation and geological processes. Active volcanism results in regional distribution of the highly erodible volcanic soils. Geographically, the study area covers the northern coasts of Iceland including its two famous fjords, the Skagafjörður, a deep fjord with a valley (19.4°W-20.0°W), and the Eyjafjörður, (18.2°W-18.6°W) one of the longest fjords in Iceland (Fig. 1). The Skagafjörður is one of Iceland's most prosperous agricultural regions, with widespread dairy, sheep farming and horse 10 Polina Lemenkova Fig. 1. Topographic map of the study area: Iceland, region of Skagafjörður and Eyjafjörður. Source: author. Fig. 1. Topographic map of the study area: Iceland, region of Skagafjörður and Eyjafjörður. Source: author. According to the recent assessment (Eckert and Engesser, 2013), the country's surface experienced a severe soil erosion on about 40%. As a result, severe land degradation and desertification are considered the most severe environmental problems in Iceland (Eddudottir et al., 2020; Gísladottir, 2001). The examples of land cover changes in Iceland include soil erosion caused by grazing pressure in the processes of sheep farming (Arnalds and Barkarson, 2003), active aeolian processes causing the spread of the sandy areas which replace rich and vegetated ecosystems by lands with low fertility and water-holding capacity (Arnalds et al., 2001). This makes the assessment of the Arctic vegetation coverage by remote sensing methods as an important field at the cutting edge of geoscience. Current environmental problems in Iceland include severe land degradation which is caused by the effects of both human and climate factors. Land degradation results in the deterioration of plant growth conditions, and the decline of land productivity in sensitive Arctic ecosystems. Land degradation is mirrored in soil deterioration, including its physical, chemical, and biological aspects. Besides, human impacts have been severe on Icelandic soils and vegetation which finally resulted in fragmentary desertification. In turn, vegetation degradation induced soil erosion which successively caused a decline in soil quality. Hence, in recent years soil erosion became an active negative process in Iceland which resulted in deterioration of the grazing areas in the central highlands of Iceland which are not suitable for grazing by sheep due to the 11 ABMJ 2020, 3(2): 10-21 poor condition. 1. Introduction The purpose of this study was to use advanced methods of the remote sensing data processing (SAGA GIS) in order to extract information on presence and conditions of vegetation and to receive models of the wetness, greenness and brightness using calculation of the selected raster bands, and to perform calculation of the enhanced vegetation index using embedded formulae in the SAGA GIS, and finally, to visualize data distribution using computed and presented histograms aimed at the environmental monitoring of the selected Arctic ecosystems in Iceland. 2. Materials and Methods This study presents the use of SAGA GIS (Böhner et al., 2006) for processing the Landsat TM image (Fig. 2). The Landsat TM is the satellite imagery of Earth, a joint program of NASA/USGS launched on July 23, 1972 and constrantly being developed since then. Landsat 7 satellite images have eight spectral bands (channels) with spatial resolution ranging from 15 to 60 meters depending on the bands, but mostly 30-meters resolution. The temporal resolution of the Landsat TM is 16 days. Each Landsat scene covers a square approximately 185185 km long and wide. 12 Polina Lemenkova Fig. 2. Applying parameters in SAGA GIS menu. Source: author. Fig. 2. Applying parameters in SAGA GIS menu. Source: author. The official website of the Landsat TM is https://landsat.gsfc.nasa.gov/the-thematic- mapper/. However, the imagery can be freely downloaded from the GloVis website: https://glovis.usgs.gov/. The methods include two approaches of the image processing: 1) Tasseled cap transformation; 2) Enhanced Vegetation index. In the algorithm, each band (B) was multiplied by a certain coefficient and the three characteristics of the vegetation, brightness, greenness and wetness, were defined as follows (Crist and Cicone, 1984a; 1984b): 1. Brightness = 0.3037 (B1) + 0.2793 (B2) + 0.4743 (B3) + 0.5585 (B4) + 0.5082 (B5) + 0.1863 (B7) 2. Greenness = −0.2848 (B1) − 0.2435 (B2) − 0.5436 (B3) + 0.7243 (B4) + 0.0840 (B5) − 0.1800 (B7) 2.1 Tasseled Cap Transformation An algorithm for the Tasseled cap transformation was developed by Kauth and Thomas (1976) to transform the spectral information of the Landsat satellite data into indicators that turned to be useful for analysis of phenological stages of vegetation. In the menu of SAGA GIS it was applied using the following path: 3. Wetness = 0.1509 (B1) + 0.1973 (B2) + 0.3279 (B3) + 0.3406 (B4) − 0.7112 (B5) − 0.4572 (B7) 2.2. Enhanced Vegetation Index For The Enhanced Vegetation Index (EVI) was approached from the SAGA GIS menu using the following path: ‘Geoprocessing>Imagery>Vegetation Indices>Enhanced Vegetation Index’. The computing of the EVI was addressed using an optimized numerical method, comparing to the traditional calculations of the vegetation indices, by enhancing the vegetation signal (Jiang et al., 2008). For The Enhanced Vegetation Index (EVI) was approached from the SAGA GIS menu using the following path: ‘Geoprocessing>Imagery>Vegetation Indices>Enhanced Vegetation Index’. The computing of the EVI was addressed using an optimized numerical method, comparing to the traditional calculations of the vegetation indices, by enhancing the vegetation signal (Jiang et al., 2008). For The Enhanced Vegetation Index (EVI) was approached from the SAGA GIS menu using the following path: ‘Geoprocessing>Imagery>Vegetation ‘Geoprocessing>Imagery>Vegetation Indices>Tasseled Cap Transformation’. Using six of seven Landsat TM bands (except for the thermal channel 6) were used for the algorithm. Indices>Tasseled Cap Transformation’. Using six of seven Landsat TM bands (except for the thermal channel 6) were used for the algorithm. Indices>Enhanced Vegetation Index’. The computing of the EVI was addressed using an optimized numerical method, comparing to the traditional calculations of the vegetation indices, by enhancing the vegetation signal (Jiang et al., 2008). Indices>Enhanced Vegetation Index’. The computing of the EVI was addressed using an optimized numerical method, comparing to the traditional calculations of the vegetation indices, by enhancing the vegetation signal (Jiang et al., 2008). As a result, three types of information were extracted based on a weighted sum of the Landsat bands: 1) Tasseled Cap Band 1 showing brightness, which is a measurement value for the ground; 2) Tasseled Cap Band 2 showing greenness, which is a measured value for the vegetation; 3) Tasseled Cap Band 3 showing wetness, which is a measured value for interactions of soil and canopy moisture. The formula of the EVI is based on the following equation (Huete et al., 2002): EVI = G ˟ (NIR – RED)/(NIR + C1 ˟ RED – C2 ˟ BLUE + L), EVI = G ˟ (NIR – RED)/(NIR + C1 ˟ RED – C2 ˟ BLUE + L), EVI = G ˟ (NIR – RED)/(NIR + C1 ˟ RED – C2 ˟ BLUE + L), where NIR is a near-infrared band of the electromagnetic spectrum (wavelength at 750 where NIR is a near-infrared band of the electromagnetic spectrum (wavelength at 750 13 ABMJ 2020, 3(2): 10-21 3) brightness (Fig. 5). 2.2. Enhanced Vegetation Index The results of the EVI model are presented in Fig. 6. Analysis of the moisture content in Fig. 3 shows the maximal values concentrated in the ice-covered regions of the glaciers stretching an as long and narrow sheet of ice and two glaciers on the southern part of the region, 64°N-65°N (bright red spots in Fig. 3). Very low values of wetness (0 to - 20) are notable for the areas of local desertification and mountainous rocks (dark grey areas in Fig. 4). Bright yellow colours correspond to moderate values (20-40) which indicates the healthy vegetation. According to the statistics (Fig. 7, upper right), the most frequent data of wetness vary from slightly negative values -28,17 to 11,8. to 2500 nm), RED is a red (wavelength at 625– 740 nm) BLUE is blue band (wavelength et 450–485 nm). Specifically, the algorithm of EVI has been updated, by focusing on the higher biomass regions using the effects of the improved sensitivity in these specific areas, by de-coupling of the canopy background signal which eventually improved vegetation monitoring, and by a reduction in the atmosphere influences. 3. Results and discussions The simulated Tasseled Cap transformations of Landsat TM data represent examples of linear combination features showing three characteristics of the vegetation: 1) wetness (Fig. 3); 2) greenness (Fig. 4); The simulated Tasseled Cap transformations of Landsat TM data represent examples of linear combination features showing three characteristics of the vegetation: 1) wetness (Fig. 3); 2) greenness (Fig. 4); Fig. 3. Wetness of vegetation, modelled by SAGA GIS. Source: author. Fig. 3. Wetness of vegetation, modelled by SAGA GIS. Source: author. 14 Polina Lemenkova Fig. 4. Greenness of vegetation, modelled by SAGA GIS. Source: author. Fig. 4. Greenness of vegetation, modelled by SAGA GIS. Source: author. Fig. 5. Brightness of vegetation, modelled by SAGA GIS. Source: author. Fig. 5. Brightness of vegetation, modelled by SAGA GIS. Source: author. 15 15 ABMJ 2020, 3(2): 10-21 Fig. 6. Enhanced vegetation index, modelled by SAGA GIS. Source: author. Fig. 6. Enhanced vegetation index, modelled by SAGA GIS. Source: author. The analysis of greenness variations (Fig. 4) clearly shows that the lowest values of greenness correspond to the ice-covered areas (dark crimson brown colours in Fig. 4) which indicates the low values on the statistical histogram (Fig. 7) ranging from -56.98 to - 18.69. On the contrary, high values correspond to the peak on the histogram with values between -23.48 to 9.12 (Fig. 7, lower left). High values of greenness are notable for the river valleys with dense vegetation coverage, clearly depicting the geomorphology of the river network. The absence or very low vegetation coverage can be interpreted from the mustard yellow colours with values 0 to -16. They are typical for the water areas and rocky terrain. correspond to the vegetation-free areas and ocean, which corresponds to the peak of 30.87 to 41.19 in Fig. 7 (upper left). In contrast, light green colours correlating with vegetation- covered areas (100-150) can be seen as depicting the river valleys (Fig. 5) and on the coastal areas. White regions show dominating ice-covered mountainous areas. The statistical analysis points at the bell-shaped distribution of the data lying in the range of 43.19 to 141.74 for the areas of vegetation with difference indicating its healthy state and density of canopy. The analysis of the EVI (Fig. 6) shows the variations of the data range between -0.14 to 0.04. 3. Results and discussions Here the lowest values corresponding to the dark brown colours are notable for the ice-covered areas and glaciers, while vegetation in valleys and coastal areas is depicted by beige. The results showed SAGA GIS to be effective for analysis of the vegetation coverage by Landsat TM bands combination. The analysis of brightness (Fig. 5) shows its variations in the range between 20 and 260 units of surface luminosity. In particular, the darkest areas (brown-coloured, Fig. 5) 16 Polina Lemenkova Fig. 7. Four statistical histograms of the data distribution: wetness, brightness, greenness, enhanced vegetation index, modelled by SAGA GIS. Source: author. Fig. 7. Four statistical histograms of the data distribution: wetness, brightness, greenness, enhanced vegetation index, modelled by SAGA GIS. Source: author. Current environmental problems of Iceland include changes in vegetation coverage and anthropogenic pressure, such as increased number of tourists which may cause some mechanical disturbances on fragile Polar landscapes (Ásgeirsdóttir and Karlsson, 2016; Óladóttir, 2019). A complex interaction between various factors affect land cover and vegetation in Iceland, for instance anthropogenic (Tverijonaite et al., 2018), climatic factors (Haraldsson and Ólafsdóttir, 2003). This can be illustrated by issues regarding the vulnerability of the Northern ecosystems, e.g. land use pressure and overgrazing. Since land degradation may affect land use sustainability in the future, these issues present the concern for the environmental monitoring in Iceland. According to previous studies (Bergþórsson et al., 1996), Iceland has experienced climatic variations over the past years which resulted in the changes in vegetation. Besides, due to the anthropogenic effects, over half of the Icelandic vegetation deteriorated from the time of Iceland's first settlement by vikings in ca. AD 830 (Hallsdóttir 1995) and over 90% of its forest cover (Þorsteinsson, 1972). In view of abovesaid, detailed studies of the selected landscapes of Iceland present a contribution to the monitoring of natural resources prevention of degradation of the Northern ecosystems. References 1. Abburu S, Golla SB (2015) Satellite Image Classification Methods and Techniques: A Review. International Journal of Computer Applications 119(8):20–25. 1. Abburu S, Golla SB (2015) Satellite Image Classification Methods and Techniques: A Review. International Journal of Computer Applications 119(8):20–25. Other studies upscaled the question of the vegetation changes and focused on the quantification of landscape fragmentation by metrics approach for environmental sustainability (Klaučo et al., 2013b, 2014, 2017). Examples of the statistical analysis applied for geosciences provide more advanced methods of data visualization (Lindh, 2004; Klaučo et al. 2013a; Lemenkova, 2019a, 2019b, 2019c). Other ways of geodata processing include machine learning, GIS (Suetova et al., 2005a, 2005b; Schenke and Lemenkova, 2008; Lemenkova, 2014). This review focused on the effective methods of Enhanced Vegetation Index and Tasseled cap transformation by SAGA GIS applied for processing of the Landsat TM scene covering the region of northern Iceland. Other studies upscaled the question of the vegetation changes and focused on the quantification of landscape fragmentation by metrics approach for environmental sustainability (Klaučo et al., 2013b, 2014, 2017). Examples of the statistical analysis applied for geosciences provide more advanced methods of data visualization (Lindh, 2004; Klaučo et al. 2013a; Lemenkova, 2019a, 2019b, 2019c). Other ways of geodata processing include machine learning, GIS (Suetova et al., 2005a, 2005b; Schenke and Lemenkova, 2008; Lemenkova, 2014). This review focused on the effective methods of Enhanced Vegetation Index and Tasseled cap transformation by SAGA GIS applied for processing of the Landsat TM scene covering the region of northern Iceland. 2. Ahmet KR, Akter S (2017) Analysis of landcover change in southwest Bengal delta due to floods by NDVI, NDWI and K- means cluster with Landsat multi-spectral surface reflectance satellite data. Remote Sensing Applications: Society and Environment 8:168–181. 3. Arnalds O, Barkarson BH (2003) Soil erosion and land use policy in Iceland in relation to sheep grazing and government subsidies. Environmental Science & Policy 6(1):105–113. 4. Arnalds O, Gisladottir F, Sigurjonsson H (2001) Sandy deserts of Iceland: an overview. J. Arid Environment 47:359– 371. The paper contributed both to the agricultural studies of vegetation coverage and the development of methods by presenting two- step methodology: computing raster bands for visualizing the Tassel Cap Transformation and EVI. The application of the Tasseled Cap Transformation and EVI in Arctic vegetation monitoring showed effective methods of data visualization. Conclusions Thanks to the advances in the remote sensing data processing by cartographic methods, the massive amount of Landsat TM images of 30-m resolution and high quality became available in agricultural studies, for instance for a crop or vegetation mapping. Reflectance curves for various land cover types on Earth, including healthy and unhealthy vegetation and its types (coniferous, broadleaf), have particular characteristics (Abburu and Golla, 2015; Knipling, 1970; Lemenkova, 2013). Remote sensing data analysis using spectral reflectance curves shown a trend in 17 ABMJ 2020, 3(2): 10-21 financial relationships that could be construed as a potential conflict of interest. land cover changes and variations in vegetation coverage of Earth (Lemenkova, 2013, 2015d). The possibility of synergism between these risk factors remains a topic that can be further researched. References With a context of the presented case study of Iceland, notable for fragile ecosystems, these two methods demonstrated usefulness for Landsat TM scene analysis of the vegetation canopy, health status and land cover parameters by cartographic means of SAGA GIS. 5. Ásgeirsdóttir T, Karlsson T (2016) International visitors in Iceland – summer 2016. 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Broge NH, Leblanc E (2001) Comparing prediction power and stability of broadband and hyperspectral vegetation indices for 18 Polina Lemenkova estimation of green leaf area index and canopy chlorophyll density. Remote Sensing of the Environment 76:156–172. 17. Haraldsson HV, Ólafsdóttir R (2003) Simulating vegetation cover dynamics with regards to long-term climatic variations in sub-arctic landscapes. Global and Planetary Change 38(3-4):313–325. 9. Brombacher J, Reiche J, Dijksma R, Teuling AJ (2020) Near-daily discharge estimation in high latitudes from Sentinel-1 and 2: A case study for the Icelandic Þjórsá river. Remote Sensing of Environment 241:111684. 18. Huete A, Didan K, Miura T, Rodriguez EP, Gao X, Ferreira LG (2002) Overview of the radiometric and biophysical performance of the MODIS vegetation indices. Remote Sensing of Environment 83:195–213. 10. Crist EP, Cicone RC (1984a) Application of the Tasseled Cap concept to simulated Thematic Mapper data. Photogrammetric Engineering and Remote Sensing 50(3):343–352. 19. Jiang Z, Huete AR, Didan K, Miura T (2008) Development of a two-band Enhanced Vegetation Index without a blue band, Remote Sensing of Environment, 112(10):3833–3845. 11. Crist EP, Cicone RC (1984b). A physically- based transformation of Thematic Mapper data – the TM Tasseled Cap. IEEE Transactions on Geoscience and Remote Sensing GE- 22(3):256–263. 20. Kim Y, Huete AR, Miura T, Jiang Z (2010) Spectral compatibility of vegetation indices across sensors: band decomposition analysis with Hyperion data. Journal of Applied Remote Sensing, 4(1):043520. 12. Eckert S, Engesser M (2013) Assessing vegetation cover and biomass in restored erosion areas in Iceland using SPOT satellite data. Applied Geography 40:179– 190. 21. Kauth RJ, Thomas GS (1976) The Tasseled Cap – a graphic description of the spectral- temporal development of agricultural crops as seen by Landsat. Proceedings of the Symposium on Machine Processing of Remotely Sensed Data, Purdue University, West Lafayette, Indiana, 4B41-4B51. 13. Eddudóttir SD, Erlendsson E, Gísladottir G (2020) Landscape change in the Icelandic highland: A long-term record of the impacts of land use, climate and volcanism. Quaternary Science Reviews 240:106363. 22. Klaučo M, Gregorová B, Stankov U, Marković V, Lemenkova P (2013a) Determination of ecological significance based on geostatistical assessment: a case study from the Slovak Natura 2000 protected area. Central European Journal of Geosciences, 5(1):28–42. 14. Conflict of Interest Gao L, Wang X, Johnson BA, Tian Q, Wang Y, Verrelst J, Mu X, Gu X (2020) Remote sensing algorithms for estimation of fractional vegetation cover using pure vegetation index values: A review. ISPRS Journal of Photogrammetry and Remote Sensing 159:364–377. 23. 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(2015a) Analysis of Landsat NDVI Time Series for Detecting Degradation of Vegetation. In: Geoecology and Sustainable Use of Mineral Resources. From Science to Practice, Belgorod, Russia, 11–13. 25. Klaučo M, Gregorová B, Stankov U, Marković V, Lemenkova P (2017) Land planning as a support for sustainable development based on tourism: A case study of Slovak Rural Region. Environmental Engineering and Management Journal, 2(16):449–458. 34. Lemenkova P (2015b) Innovations in the Geoscience Research: Classification of the Landsat TM Image Using ILWIS GIS for Geographic Studies. In: Prospects for the Higher School Development. Grodno, Belarus, May 28–29, 2015, 60–63. 26. Knipling EB (1970) Physical and physiological basis for the reflectance of visible and near-infrared radiation from vegetation. Remote Sensing of Environment 1:155-159. 35. Lemenkova P (2015c) To the Question of the Environmental Education: how Landsat TM, ETM+ and MSS Images can be Processed by GIS-Techniques for Geospatial Research. Trends and Perspectives in the Creating Regional Systems of the Additional Adults Education. Vitebsk, Belarus. 27. Lemenkova P (2020a) GMT Based Comparative Geomorphological Analysis of the Vityaz and Vanuatu Trenches, Fiji Basin. Geodetski List, 74(1):19–39. 28. Lemenkova P (2020b) Variations in the bathymetry and bottom morphology of the Izu-Bonin Trench modelled by GMT. Bulletin of Geography. Physical Geography Series 18(1): 41–60. 36. Lemenkova P (2015d) Processing Remote Sensing Data Using Erdas Imagine for Mapping Aegean Sea Region, Turkey. Informatics. Problems, Methodology, Technologies, 3, 11–15. 37. Conflict of Interest Lemenkova P (2014) Opportunities for Classes of Geography in the High School: the Use of ’CORINE’ Project Data, Satellite Images and IDRISI GIS for Geovisualization. In: Perspectives for the Development of Higher Education. Belarus, Grodno, 284–286. 29. Lemenkova P (2020c) GEBCO Gridded Bathymetric Datasets for Mapping Japan Trench Geomorphology by Means of GMT Scripting Toolset. Geodesy and Cartography 46(3):98–112. 30. Lemenkova P (2019a) Statistical Analysis of the Mariana Trench Geomorphology Using R Programming Language. Geodesy and Cartography 45(2):57–84. 38. Lemenkova P (2013) Monitoring Changes in Agricultural Landscapes of Central Europe, Hungary: Application of ILWIS GIS for Image Processing. Geoinformatics: Theoretical and Applied Aspects. Ukraine, Kiev, 13–16 May, 2013. 31. Lemenkova P (2019b) Testing Linear Regressions by StatsModel Library of Python for Oceanological Data Interpretation. Aquatic Sciences and Engineering 34:51–60. 39. Lemenkova P (2011) Seagrass Mapping and Monitoring Along the Coasts of Crete, Greece. M.Sc. Thesis. Netherlands: University of Twente. 158 pp. 32. Lemenkova P (2019c) AWK and GNU Octave Programming Languages Integrated with Generic Mapping Tools for 20 Polina Lemenkova 40. Lindh P (2004) Compaction- and strength properties of stabilised and unstabilised fine-grained tills. PhD Thesis, Lund University, Lund. 41. Óladóttir OT (2019). Tourism in Iceland in Figures. Icelandic Tourist Board, 28 p. 42. Þorsteinsson, I. 1972. Gróðurvernd: byggð á hóflegri nýtingu og ræktun lands [Carrying capacity of Icelandic rangelands]. Rit landverndar, 2. Landvernd, Reykjavik, 128 pp. 43. Schenke HW, Lemenkova P (2008) Zur Frage der Meeresboden-Kartographie: Die Nutzung von AutoTrace Digitizer für die Vektorisierung der Bathymetrischen Daten in der Petschora-See. Hydrographische Nachrichten 81:16–21. 44. Suetova IA, Ushakova LA, Lemenkova P (2005a) Geoinformation mapping of the Barents and Pechora Seas. Geography and Natural Resources 4:138–142. 45. Suetova IA, Ushakova LA, Lemenkova P (2005b) Geoecological Mapping of the Barents Sea Using GIS. International Cartographic Conference. 46. Taufik A, Ahmad SSS, Ahmad A (2016) Classification of Landsat 8 satellite data using NDVI thresholds. Journal of Telecomunication Electronic and Computer Engineering 8(4):37–40. 47. Tverijonaite E, Ólafsdóttir R, Thorsteinsson T (2018). Accessibility of protected areas and visitor behaviour: A case study from Iceland. Journal of Outdoor Recreation and Toursim, 24:1–10. 48. Zaitunah A, Ahmad AG, Safitri RA (2018) Normalized difference vegetation index (ndvi) analysis for land cover type using landsat 8 oli in besitang watershed, Indonesia. IOP Conf. Series: Earth and Environmental Science 126:012112. 21 21
https://openalex.org/W3172198909	https://wnus.edu.pl/aie/file/article/view/18950.pdf	English	null	Medical Futility or Persistent Therapy? A Dispute over the Terms and Definitions in the Polish Context	Analiza i Egzystencja	2,021	cc-by-sa	9,623	„Analiza i Egzystencja” 53 (2021) ISSN 1734-9923 DOI: 10.18276/aie.2021.53-03 „Analiza i Egzystencja” 53 (2021) ISSN 1734-9923 DOI: 10.18276/aie.2021.53-03 „Analiza i Egzystencja” 53 (2021) ISSN 1734-9923 DOI: 10.18276/aie.2021.53-03 MARCIN FERDYNUS* ORCID: 0000-0003-0176-1023 * Marcin Ferdynus – PhD, is a lecturer and researcher in the Department of Ethics at the John Paul II Catholic University of Lublin. He received the award of the Pres- ident of the Council of Ministers for his doctoral dissertation Prolonging life seen as a moral problem, which was also selected as the best philosophical work in 2017 by the Polish Academy of Sciences. His work covers bioethics, medical ethics and nursing philosophy. Address for correspondence: John Paul II Catholic University of Lublin, Faculty of Phi- losophy, Institute of Philosophy, Department of Ethics, Al. Racławickie 14, 20-950 Lublin, Poland. E-mail: marcin.ferdynus@kul.pl. ** The project is funded by the Minister of Science and Higher Education within the programme under the name “Regional Initiative of Excellence” in 2019–2022, project number: 028/RID/2018/19, the amount of funding: 11 742 500 PLN. * Marcin Ferdynus – PhD, is a lecturer and researcher in the Department of Ethics at the John Paul II Catholic University of Lublin. He received the award of the Pres- ident of the Council of Ministers for his doctoral dissertation Prolonging life seen as a moral problem, which was also selected as the best philosophical work in 2017 by the Polish Academy of Sciences. His work covers bioethics, medical ethics and nursing philosophy. Address for correspondence: John Paul II Catholic University of Lublin, Faculty of Phi- losophy, Institute of Philosophy, Department of Ethics, Al. Racławickie 14, 20-950 Lublin, Poland. E-mail: marcin.ferdynus@kul.pl. The project is funded by the Minister of Science and Higher Education within the programme under the name “Regional Initiative of Excellence” in 2019–2022, project b 028/RID/2018/19 th t f f di 11 742 500 PLN MEDICAL FUTILITY OR PERSISTENT THERAPY? A DISPUTE OVER THE TERMS AND DEFINITIONS IN THE POLISH CONTEXT Keywords: persistent therapy, medical futility, extraordinary medical procedures, terminally ill patient Słowa kluczowe: uporczywa terapia, medyczna daremność, nadzwyczajne procedury medyczne, pacjent terminalnie chory Keywords: persistent therapy, medical futility, extraordinary medical procedures, terminally ill patient Słowa kluczowe: uporczywa terapia, medyczna daremność, nadzwyczajne procedury medyczne, pacjent terminalnie chory 44 Marcin Ferdynus 1 Although the term ‘over-zealous treatment’ derives from the Catholic Church’s moral teaching (I agree with the anonymous reviewer and I am aware of this context), it does not mean that this term is limited only to theological debate. Polish authors translate the term “uporczywa terapia” as “over-zealous treatment” (mainly theologians, but not limited to theologians; Machinek, 2015; Dowgiałło-Wnukiewicz, Kozera, Lech, Rym- kiewicz, Michalik, 2019) or as “persistent therapy” (mainly medical professionals, but not limited to them; Krucińska, Saran, Czyżewski, 2018; Cebulska, Koźlak, Dybalski, 2019; Wach, 2020, pp.78–79). Moreover, some authors emphasize that the term „per- sistent therapy” is used as an equivalent to the term „medical futility” (Szewczyk, 2016; Suchorzewska, Basińska, Olejniczak, 2008). I translate the term „uporczywa terapia” as „persistent therapy” for two reasons. The first is that more and more Polish authors translate the term „uporczywa terapia” as „persistent therapy” instead of as „over-zealous treatment” in the medical literature. The second reason is more important. I think that the term „persistent therapy” more strongly emphasizes the role of the patient (his personal preferences) in making decisions on the continuation or discontinuation of treatment than the term „over-zealous treatment” (sometimes it is described as an „overtreatment” in the international literature), which focuses more on the physician’s role in decision making. Introduction In contemporary society, medicine has become almost omnipotent, and the doctor, like the mythical Asclepius, has turned into a god who can withhold or at least delay death. When a person is in critical condition, the decision to hospitalize is made almost immediately. Conviction in the high effective- ness of resuscitation procedures results in the use of intensive care in severely ill patients, whose chances of survival are low. These actions may prolong life, but they may also prolong the agony, delaying death in an unnatural manner (Randall, Downie, 2010, p. 100). In some cases, these interventions may be qualified as persistent therapy.1 Due to the absence of bioethical and medical regulations, Polish ex- perts in bioethics have started a discussion about the need to settle issues surrounding prolonging the lives of the terminally ill. This discussion is based on the dispute of whether the term “persistent therapy”, which is popular in Poland, should be rejected and replaced with the term “medical futility” in regulations. The dispute also encompasses the way of defining these terms. Until recently, “persistent therapy” has been the leading term in Polish bioethical and medical literature. Although many authors in Poland still refer to this term (Bazaliński, Marciniec, Sałacińska, Przybek-Mita, Więch, 2018; Budziński, 2016; Szeroczyńska, 2013; Wach, 2013), we 45 Medical Futility or Persistent Therapy? A Dispute over the Terms... could observe a clear change in this respect in the past ten years. The term “medical futility” has become increasingly popular (Kübler, Siewiera, Durek, Kusza, Piechota, Szkulmowski, 2014; Siewiera, Kübler, 2015; Szabat, 2013, pp. 70–75; Szewczyk, 2009, pp. 303–307). Among other reasons, the need to change terms and introduce them into bioethical and medical documents is justified by the need for language standardisation and better international co-operation (Szewczyk, 2016). Participants in the discussion have raised various arguments for the rejection of the term “persistent therapy” and the introduction of the term “medical futility”. The dispute is fairly complex and entangled in various contexts: philosophical, medical, cultural, axiological, and religious. This article aims to present the most important aspects of the indicated dispute regarding the two terms and their definitions. I show that both positions can be reconciled under certain conditions. These conditions manifest themselves within a broad and a narrow semantic scope in the modified definition developed by the Polish Working Group on End-of-Life Ethics (PWG). Introduction In other words, I argue for modifying the PWG’s defini- tion of persistent therapy. Moreover, I stress that the concept of persistent therapy of the PWG deserves attention because it is often quoted in the Polish literature on the subject (Pawlikowski, Muszala, Gajewski, Krajnik, 2021; Cebulska, Koźlak, Dybalski, 2019; Krucińska, Saran, Czyżewski, 2018), and, thus, may have an impact on the shaping of medical practice. I also assert that the term “medical futility” can be useful for bioethics only when its definition is limited to a narrow semantic scope. The definition of medical futility Reflecting on the meaning of medical futility, it is worth noting that the word “futile” is derived from the Latin futtilis (Jenal, Moreno, 2017, p. 103) and refers to actions or instruments that are inherently leaky and, therefore, ill-suited to achieving desired ends. The implication is that the use of leaky means will always be in vain as the leak is an intrinsic defect that will make failure inevitable (Rubin, 1998, p. 42). The ordinary meanings of futile include ineffective, useless, unsuccessful, and meritless (Robinson, 2010). Merriam-Webster’s Dictionary defines futility as “serving no useful purpose; completely ineffective” (Swetz, Burkle, Berge, Lanier, 2014, p. 943), and the Oxford English Dictionary defines the word “futile” defines as “leaky, vain, Marcin Ferdynus 46 failing of the desired end through intrinsic defect” (Schneiderman, Jecker, 2011, p. 20). According to Aghabarary and Nayeri, medical futility occurs when: (1) there is a goal, (2) there is an action or activity for achieving that goal, and (3) there is a virtual certainty that the action or the activity fails to achieve the goal (2016). The original meaning of the word “futile” indicates that a futile action is one that cannot achieve its goal, no matter how often it is repeated (Schneiderman, Jecker, Jonsen, 1990, p. 950). Debates over futile care emerged in medical ethics in the late 1980s and early 1990s (Kearns, Gordijn, 2018, p. 12; Veatch, 2013, p. 12; Moratti, 2009, p. 369; Youngner, 2004, p. 1718). The idea was, as Wilkinson and Savulescu emphasise, that if physicians identified a particular treatment as futile, this would solve the problem of conflicts. Physicians had no obli- gation to provide futile therapy, and refusing to do so would, thus, not be paternalistic (Wilkinson, Savulescu, 2019, p. 22).2 Over the next decade, many doctors and ethicists sought to define medical futility in a way that would be practically applicable.3 For example, the most-cited and perhaps one of the broadest definitions of medical futility in the literature is the one provided by Schneiderman, Jecker, and Jonsen. Their definition refers to two parameters: a quantitative one and a qualitative one. Quantitatively, when doctors (based on their own experience, their colleagues’ experience, or em- pirical data) state that the therapy was useless in the last 100 cases, it can be deemed medically futile. 2 It is worth noting that alternative terms for “medical futility” can be found in the literature: “non-beneficial treatment”, “not clinically appropriate”, “not medically indicated”, “clinical futility”, “medically inappropriate”, “medically inadvisable” and “potentially inappropriate” (Wilkinson, Savulescu, 2011). 3 I do not discuss many concepts of medical futility; such a discussion is neither possible nor necessary. I only highlight a few important aspects of medical futility that will be helpful for the further discussion. The definition of medical futility Qualitatively, these authors suggest that any therapy that only sustains a state of permanent unconsciousness or fully prevents a patient from becoming independent of intensive medical care should be regarded as useless – which means futile – therapy (Schneiderman, Jecker, Jonsen, 1990, pp. 951–952; Schneiderman, Jecker, 2011, pp. 14–19). This understanding of the term has been heavily criticised (Burt, 2002; Lantos, 2006). Some authors have suggested that the concept of futility obscures many ambiguities and assumptions (Truog, Brett, Frader, 1992), while others have argued that the attempt to define futile treatment is itself futile (Brody, Medical Futility or Persistent Therapy? A Dispute over the Terms... 47 Halevy, 1995). The available definitions do not succeed in justifying the unilateral withholding of treatment. Furthermore, the quantitative and quali- tative aspects of futility have often been challenging for clinicians to parse out because these aspects rely on value judgements on the quality of life as well as its role in assessing the virtue of longevity (Youngner, 1988). Some doctors have suggested that what a patient or surrogate defines as quality or quantity may differ from the clinician’s perspective, and one can argue that qualitative futility is only met if a treatment does not allow patients to live their lives according to their goals, preferences, and values, which cannot be determined clinically or by how the last 100 patients responded in a given situation (Swetz, Burkle, Berge, Lanier, 2014, p. 944). Many studies have demonstrated that physicians disagree about quantitative and qualitative thresholds for futility (Curtis, Park, Krone, Pearlman, 1995; Van McCrary, Swanson, Youngner, Perkins, Winslade, 1994).4 In the literature, apart from the conception proposed by Schneider- man et al., two forms of futility can be distinguished: physiological and normative (Youngner, 1988; Veatch, 2013, p. 14). The term “futility” means that the goal for which the therapy is proposed is either unachievable or insufficiently worthwhile. In other words, the treatment is described as physiologically, factually futile (physiological aspect), or as evaluatively futile (normative aspect). In the first instance, a judgement that treatment is physiologically futile implies that it is not physiologically possible to achieve the goal, and the treatment is, thus, futile. In the second instance, a judgement that treatment is evaluatively futile implies that the goal itself would not be worth pursuing (Rubin, 1988, pp. 53–54). 4 I will present more critical opinions on this matter in the further discussion. The definition of medical futility Rubin explains, “[f]rom a factual perspective, we can investigate whether the treatment in question is a possible means of achieving the designated goal. From an evaluative perspective, we can judge the worthiness of a designated goal and hence the worthiness of employing the treatment as a means of achieving it” (Rubin, 1998, p. 54). The most narrowly defined type of medical futility is referred to as “physiological futility” (Vivas, Carpenter, 2020), wherein physicians do not make any evaluative assessment that a treatment’s effect is not worth- while. There is no normative (evaluative) disagreement; the physicians can ascertain physiological futility based on their clinical knowledge. The basis 48 Marcin Ferdynus for refusing treatment is a scientific and empirical one: the treatment does not work.5 This use of the term “futility” is true to the word’s core meaning (White, Pope, 2016, p. 72). ( , p , , p ) It cannot be ruled out that the crisis being experienced by the concep- tions of medical futility today refers to the violation of methodological rigours. Such conceptions that take into account not only medical factors (physiological judgements) but also non-medical factors (valuing judge- ments) are not correct, and the error lies in the excessively broad interpreta- tion of the definition of medical futility.6 The semantic scope of many concep- tions of this term has been enriched in an unauthorised manner. I presume that the consequences of this error are reflected in the terminological chaos that has arisen around defining medical futility in the last few years and in the impossibility of reaching a consensus on this matter. I also believe that the most reasonable approach to “rescuing” the understanding of medical futility is to return to its original meaning: forming a definition based on strictly medical factors. I believe this is a strong argument against the broader scope of the term. Therefore, I propose relating medical futility only to medi- cal factors. I will return to the definition of medical futility later in the article (in the section titled “An attempt to overcome the stalemate – Modifying the PWG’s definition) because it requires some clarification. Now, this paper will turn its focus to the discussion surround the concept of persistent therapy. 5 Loretta Kopelman states that judgments about the futility of treatments in the contested cases must be supported by evidence and modified as the relevant evidence changes (Kopelman, 1995, p. 111).i 6 Definitions of medical futility that address factors beyond medical ones are inadequate in the sense that the scope of definiens becomes superior to the scope of definiendum. 5 Loretta Kopelman states that judgments about the futility of treatments in the contested cases must be supported by evidence and modified as the relevant evidence changes (Kopelman, 1995, p. 111). 6 Definitions of medical futility that address factors beyond medical ones are inadequate in the sense that the scope of definiens becomes superior to the scope of definiendum. The Polish Working Group on End-of-Life Ethics’ definition and interpretation of persistent therapy In Poland, the discussion concerning persistent therapy and medical futil- ity began in 2008, when the PWG’s definition of persistent therapy was published. According to the PWG, persistent therapy is the use of medical procedures to maintain the life function of the terminally ill in a way that Medical Futility or Persistent Therapy? A Dispute over the Terms... 49 prolongs their dying, introducing excessive suffering or violating their dignity (Bołoz, Krajnik 2008, p. 77).i j The comment to the definition of persistent therapy proposed by PWG includes a suggestion that this definition reduces the use of „persistent” to denote medical procedures applied during the “dying” of a person regarded as “terminally ill” (Krajewski, 2008, p. 78). Moreover, “dying” should be understood as the terminal period of a disease, in which the patient’s condi- tion continuously deteriorates, that leads to death within a short and pre- dictable time. A terminally ill patient is considered to be a person on whom all therapeutic attempts to give a real chance for recovery or to inhibit the disease process have been exhausted. “Terminally ill” also applies to patients for whom none of such therapies exist. It is also stressed that a terminally ill person in a state of dying is subjected to procedures that, as a mat- ter of fact, sustain the patient’s life and prolong his dying (Krajewski, 2008, p. 78). The next section delineates the purposes of limiting the definition. According to the aforementioned comment, the limitation of the time to which persistent therapy refers is meant to help avoid difficult discussions on the possible acceleration of death. Additionally, the very statement that a terminally ill person is in a state of dying indicates that life-sustaining treatments would only prolong the dying process (Krajewski, 2008, 78). It is not known, however, why the PWG’s definition narrows the term “per- sistence” by establishing one criterion: excessive suffering. Moreover, it is not clear whether this means only physical suffering caused by pain or suffering in a broader context, such as suffering that can be interpreted as a group of complex unpleasant feelings resulting from various physical and mental factors. This complex group of feelings might be the result of both the ailments of the human body and external factors coming from the world that surrounds us (Suchorzewska, 2013). 7 At this point, I emphasise only the narrow nature of the definition proposed by PWG. I believe that even though the therapy may sometimes have little chance of suc- cess, the patient may find it persistent for some reasons (for example, due to excessive suffering). This position will become clearer when I propose a modification to PWG’s definition of persistent therapy. The Polish Working Group on End-of-Life Ethics’ definition and interpretation of persistent therapy Because of limitations imposed on the definition of persistent therapy, scholars discussing this issue have doubts about the justifiability of narrowing the meaning of persistent therapy to only concern the terminally ill and dying persons (Orłowski, 2009). To resolve this question, we should begin by asking how “persistence” is interpreted. 50 Marcin Ferdynus The meaning and scope of “persistence” The adjective “persistent” derives from the noun “persistence” and expresses fierceness, tenacity, perseverance, endurance, and something that lasts continuously (Bartoszek, 2000, p. 268; Bartoszek, 2012). If something is persistent, we say that it cannot be suppressed, removed, or liquidated too easily or that it may be burdensome (Ferdynus, 2017, p. 125; Suchorzewska, Basińska, Olejniczak, 2008, p. 65). This term specifies the duration and inten- sification of a phenomenon. In medicine, permanent pain is often described as persistent (Aszyk, 2006, pp. 150–151). If it exceeds the intensity limit, it causes huge suffering that sometimes becomes unbearable (Ferdynus, 2017, p. 126). Thus, we can suppose that calling a therapy persistent means that the continuation of the therapy may not be completely ineffective and that it causes excessive suffering for the patient.7 The patient may resign him- self to another chemotherapy if previous ones failed to improve his health considerably and caused great suffering. As it remains possible that further chemotherapies could improve his health, we cannot state that the continu- ation of chemotherapy would only prolong the dying process (Chyrowicz, 2015, p. 312). This also applies to patients suffering from amyotrophic lat- eral sclerosis (ALS). We cannot determine whether the scope of “excessive suffering” encompasses situations in which patients suffering from ALS express a strong unwillingness to be permanently connected to a respirator. After being permanently connected to a respirator, ALS patients are not in the situation of a person in a state of dying, even though they are terminally ill (Andersen, Abrahams, Borasio, Carvalho de, Chio, Damme Van, Weber, 2012). Thus, it seems right to believe that if an ALS patient or any other patient is in a state of dying, the doctor should not focus on providing treat- ment, as patients in states of agony are not treated, but rather on making every effort to alleviate suffering and ensuring that the patient is supported until the end of the dying process (Ferdynus, 2018, p. 422). If starting, con- tinuing, or ceasing a therapy becomes morally problematic, it is not because Medical Futility or Persistent Therapy? A Dispute over the Terms... 51 the therapy proves completely ineffective from a medical perspective but because of the aforementioned cases. The examples mentioned here by no means exhaust the scope of patients for whom therapeutic persistence could be ascertained. The meaning and scope of “persistence” The aim of the examples is only to remind us that the defini- tion of persistent therapy proposed by the PWG does not encompass many cases of seriously ill patients because it is restricted only to cases of termi- nally ill and dying patients who excessively suffer. More arguments against persistent therapy The debate about persistent therapy and medical futility in Poland has led to a polarisation of opinions. Some opponents of persistent therapy believe that the term is an anachronistic legal construction that expresses far-reaching mistrust towards the medical profession and its patients. Because of the correspondence between persistent therapy and medical futility, it has been suggested that the term “persistent therapy” – a popular expression in Po- land – should be rejected (Szewczyk, 2016). It is important to note that the definition of persistent therapy that is being contested is the definition for- mulated by the PWG. Nevertheless, the postulate about the elimination of the term “persistent therapy” from Polish bioethical literature is not limited to the PWG’s definition but to the term “persistent therapy” in general. Argu- ments against “persistent therapy” are as follows: 1. Leaving the term “persistent therapy” in Polish bioethical literature leads to terminological chaos. Previously mentioned ambiguities in the term’s definition make it difficult or impossible for proper communication to happen between personnel in intensive care units and between the personnel and the patient, his family, or legal representative.i 2. Years of discussion on medical futility show that the PWG’s defini- tion of persistent therapy, which is similar to the definition of medi- cal futility, is hardly a justifiable anachronism.ii 3. Remaining at the stage of definition means that the PWG’s defini- tion can be accused of unjustified paternalism.i 4. The inclusion of “dignity” in the definition of persistent therapy means that a doctor must decide whether the therapy they are considering violates this value. The presence of “dignity” in the 52 Marcin Ferdynus definition of persistent therapy becomes a source of fears concerning the excessive paternalism of doctors in the determination of persis- tence. Moreover, “dignity” as an ambiguous term makes it impos- sible to reach a consensus on the meaning of therapeutic persistence.i definition of persistent therapy becomes a source of fears concerning the excessive paternalism of doctors in the determination of persis- tence. Moreover, “dignity” as an ambiguous term makes it impos- sible to reach a consensus on the meaning of therapeutic persistence.i 5. The PWG’s definition of persistent therapy burdens the doctor and the patient or his legal representative with the task of estimating the dying patient’s degree of suffering, which is a difficult, morally questionable, and highly subjective matter.i 6. More arguments against persistent therapy A negative aspect of the PWG’s definition is its complexity, which has been indicated by clinicians.i 7. The relationship between the definition of persistent therapy and the teaching of the Catholic Church (CC) raises concerns that the adoption of the former violates the principles of a democratic state of law with a variety of worldviews. 8. The term “persistent therapy” has provincial connotations that limit its use to Poland, thereby hindering the discussion on the interna- tional bioethical and medical forum. 9. The fact that the term “persistent therapy” is commonly used in Polish literature is not a sufficient reason to leave this term in it.8 In connection with the aforementioned arguments, there is a postulate that “persistent therapy” should be replaced with “medical futility” in the bioethical and medical context. This opinion is shared by some scholars (Suchorzewska, 2016; Kübler, 2016). In connection with the aforementioned arguments, there is a postulate that “persistent therapy” should be replaced with “medical futility” in the bioethical and medical context. This opinion is shared by some scholars (Suchorzewska, 2016; Kübler, 2016). 8 These arguments were collected by Szewczyk (2016). Potential replies to claims When replying to the first claim (1), we could ask why using the term “persistent therapy” would contribute to bigger terminological chaos than using the term “medical futility”. A few years ago, the authors of the article “Medical futility and its challenges: a review study” analysed 89 publica- tions about “medical futility” and found that it is a complex, ambiguous, subjective, case-specific, value-based, and goal-dependent concept that almost always involves some degree of uncertainty. In addition, the ar- ticle’s overview of various definitions of medical futility has shown that Medical Futility or Persistent Therapy? A Dispute over the Terms... 53 the formation of such definitions is influenced by a wide range of factors: doctors’ and patients’ value systems; medical uses; social, cultural, and religious contexts; and individuals’ emotions and personality traits. The au- thors of the study concluded that it is not possible to reach a consensus on the concept of medical futility because there is no objective and valid criterion that would serve as a basis for defining this term (Aghabarary and Nayeri, 2016). This opinion is shared by other authors discussing the issue of medi- cal futility (Bosslet, Pope, Rubenfeld, Lo, Truog, Rushton, White, 2015; Nates, Nunnally, Kleinpell, Blosser, Goldner, Birriel, Sprung, 2016; Paris, Hawkins, 2015, p. 51). Therefore, many scholars argue that medical futility should be defined based on the unique condition of each patient (Heland, 2006; Jox, Schaider, Marckmann, Borasio, 2012). Recent research, which has indicated that medical personnel properly understand the term “persistent therapy”, speaks against the opinion that leaving the term “persistent therapy” in Polish bioethical literature causes terminological chaos or makes communication between the doctor and the patient impossible (Bazaliński, Marciniec, Sałacińska, Przybek-Mita, Więch, 2018). While it is true that authors often use the term “persistent therapy” in Polish literature, definitions of the term do not differ widely. The PWG’s definition is only one of a limited number of examples, although its defini- tion is commented upon most frequently.i The second claim (2) could be valid if there were as many defi- nitions of persistent therapy in Polish scholarship as there are defini- tions of medical futility in English scholarship. Since the publication of the PWG’s definition in 2008, no new definition of persistent therapy has ap- peared in Poland. Potential replies to claims Thus, transposing this opinion onto persistent therapy is wrong despite the seemingly valid opinion about the impossibility of agree- ing on the term “medical futility”. While it seems premature to exclude the possibility of reaching such a consensus someday, we also cannot state that an agreement will ever be reached. In addition, the second claim could be valid if we assumed that medical futility and persistent therapy are identical terms. In fact, some authors regard these terms as identical (Szewczyk, 2016). While it can be said that these authors narrow the PWG’s definition of the meaning of medical futility, we cannot think that such a narrow interpreta- tion of persistence is legitimated. I think that medical futility should not be identified with persistent therapy from a methodological viewpoint. This is an issue of key importance that I will expand upon in the next point. 54 Marcin Ferdynus The next two claims (3 and 4) refer to unjustified or excessive pater- nalism and the term “dignity”. We agree with the statement that the term “dignity” has many facets, which means that it is ambiguous and, therefore, unclear (Lombard, 2018, pp. 98–100; Napier, 2020, pp. 83–105). There are, however, also opinions such as the one expressed by Immanuel Kant, who argued that dignity has a concrete and absolute character. According to Kant, dignity is a value that has no price and no equivalent for which it could be exchanged. Kant writes: “In the kingdom of ends everything has either a price or a dignity. What has a price can be replaced by something else as its equivalent; what on the other hand is above all price and therefore admits of no equivalent has a dignity [...]; that which constitutes the condi- tion under which alone something can be an end in itself has not merely a relative worth, that is, a price, but an inner worth, that is, dignity” (Kant, 2012, pp. 434–435).9 If, for example, we assume that dignity is an irreducible and unlosable value that cannot be granted or taken away by valuing subjects (other per- sons), it could not be violated in any way. Therefore, the claim of unjustified or excessive paternalism could be annulled, at least from a theoretical view- point (from the definition level). If, however, dignity was identified with au- tonomy, the claim of unjustified paternalism could be legitimate. 9 See also: Kerstein (2019). 10 See also: Schneiderman (2011). Potential replies to claims The PWG’s definition of persistent therapy indicates that the decision on the discontinu- ation of treatment is made by the doctor. However, the problem lies in the fact that members of the PWG do not clarify the term “dignity” used in the definition (Krajewski, 2008). It undoubtedly forms a weak link in this definition. It does not facilitate the discussion either, because it inevitably leads to the valuing of human life. However, we must acknowledge that among conceptions of medical futility there are also a few definitions with a tendency to value life. One such postulate appears, among others, in the conception of medical futility proposed by Schneiderman et al. (1990).10 In the Polish context, similar understandings are voiced by Kübler et al. (2014, p. 230). They stress that a patient benefits from a therapy only when it allows him to survive and be released from the intensive care unit. If this condition is not fulfilled, we can regard such therapy as medically futile. Some ethicists find such Medical Futility or Persistent Therapy? A Dispute over the Terms... 55 criteria confusing and question whether it can be claimed that someone who is dependent on intensive care even for a while cannot live a relatively valuable life (Macauley, 2018). The critics of the qualitative approach in the determination of medical futility stress that physically and mentally disabled persons can obtain much satisfaction in their life, although others may not recognise the potential benefits associated with the therapy undertaken by these patients (White, Pope, 2016). The quantitative criterion is criticised mainly for three reasons: (1) it is unclear at which point the percentage threshold should be fixed, (2) using measurements taken from research on the population level is imprecise, and (3) disease and mortality rates are often based on uncertain predictions (White, Pope, 2016, p. 73).11 Others add that no two identical cases occur in medicine and argue that statistics may prove to be incorrect (Chyrowicz, 2015, p. 317). The next two claims (5 and 6) can be addressed jointly. Although some clinicians say that the PWG’s definition of persistent therapy is burdened with a certain “degree of complexity”, they do not explain where this dif- ficulty lies (Siewiera, Kübler, 2015, pp. 9–10). It is possible that estimating the level of suffering of a dying patient may prove complicated (claim 6). 11 Other authors identify certain problems connected with the estimation of medical futility: Sørensen and Andersen (2019). Potential replies to claims Although the PWG’s definition specifies excessive suffering as the main criterion for therapeutic persistence, some conceptions of medical futility also refer to suffering that requires estimation (Salter, 2020; Aghabarary, Nayeri, 2016; Robinson, 2010; Sibbald, Downar, Hawryluck, 2007). It is, however, beyond doubt and dispute that the estimation of suffering is a sub- jective matter and, therefore, may arouse moral doubts: To what extent is therapeutic persistence related to suffering that is actually persistent? I think that the verification of such situations is not always possible for external observers (even for doctors). Sometimes, this question can be resolved only by the patient (Cassell, 2016). But does the patient’s estimation of his own suffering not argue in favour of the fact that he possesses autonomy?i The seventh claim suggests that the PWG’s definition of persistent therapy is connected with the CC’s moral teachings, which raises concerns that the adoption of this definition will violate the principles of a democratic state of law with a variety of worldviews. To verify the correctness of this claim, we must refer to the content of a representative document in this Marcin Ferdynus 56 respect. The Catechism of the Catholic Church (CCC) seems to fulfil such a role. It reads: “Discontinuing medical procedures that are burdensome, dangerous, extraordinary, or disproportionate to the expected outcomes can be legitimate; it is the refusal of »persistent therapy«. Here, one does not will to cause death; one’s inability to impede it is merely accepted. The de- cisions should be made by the patient if he is competent and able or, if not, by those legally entitled to act for the patient, whose reasonable will and legitimate interests must always be respected” (Catechism of the Catholic Church, no. 2278).12 If we consider this document to be representative with regard to the scope of “persistent therapy”, we must admit again that the PWG’s definition of persistent therapy is close to the definition of medical futility. Thus, we must also assume that the interpretation of “therapeutic persis- tence” by the PWG’s members deviates from the official teachings of the CC. The scope of the definition contained in the CCC is incomparably broader than in the PWG’s definition. Therefore, the impact of the CC’s teachings on the emergence of this definition can be regarded as minor or even non-existent.i I fully agree with the last claims (8 and 9). 12 I am aware that the English version of the Catechism of Catholic Church (CCC) contains the term “over-zealous treatment” and the Polish version of the CCC contains the term “uporczywa terapia”. According to earlier remarks and for consistency of my argumentation I use here the term “persistent therapy”. 13 It must be stress the terms “persistence” and “persistent therapy” are not entirely alien; they appear in international literature (Groarke, 2018; Paris, Cummings, Moore, 2019; Hoehn, 2019), although they are rarely discussed. 12 I am aware that the English version of the Catechism of Catholic Church (CCC) contains the term “over-zealous treatment” and the Polish version of the CCC contains the term “uporczywa terapia”. According to earlier remarks and for consistency of my argumentation I use here the term “persistent therapy”. 13 It must be stress the terms “persistence” and “persistent therapy” are not entirely alien; they appear in international literature (Groarke, 2018; Paris, Cummings, Moore, 2019; Hoehn, 2019), although they are rarely discussed. 15 The term “extraordinary” is included in the definition because it is often used in Polish bioethical literature quoted in this article. However, we can wonder whether the term “extraordinary” could be replaced with the term „burdensome”. 14 The definition of “terminally ill” in Poland is different from the definition used in current medical practice, at least in the US. In the US, it is six months or less (Macauley, 2018, p. 72; Smith, Glare, 2016, p. 172).i 16 The highlighted aspects are established on the basis of the following publication: Pellegrino (2000). Potential replies to claims With regard to the first claim (8), critics are right to say that the term “persistent therapy” is present mainly in Polish literature, thereby hindering the discussion with experts on the international bioethical and medical forum.13 The problem is, however, that Polish ethicists and critics have not joined the international discussion on medical futility until now. This article may serve as a response to the claim quoted here, which can be labelled as provincialism.i The last claim (9) is fully justified. I agree that the popularity of a term in literature – in this specific case, “persistent therapy” – is not a sufficient argument for leaving the term there. However, I think that there is a deeper reason for which the term “persistent therapy” can still be useful. Medical Futility or Persistent Therapy? A Dispute over the Terms... 57 An attempt to overcome the stalemate – Modifying the PWG’s definition 17 Waisel and Troug also emphasise that “The adoption of physiologic futility cannot substantially change practices, if the decision to halt therapy must wait until the physi- ologic objective cannot be achieved, then the patient, for all practical purposes, will be dead nearly immediately after the declaration of physiologic futility. This strength of phys- iologic futility minimises the chance of a physician error in quantitative assessment limiting the length of life.” (Waisel, Troug, 1995, p. 307). An attempt to overcome the stalemate – Modifying the PWG’s definition The PWG’s definition of persistent therapy involves many limitations. Its main shortcoming is the excessive narrowness of its scope, which does not include many real case studies of occurrences in intensive care units (Ferdynus, 2017, pp. 121–127). Such a definition of persistent therapy is, thus, not useful in medicine. Therefore, I propose modifying PWG’s defini- tion of persistent therapy as follows: Persistent therapy is the application of extraordinary or causally inef- ficacious medical procedures to sustain the life of a terminally ill patient.i Wording the definition in this way does not narrow persistent therapy to only terminally ill persons who are considered to be in the state of dy- ing. In this definition, a terminally ill person can be interpreted as a patient who will die within twelve months or less (Szewczyk, 2009, p. 291; Fer- dynus, 2017, p. 122),14 and the expert in the ascertainment of the terminal state is the physician.ii This modified version of the PWG’s definition also states that persistent therapy is related to the application of extraordinary medical procedures.15 This extraordinariness is determined by various aspects that directly con- cern the patient: (a) physical, (b) emotional (psychological), (c) axiologi- cal, and (d) economic.16 The last aspect does not suggest that human life should be subject to economic calculations, but that costs related to the ap- plication of a therapy cannot be completely ignored (Hughes, 2020). Thus, regarding the physical aspect (a), the recognition of a therapy as persistent may involve pain that is difficult to eliminate or, more broadly, excessive suffering. With respect to the emotional aspect (b), persistence may involve fear or a strong reluctance to undergo the therapy (e.g., an ALS patient who no longer wishes to be permanently connected to a respirator). The patient’s 58 Marcin Ferdynus subjective preferences regarding values and beliefs (e.g., moral and religious ones; Pellegrino, 2005) can be regarded as persistent in the axiological sense (c). Persistence in an economic sense (d) will involve significant effort to acquire a medical device or costs that prove burdensome for the patient (or their family; Ferdynus, 2017, p. 143). I do not claim that this is the only set of criteria that allows us to regard a therapy as persistent. An attempt to overcome the stalemate – Modifying the PWG’s definition However, I as- sert that the scope of therapeutic persistence is not limited to the “excessive suffering” that the PWG indicates in its definition.fi Referring to medical procedures as “causally inefficacious” means that a therapeutic action undertaken by a physician cannot achieve the intended physiological goal, and, thus, it is medically futile (Bosslet, Pope, Rubenfeld, Lo, Truog, Rushton, White, 2015; Waisel, Troug, 1995, p. 306). We can also say that if the treatment cannot restore irreversibly lost functions of the body (e.g., a patient has irreversibly lost their capacity to breathe independently) or cannot stop the progression of the disease (e.g., the ineffectiveness of aspirin in managing cancer, of defibrillation on asystole, or of conventional cardiopulmonary resuscitation for a patient with myo- cardial rupture; Aghabarary and Nayeri, 2016), then it is medically futile. I agree with the opinion of Waisel and Troug, who emphasise the following: “If a treatment cannot achieve a physiologic objective, and thus, no benefit is being offered to the patient, then a physician is not obligated to offer this treatment. Physiologic futility appears to have the least risk for unilaterally imposed physician value judgments. Admittedly, problems may occur in determining precise definitions of physiologic functions (such as circulation and ventilation), but these are more technical in nature and do not involve substantial value judgments” (Waisel, Troug, 1995, p. 306).17 I think that physicians as professionals can ascertain physiological futility based on their current clinical knowledge. Moreover, the basis for refusing treatment is a scientific and empirical one: the therapy either works or it does not. I do not depreciate statistical data in clinical practice – data can help determine medical management. However, I argue that the Medical Futility or Persistent Therapy? A Dispute over the Terms... 59 understanding of medical futility presented above avoids unnecessary ques- tions such as the following: Is an effect so small that it is not worth pursuing? How small should the chance of successful treatment be to qualify as futile (i.e., one in a hundred, a thousand, a million; Wilkinson, Savulescu, 2019, p. 27)? Furthermore, from a methodological point of view, it seems right to ascertain medical futility only with regard to strictly medical factors (i.e., to consider the physiological aspect). 18 It must be stressed that while the persistent therapy in the narrow sense (medical futility) relies on causal links and efficacy, persistent therapy in the broad sense involves evaluative judgements on the relationship between the burdens and benefits of medical intervention. An attempt to overcome the stalemate – Modifying the PWG’s definition Only with such an interpretation of medi- cal futility can I agree with the authors of “Principles of biomedical ethics” that doctors are not obliged to initiate or continue such therapy (Beauchamp, Childress, 2012, p. 169), even when the patient, their family, or their rep- resentative exerts strong pressure in this respect (Caplan, 2012). The deter- mination of medical futility is a medical decision; therefore, it falls within the competence of the doctor (Chańska, 2009, p. 212), not an ethicist, the patient, their family, or their representative (Terra, Powell, 2012, p. 104). Furthermore, the scholarship on the subject emphasises that the continua- tion of medical futility is a medical error (Kübler, Siewiera, Durek, Kusza, Piechota, Szkulmowski 2014, p. 230). Considering what has been said so far, we can suppose that persistent therapy should be understood within both a narrow and a broad semantic scope. In other words, I maintain that “persistent therapy” (as a single term) should be given with dual interpretations. In the broad sense, the content of persistent therapy may overlap with the modified version of the PWG’s definition as presented in this paper (or with a similar version). In the narrow sense, persistent therapy is a form of medical futility (i.e., causally inefficacious therapy). The essential difference between the broad and nar- row understanding of persistent therapy is that in the former, apart from medical factors, the patient’s (or surrogate’s) subjective preferences take precedence.18 Moreover, medical futility is reduced only to strictly medical factors, and the expert in ascertaining medical futility (or persistent therapy in the narrow sense) is the physician, not the patient. It seems that we can consider leaving both terms – “persistent therapy” and “medical futility” 60 Marcin Ferdynus – to bioethical and medical regulations only if the indicated methodological reservations are considered. – to bioethical and medical regulations only if the indicated methodological reservations are considered. Concluding remarks As indicated at the beginning, the focus of the analyses was on the dispute surrounding definitions and terms. This means that many other issues have been intentionally excluded because their analysis would largely go beyond the limits of this paper. Finally, I would like to add a few further remarks.l First, a large proportion of the conflicts emerging in intensive care units relates to the commencement, continuation, or discontinuation of persistent therapy. On the one hand, patients or their representatives sometimes attempt to persuade doctors to apply persistent therapy within a narrower scope (i.e., medical futility). However, as I have demonstrated, a patient’s demands are completely unjustified when “persistent therapy” is understood in a narrow sense, and the patient has no right to demand the continuation or commence- ment of medical futility from the doctor. The expert in deciding on such a therapy is the doctor (or the medical team). On the other hand, the situation is different when a therapy is considered to be persistent within a broader scope. With regard to the ascertainment of discontinuing such therapy, pa- tients have the right to demand that their subjective decisions be respected. I am far from arguing that the ascertainment of persistence within both scopes is simple and that each doctor discontinuing therapy is free of doubts. How- ever, I wish to reiterate the aforementioned opinion that no two identical cases in medicine exist, and statistics may prove to be incorrect. Therefore, we must agree with those who believe that the patient’s individual condition should be taken into account in any decision on discontinuing therapy. Nev- ertheless, doctors require criteria to decide on discontinuation, even if these criteria are not unequivocal and individual cases are subject to discussion. Second, it seems important to solve all conflicts between the patient and the physician through dialogue (Quill, Holloway, 2012; Burkle, Benson, 2012; Brody, 1997, p. 14). Building a warm relationship based on openness and trust between the doctor (or medical personnel) and the patient (or family or representative) may prove to be a key issue in making a decision on discon- tinuing therapy (McAndrew, Hardin, 2020; Andersen, Abrahams, Borasio, Carvalho de, Chio, Damme Van, Weber, 2012). Third, palliative care as an Medical Futility or Persistent Therapy? A Dispute over the Terms... 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In: S.G. Post (ed.), Encyclopedia of Bioethics (pp. 1718–1721). Vol. 3. New York: Macmillan Reference. Summary This article presents the current discussion around the terms “medical futility” and “persistent therapy” and their definitions. This discussion is based on the dis- pute of whether the term “persistent therapy” should be rejected and replaced with the term “medical futility” in Polish bioethical and medical regulations. The dispute started after the Polish Working Group on End-of-Life Ethics (PWG) had published its definition of persistent therapy in 2008. To settle the dispute, the author proposes a modified version of the PWG’s definition of persistent therapy that combines persis- tence and futility. He argues that persistent therapy is the application of extraordinary or causally inefficacious medical procedures to sustain the life of a terminally ill patient. He also asserts that medical futility can be useful for bioethics only when its definition is limited to medical factors.
https://openalex.org/W3204562826	https://ora.ox.ac.uk/objects/uuid:2c5ca876-fa06-4dea-8fe3-b57d1ada937e/files/sdb78td15p	English	null	Optima TB: A tool to help optimally allocate tuberculosis spending	PLOS computational biology/PLoS computational biology	2,021	cc-by	13,422	PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY OPEN ACCESS Citation: Gosce´ L, Abou Jaoude GJ, Kedziora DJ, Benedikt C, Hussain A, Jarvis S, et al. (2021) Optima TB: A tool to help optimally allocate tuberculosis spending. PLoS Comput Biol 17(9): e1009255. https://doi.org/10.1371/journal. pcbi.1009255 Abstract Approximately 85% of tuberculosis (TB) related deaths occur in low- and middle-income countries where health resources are scarce. Effective priority setting is required to maxi- mise the impact of limited budgets. The Optima TB tool has been developed to support ana- lytical capacity and inform evidence-based priority setting processes for TB health benefits package design. This paper outlines the Optima TB framework and how it was applied in Belarus, an upper-middle income country in Eastern Europe with a relatively high burden of TB. Optima TB is a population-based disease transmission model, with programmatic cost functions and an optimisation algorithm. Modelled populations include age-differentiated general populations and higher-risk populations such as people living with HIV. Populations and prospective interventions are defined in consultation with local stakeholders. In partner- ship with the latter, demographic, epidemiological, programmatic, as well as cost and spending data for these populations and interventions are then collated. An optimisation analysis of TB spending was conducted in Belarus, using program objectives and con- straints defined in collaboration with local stakeholders, which included experts, decision makers, funders and organisations involved in service delivery, support and technical assis- tance. These analyses show that it is possible to improve health impact by redistributing cur- rent TB spending in Belarus. Specifically, shifting funding from inpatient- to outpatient- focused care models, and from mass screening to active case finding strategies, could reduce TB prevalence and mortality by up to 45% and 50%, respectively, by 2035. In addi- tion, an optimised allocation of TB spending could lead to a reduction in drug-resistant TB infections by 40% over this period. This would support progress towards national TB targets RESEARCH ARTICLE Optima TB: A tool to help optimally allocate tuberculosis spending Lara Gosce´ ID1☯, Gerard J. Abou JaoudeID1☯, David J. Kedziora2☯, Clemens BenediktID3, Azfar HussainID2, Sarah JarvisID2, Alena Skrahina4, Dzmitry Klimuk4, Henadz Hurevich4, Feng Zhao3‡, Nicole Fraser-HurtID3, Nejma CheikhID3, Marelize Gorgens3, David J. Wilson3, Romesh AbeysuriyaID2, Rowan Martin-HughesID2, Sherrie L. KellyID2, Anna RobertsID2, Robyn M. StuartID2,5, Tom PalmerID1, Jasmina Panovska-GriffithsID1,6, Cliff C. Kerr2, David P. Wilson2‡, Hassan Haghparast-BidgoliID1, Jolene SkordisID1, Ibrahim AbubakarID1 1 University College London, London, United Kingdom, 2 Burnet Institute, Melbourne, Australia, 3 World Bank, Washington, District of Columbia, United States of America, 4 The Republican Scientific and Practice Centre for Pulmonology and Tuberculosis, Minsk, Belarus, 5 University of Copenhagen, Copenhagen, Denmark, 6 University of Oxford, Oxford, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ‡ FZ is Unavailable. DPW’s contributions for this paper were conducted at Burnet Institute where ideas for the Optima TB initiative originated, prior to joining the Bill & Melinda Gates Foundation (BMGF). He has since removed association with Optima TB. D.P. Wilson does not currently specifically endorse Optima TB nor is this manuscript represented by the BMGF. l @ l k Author summary Tuberculosis (TB) remains a leading global cause of death and morbidity, and 85% of deaths occur in countries where resources for TB care and control are limited. Many countries cannot finance all TB interventions or technologies, which means difficult deci- sions on what to prioritise and publically finance. Modelling tools can help decision-mak- ers set priorities based on evidence, in a systematic and transparent way. This study presents Optima TB, a tool that estimates which allocations of spending across interven- tions will most likely maximise specified objectives—such as minimising TB deaths, prev- alence and incidence. In partnership with local decision-makers and stakeholders, Optima TB was applied in Belarus. Recommendations from the model findings include focussing investment on outpatient rather than inpatient care and actively finding people with TB (e.g. through contact tracing) rather than mass testing of the population. The rec- ommended reallocations of spending could reduce TB prevalence and deaths by up to 45% and 50%, respectively, by 2035 for the same amount of spending. Key stakeholders were engaged throughout the analysis and findings and uncertainty around the results were clearly communicated with decision-makers. The timeliness of the results helped inform national dialogue on TB care reform, among other key policy discussions. PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending without additional financial resources. The case study in Belarus demonstrates how reallo- cations of spending across existing and new interventions could have a substantial impact on TB outcomes. This highlights the potential for Optima TB and similar modelling tools to support evidence-based priority setting. without additional financial resources. The case study in Belarus demonstrates how reallo- cations of spending across existing and new interventions could have a substantial impact on TB outcomes. This highlights the potential for Optima TB and similar modelling tools to support evidence-based priority setting. without additional financial resources. The case study in Belarus demonstrates how reallo- cations of spending across existing and new interventions could have a substantial impact on TB outcomes. This highlights the potential for Optima TB and similar modelling tools to support evidence-based priority setting. Competing interests: The authors have declared that no competing interests exist. Author Feng Zhao was unavailable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge. Editor: Roger Dimitri Kouyos, University of Zurich, SWITZERLAND Editor: Roger Dimitri Kouyos, University of Zurich, SWITZERLAND Editor: Roger Dimitri Kouyos, University of Zurich, SWITZERLAND Received: December 3, 2020 Accepted: July 7, 2021 Published: September 27, 2021 Copyright: © 2021 Gosce´ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data and summary of results are openly available in the World Bank Open Knowledge Repository http://hdl.handle.net/10986/ 27475. Funding: This study was supported by the World Bank Group (www.worldbank.org). The Bank team provided input in study design, assisted with data collation efforts, in interpreting and disseminating results, and were involved in the decision to submit this paper for publication. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 1 / 24 PLOS COMPUTATIONAL BIOLOGY Introduction The past decade has seen global improvements in key TB indicators, including incidence and notifications reported by National Tuberculosis Programmes (NTPs). However, while global active TB incidence has decreased at an annual rate of 1.5–1.8%, this fell short of the 4–5% decline required by 2020 to meet the End TB strategy milestones [1, 2]. Furthermore, to meet the End TB 2035 targets of treating at least 90% of all incident cases [3], the rate of active TB notification (estimated at 69% of incidence in 2018) [4] must increase substantially. Diagnosed people must then be linked to effective care and treatment. Approximately 85% of TB-related deaths occur in low- and middle-income countries, where available resources for TB programmes are scarce [4]. The emergence of new drugs and technologies, such as bedaquiline, GeneXpert tests and geospatial mapping to inform targeted screening, offer additional options in the TB response. However, governments and NTPs are not able to fully finance all available interventions. As such choices must be made regarding which interventions to prioritise and at what level of coverage for populations in need. Best practice for priority setting involves evidence-based, systematic and transparent deci- sions that reflect trade-offs, with a high level of stakeholder involvement [5, 6]. When setting priorities, governments of low- and middle-income countries are faced with significant chal- lenges including financial barriers and limited experience with decision-science [6–8]. Model- ling tools can support analytical capacity to inform evidence-based decision-making [9, 10]. Allocative-efficiency modelling tools in particular enable the health impact of different inter- ventions or packages of services to be estimated for a given level of spending [10, 11]. In PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 2 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending addition, such analyses can support transparent decision making, provided they are carried out with appropriate consultation [10]. Common objectives for TB responses include minimis- ing new TB infections, TB-related deaths, disability-adjusted life years (DALYs), and current and future TB-related costs. addition, such analyses can support transparent decision making, provided they are carried out with appropriate consultation [10]. Common objectives for TB responses include minimis- ing new TB infections, TB-related deaths, disability-adjusted life years (DALYs), and current and future TB-related costs. A number of tools currently provide evidence on allocative efficiency for TB responses, including TIME Impact, AuTuMN, SEARO and EMOD [12–16]. Introduction These have already been applied in a range of countries and typically simulate (a) TB transmission within and between population groups, (b) TB disease progression, (c) the effects of TB prevention, testing and treatment programmes, and (d) the economic effects of policy choices. However, if developed to enable linking with cost functions, which represent the relationship between intervention spending and corresponding coverage levels, and an optimisation algorithm, mathematical modelling tools can be helpful in determining an optimised resource allocation for defined objectives [17]. Resource optimisation models can also be used to estimate the minimal amount of resources required to achieve specific targets. This paper presents Optima TB, an open-source tool to support and inform decision-mak- ing to improve the TB response. Optima TB is part of the modelling suite of the Optima Con- sortium for Decision Science (OCDS) [18–20], which has developed and applied disease- specific resource optimisation models in collaboration with governments in over 60 countries, the World Bank, non-governmental organisations, local stakeholders and academic institu- tions. Optima TB draws on this experience and builds on existing interdisciplinary dialogues between modelers, epidemiologists and government officials. The paper describes how Optima TB was specifically designed to support countries in prioritising available resources for TB control through allocative efficiency analyses. To illustrate the use of Optima TB in country decision-making, a case study in Belarus is presented. Belarus has the highest proportion of TB drug-resistance worldwide, comprising 38% and 67% of new and retreated cases, respectively. Globally, the median cost of treating drug-resis- tant TB (DR-TB) is often at least six times higher than treating drug-susceptible TB (DS-TB), and treatment outcomes are less successful (at a rate of 55%) compared with those for DS-TB (82%) [21]. At the time of analysis, the provision of TB treatment in Belarus relied on an ageing infrastructure of costly tertiary care facilities, and ineffective practices such as population-wide mass-screening using chest X-rays [22]. There was thus a need to investigate the cost and impact of the TB response in Belarus, and to revisit the package of services provided by the NTP. These TB care practices and challenges are common elsewhere in Eastern Europe and as such, the findings of this case study will have relevance to other countries [23–27]. Introduction The following sections describe how the Optima TB tool was used to determine a recom- mended package of priority interventions and associated spending allocations, as well as esti- mate the potential impact on key TB indicators if there were to be a change in policy based on these recommendations. Optima TB methodology The Optima TB incorporates four main components: (a) an underlying epidemiological dynamic disease transmission model to which intervention outcomes are linked; (b) cost-func- tions that combine data on intervention expenditure and coverage to estimate and project intervention outcomes; (c) objective functions often reflecting national strategic targets, along- side constraints to reflect logistic, ethical, political and financial considerations; and (d) a mathematical optimisation algorithm that combines (a)-(c) to identify the most efficient allo- cation of resources. These components are depicted schematically in Fig 1. 3 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Fig 1. Schematic of an Optima TB analysis (source: OCDS). https://doi.org/10.1371/journal.pcbi.1009255.g001 Fig 1. Schematic of an Optima TB analysis (source: OCDS). https://doi.org/10.1371/journal.pcbi.1009255.g001 https://doi.org/10.1371/journal.pcbi.1009255.g001 The epidemiological model at the core of the Optima TB tool is represented in Fig 2 and is described below. The mathematical optimisation algorithm used is detailed elsewhere [28]. The version of Optima TB code used for the Belarus analysis is open source and open access (https://github.com/optimamodel/optima-tb). The latest epidemiological model is implemented within the open access Atomica tool (https://github.com/atomicateam/ atomica). The user interface and webserver components are written using Sciris (https:// github.com/sciris/sciris). The tool is available as part of the “Atomica Applications” suite (https://github.com/sciris/atomica_apps). The interface itself is available at http://tb. ocds.co. Model overview. The model is a compartmental model of disease transmission. While individual based models (IBMs) are best at capturing factors that influence infection on a per- son-by-person base, this structure was chosen because of the advantages it offers when study- ing disease transmission in a population on a large scale. Furthermore, although infection progression towards TB disease is a heterogeneous process [29] where the 10% lifetime risk of disease following infection [30] can vary according to age and co-infections [31], Optima TB is able to capture these differences. p Specifically, Optima TB allows for several populations to be defined, each population with its respective structure shown in Fig 2 and each with distinct parameter values. These popula- tion structures are then simulated to interact. This includes people with co-morbidities; these populations are modelled with modified parameters for TB disease progression, mortality risk, and co-morbidity treatment coverage such as antiretroviral therapy for people living with HIV. Accordingly, Optima TB is able to readily capture the following aspects with deterministic modelling: • Development of TB disease and its severity is age dependent. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 Optima TB methodology Children aged under 5 and those infected with latent TB are at higher risk of progressing to active TB [32–34]. • Incidence of TB is higher in individuals with impaired immunity [35]. Consequently, co- morbidities with other illnesses that suppress immunity (e.g. HIV infection, diabetes etc.) often entail a higher probability of developing active TB [36–39]. • TB transmission is much higher in closed and crowded environments such as prisons [40] and mines [41, 42]. 4 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Fig 2. Structure of the epidemiological model at the core of Optima TB. Square boxes in the figure represent compartments, or population sizes, at a given point in time. Ellipses represent junctions, which unlike compartments do not represent population sizes, and only enable the ‘filtering’ of flows of latent activations into either smear-positive or smear-negative, and subsequently into drug-susceptible or a form of drug-resistant TB. Solid lines between boxes and ellipses represent transition rates, or the probability of moving from one compartment to another, within a given timeframe. Dashed lines represent specific transition rates relating to the probability of interrupted treatment or developing multi-drug resistant or extensively drug-resistant TB while on-treatment. https://doi.org/10.1371/journal.pcbi.1009255.g002 Fig 2. Structure of the epidemiological model at the core of Optima TB. Square boxes in the figure represent compartments, or population sizes, at a given point in time. Ellipses represent junctions, which unlike compartments do not represent population sizes, and only enable the ‘filtering’ of flows of latent activations into either smear-positive or smear-negative, and subsequently into drug-susceptible or a form of drug-resistant TB. Solid lines between boxes and ellipses represent transition rates, or the probability of moving from one compartment to another, within a given timeframe. Dashed lines represent specific transition rates relating to the probability of interrupted treatment or developing multi-drug resistant or extensively drug-resistant TB while on-treatment. https://doi.org/10.1371/journal.pcbi.1009255.g002 Equations and parameters. The model structure is depicted in Fig 2 and this section pro- vides mathematical labels for the number of people in each compartment. All individuals are born susceptible, S; some of them (usually children) get vaccinated and move into the vacci- nated compartment V. People who get infected with Mycobacterium Tuberculosis first move into the early latent untreated compartment, Lu e. Optima TB methodology If they do not develop active TB in the first 5 years after infection, they move into the late latent untreated compartment, Lu l . The model also considers treatment for latent tuberculosis, represented by the early latent on-treatment and late latent on-treatment compartments, Lt e and Lt l, respectively, along with those successfully treated from latency, J. People who received preventive treatment (in the form of vaccination or latency treatment) once reinfected move to their own latency pathway, Lp e and L p l , where they do not generally have the possibility of getting treated again (except for cases such as peo- ple living with HIV (PLHIV) and specific scenarios). This is the ‘diagnosis restricted’ pathway in Fig 2. If active TB arises, people move into the undiagnosed active disease set of compartments, Du (via a single intermediate subclinical compartment, Du e; see Eq A in S1 File). There are six undiagnosed compartments; the active TB pathway is divided by smear, SP for positive and SN for negative, as well as by strain type, i.e. drug sensitive (DS), multi drug resistant (MDR) and extensively drug resistant (XDR). A proportion of these individuals get tested and move into a diagnosed compartment set, Dd, similarly divided by SP/SN and DS/MDR/XDR. Those that start treatment move into active-disease on-treatment compartments, subdivided in the same way. Last, individuals who complete active TB treatment move into compartment R, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 5 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending where they remain for two years. If they experience relapse, they move back to compartments Du, otherwise they move to compartment J from which reinfection is possible. where they remain for two years. If they experience relapse, they move back to compartments Du, otherwise they move to compartment J from which reinfection is possible. The epidemiological motivations behind this modelling design, as well as its equations and parameter definitions, are provided in S1 File, where in-depth discussions about vaccination, latency, reactivation, drug-resistance and TB recurrence can be found. Naturally, data are not always available to inform the compartment sizes and the transition rates for specific country contexts. Assumptions can be made, but these can arguably lead to more uncertainty and spurious claims than would have arisen if a dynamic feature were entirely excluded. Optima TB methodology Optima TB was designed from the start to circumvent rigidity; coded in Python and leveraging its object-oriented paradigm, it is easy to include or exclude compart- ments and transitions as required. For instance, the model uses parameters δ1 and δ2 to include the possibility of assuming different fitness for MDR and XDR strains respectively, and also six parameters ^ti to study the possible escalation of drug-resistance following incomplete treat- ment or treatment with non-protocol based regimens. Moreover, the standard form of our TB model treats latency far more comprehensively than is commonly done in other models because of the scarcity of data on latent infections. However, estimates of disease progression from latent to active TB and of untreated active TB outcomes are calculated from very old studies, for which reproducibility would be impossible nowadays, and they are therefore sub- ject to adaptation through calibration (more details are available in S1 File). Calibration is a multi-step, iterative process. First the model is calibrated against population demographics. Afterwards model estimations are compared with existing data, including esti- mations of incidence and prevalence from World Health Organisation (WHO) or national data sources [21] disaggregated by sub-populations such as age groups, smear status and/or drug sensitivity. Cost and impact. Optima TB accommodates interventions that directly or indirectly tar- get TB. The former includes prevention, diagnosis and treatment interventions and the latter includes interventions such as behavioural change and awareness campaigns. To include an intervention in an Optima TB analysis, the following must be specified or informed by data: (a) populations served; (b) intervention impact (e.g. diagnostic yield, or probability of success- fully completing treatment); (c) unit cost; and (d) intervention coverage. Non-targeted TB programmes, for which a direct impact cannot be assigned, such as management and adminis- tration activities, are still costed and included in the analyses but are not included within the optimisation process. National expenditure on TB is rarely tracked and reported by intervention, but there is often an estimate of total spending on TB and occasionally a disaggregation by broad interven- tion category such as prevention, diagnosis or treatment. Intervention spending is therefore often estimated using either a top-down approach or a bottom-up calculation, using unit costs and program coverage. Application in Belarus This section details the process of applying Optima TB in Belarus, including data collation, model calibration, interventions modelled, and an optimisation analysis of TB spending. This section details the process of applying Optima TB in Belarus, including data collation, model calibration, interventions modelled, and an optimisation analysis of TB spending. A request for technical support to prioritise TB interventions was made by the Ministry of Health of the Republic Belarus to the World Bank. In addition, an NTP review had found that the Belarus TB response required further alignment with WHO recommendations and guide- lines [45]. Following discussions with relevant stakeholders, an allocative efficiency analysis was selected as the preferred approach to inform national priority setting and TB reform pro- cesses. A group of key stakeholders was then formed, alongside a smaller working group of local experts from The Republican Research and Practical Centre for Pulmonology and TB. A full list of the stakeholders involved, is included in S2 File. Stakeholders were kept informed throughout the process and reviewed optimisation objectives and results, while the expert working group provided input throughout the analysis, generated and validated assumptions, and reviewed results. Results were presented in a dissemination workshop and an application report was generated and validated by the national team, funders, and stakeholders. The appli- cation report for Belarus is posted on the funder’s website (https://openknowledge.worldbank. org/handle/10986/27475) and on the Optima Consortium for Decision Science website (http://ocds.co/tb/applications.html). Data collation, calibration, and validation. Six populations were defined for Belarus as follows: (1) people age 0–4 years, (2) 5–14 years, (3) 15–64 years (without HIV), (4) 65+ years (without HIV), (5) people living with HIV aged 15 years or more, and (6) inmates aged 15 years or more. For Belarus, United Nations (UN) Population Division World Prospects data, local census data and national reports were used to inform population sizes, migration rates, birth rates and non-TB death rates [46–49]. Data on the number of notified TB infections were obtained from the National TB Programme database [50], and other epidemiological data were compiled from local and international publications or reports [51–54]. Key population statistics are provided in Table 1. Intervention cost and expenditure data were sourced from the Belarus TB sub-accounts within the WHO National Health Accounts and other secondary sources [22, 55–57]. Optima TB methodology By default, the number of people that can be covered by a program can be defined either to scale linearly with expenditure, with a capacity constraint for the maxi- mum number of people that can be covered in a year, or with a saturation value to represent demand constraints such as hard to reach populations. The scaling to reach this saturation value as a percentage of the target population is non-linear, with the marginal unit cost increasing as coverage approaches saturation. Optimisation. Optimisation aims to identify the combination of funded interventions (investments) that achieves the best possible outcome with respect to the optimisation objec- tives and subject to the optimisation constraints. The objective for optimisation can be to min- imize TB-related DALYs, TB-related deaths, total new active TB infections, the prevalence of DS-TB, MDR-TB, or XDR-TB, or any (user-specified) weighted combination thereof. For each intervention, minimum and maximum spending constraints can be specified. 6 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Optimisation can be performed using one of three built-in optimisation algorithms. By default, Optima TB uses Adaptive Stochastic Descent (ASD) [28] implemented by the “Sciris” Python package; this is a gradient-based descent algorithm, which makes stochastic downhill steps in parameter space from an initial starting point, choosing future step sizes and direc- tions based on the outcome of previous steps. Other optimisation algorithms available in Optima TB are particle swarm optimisation [43] via the “Pyswarm” package, which is more computationally expensive but is better able to find global minima if parameter space is com- plex, and a sequential model-based optimisation algorithm via the “Hyperopt” package [44], which balances the exploration of global and local minima. Application in Belarus Data on the coverage and impact of interventions were sourced from the National TB Programme dataset, and a comprehensive review of local and international litera- ture provided additional data where required [50, 52, 58–71]. Local stakeholders and experts were consulted throughout the analysis to provide input on missing or inconsistent data as well as on epidemic projections. Subsequently, the model was calibrated through manual fitting to match closely to the annual numbers of notified TB infections, as well as estimates of key TB indicators such as active TB incidence/prevalence (Fig 3) and latent TB prevalence. Parameters with the greatest uncertainty were selected for adjustment during the calibration process. These were the: (1) transmission rate, (2) probability of progressing from early- or late-latent TB to active TB, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 7 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Table 1. Key population statistics. Population 2005 2010 2015 Population Sizes 0–4 429,281 509,595 577,740 5–14 1,007,770 865,489 921,333 15–64 6,718,570 6,660,400 6,393,500 65+ 1,414,080 1,306,960 1,318,510 PLHIV (15+) 15,013 21,040 34,089 Prisoners (15+) 36,948 37,352 33,388 TB Prevalence 0–4 0.03% 0.01% 0.01% 5–14 0.06% 0.06% 0.04% 15–64 0.18% 0.16% 0.15% 65+ 0.15% 0.15% 0.14% PLHIV (15+) 2.34% 2.00% 1.31% Prisoners (15+) 1.41% 0.70% 0.41% TB Incidence 0–4 50 24 13 5–14 241 177 138 15–64 3,223 2,980 2,730 65+ 594 547 554 PLHIV (15+) 97 110 155 Prisoners (15+) 142 73 42 TB-related mortality 0–4 16 9 4 5–14 62 49 33 15–64 1,131 1,128 884 65+ 192 189 165 PLHIV (15+) 59 72 71 Prisoners (15+) 56 29 14 https://doi.org/10.1371/journal.pcbi.1009255.t001 https://doi.org/10.1371/journal.pcbi.1009255.t001 (3) re-infection rate of people recovered from active TB, and (4) the proportion of early- versus late-latent TB infection. Model inputs, calibration parameter values, and epidemic projections were compared against peer-reviewed publications, parameter values used in other modelling studies, and secondary data and estimates and epidemic projections as part of the calibration process. Further details on the process as well as the estimates used, are contained in the pub- lished report [72]. Interventions modelled. Different types of interventions can be included in Optima TB, such as preventative therapy, screening and diagnosis, active TB treatment, and other TB- related activities such as management, procurement, or human resources. Table 2 details the interventions modelled in the Belarus analysis. Application in Belarus To derive the unit costs for each intervention listed in Table 2, a mixture of top-down and bottom-up costing was undertaken using a calcu- lated average cost per diem of inpatient or outpatient visits (see S2 File). National drug pro- curement records for 2016 and other secondary data were consulted, as well as expert opinion in cases such as mass-screening [22, 55–57]. Coverage and impact data were then used to gen- erate a cost-function for each intervention, with the exception of non-targeted interventions not considered in the optimisation analysis such as management or procurement activities. Interventions that were not targeted in the optimisation analysis either do not have a direct PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 8 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Fig 3. Calibration output graphs. (a) Datapoints are UN Population Division estimates (2000–2015) [49], (b) and (c) datapoints are WHO active TB prevalence upper-bound estimates disaggregated by population group (2000–2014) [53] and (d) shaded area represents confidence intervals of WHO active TB prevalence estimates for total population (2000–2014) [53]. https://doi org/10 1371/journal pcbi 1009255 g003 Fig 3. Calibration output graphs. (a) Datapoints are UN Population Division estimates (2000–2015) [49], (b) and (c) datapoints are WHO active TB prevalence upper-bound estimates disaggregated by population group (2000–2014) [53] and (d) shaded area represents confidence intervals of WHO active TB prevalence estimates for total population (2000–2014) [53]. https://doi.org/10.1371/journal.pcbi.1009255.g003 https://doi.org/10.1371/journal.pcbi.1009255.g003 https://doi.org/10.1371/journal.pcbi.1009255.g003 measurable impact on the epidemic (non-targeted interventions), such as procurement activi- ties, or did not have sufficient data at the time as was the case for alcohol interventions and pal- liative care. Optimisation analysis. After extensive consultation with a TB working group of stake- holders in Belarus, three key output indicators were identified for the optimisation analysis: (1) TB-related deaths, (2) all TB infections (i.e. prevalence), and (3) new TB infections (i.e. incidence). To determine the optimised allocation of resources, the working group agreed to define an objective function for the ASD model algorithm to minimise these three key output indicators, given the local epidemic parameters and data, cost of delivering services, and sub- ject to defined constraints. Before running the optimisation analysis, constraints shown in Table 3 were therefore defined by key stakeholders and local experts to reflect logistic, ethical, political, and financial barriers for scaling-up or defunding specific interventions. PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Table 2. Interventions included in the Belarus analysis. Intervention Population(s) targeted Description Unit cost (US $, 2015) Spending (US$, 2015) Screening and diagnosis interventions Active Case Finding 15–64; 65+; PLHIV Screening of high-risk groups that are considered at high risk of developing active TB (e.g. PLHIV, homeless, people who inject drugs etc.) with chest X-ray or fluorography $1.00 $942,159 Incentivised Active Case Finding 15–64; 65+; PLHIV Active case finding with subsided transportation for healthcare workers and incentives for each case identified $5.20 Prospective intervention Contact Tracing All populations (contacts of people with active TB) Tracing of household contacts of people diagnosed with active TB, and screening using symptomatic questionnaire, smear-sputum microscopy, and GeneXpert $1.00 $7,046 Incentivised Contactı` Tracing All populations (contacts of people with active TB) Contact tracing with subsided transportation for healthcare workers and incentives for each case identified $5.20 Prospective intervention Symptomatic Diagnosis (including Xpert) All populations screen questions followed by smear-sputum microscopy, Gene Xpert and liquid culture for patients that present at a health facility with symptoms of TB $39.98 $770,489 Mass-Screening All populations Yearly general population-wide mass-screening using chest X-rays and fluorography $1.00 $9,758,596 Treatment for active TB Hospital Focused DS-TB People receiving treatment for DS-TB Treatment for DS-TB with hospitalisation for 60 days out of a total treatment duration of 180 days $2,609.72 $7,283,723 Hospital Focused MDR-TB People receiving treatment for MDR-TB Standardised treatment regimen for multidrug-resistant TB (MDR-TB), with hospitalisation for 180 days out of a total treatment duration of 540 days $14,158.05 $12,884,772 Hospital Focused XDR-TB People receiving treatment for XDR-TB Treatment for extensively drug-resistant TB (XDR-TB) with available second and third-line drugs, with hospitalisation for 240 days out of a total treatment duration of 660 days $20,483.19 $7,445,214 Ambulatory DS-TB People receiving treatment for DS-TB WHO recommended outpatient service delivery, with hospitalisation only during the intensive phase of a given regimen or until smear conversion. Involves hospitalisation for 14 days out of a total treatment duration of 180 days $1,877.83 Prospective intervention Ambulatory MDR-TB People receiving treatment for MDR-TB WHO recommended outpatient service delivery, with hospitalisation only during the intensive phase of a given regimen or until smear conversion. Standardised MDR-TB regimen with hospitalisation for 45 days out of a total treatment duration of 540 days $10,196.29 Prospective intervention Ambulatory MDR-TB Short-Course People receiving treatment for MDR-TB Short-course MDR-TB regimen. Application in Belarus Addition- ally, time-dependent constraints were included to reflect realistic timings for changes in the implementation of interventions. Changes in intervention spending between 2015 and target spending levels were capped either at a maximum change of 30% per year for existing inter- ventions, or at a maximum of US$1M for new interventions for the first year and 30% in sub- sequent years, until the optimised level of spending on an intervention is reached. Interventions for which data on impact were not available, in the form of yields and sensi- tivities for screening or diagnosis and relative risks for treatment outcomes, were treated as PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 9 / 24 PLOS COMPUTATIONAL BIOLOGY All treatment unit costs are per fixed costs and excluded from the optimisation analysis. To achieve the optimisation objective under set constraints, the optimisation analysis aimed to address the following two questions: fixed costs and excluded from the optimisation analysis. To achieve the optimisation objective under set constraints, the optimisation analysis aimed to address the following two questions: 1. What is the optimised allocation of TB spending and associated programme coverage levels, to minimise TB mortality, prevalence, and incidence in Belarus between 2017 and 2035? 2. How much progress will be made toward national and international targets under opti- mised resource allocations in Belarus compared with continuation of the 2015 response? 2. How much progress will be made toward national and international targets under opti- mised resource allocations in Belarus compared with continuation of the 2015 response? https://doi.org/10.1371/journal.pcbi.1009255.t002 PLOS COMPUTATIONAL BIOLOGY Involves hospitalisation for 30 days, out of a total treatment duration of 315 days $4,520.46 Prospective intervention Ambulatory XDR-TB People receiving treatment for XDR-TB WHO recommended outpatient service delivery, with hospitalisation only during the intensive phase of a given regimen or until smear conversion. Treatment for XDR-TB with available second and third-line drugs, with hospitalisation for 60 days out of a total treatment duration of 660 days $15,440.95 Prospective intervention Incentivised Ambulatory DS-TB People receiving treatment for DS-TB Similar to the ambulatory DS-TB intervention, but incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $2,215.36 Prospective intervention Incentivised Ambulatory MDR-TB People receiving treatment for MDR-TB Similar to the ambulatory MDR-TB intervention, but incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $11,324.79 Prospective intervention Incentivised Ambulatory MDR-TB Short-Course People receiving treatment for MDR-TB Similar to the ambulatory MDR-TB short-course intervention, but incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $5,099.96 Prospective intervention Incentivised Ambulatory New Drugs MDR-TB People receiving treatment for MDR-TB Similar to the ambulatory MDR-TB intervention but with new and repurposed drugs, including bedaquiline and linezolid, added to the background regimen. Incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $14,797.00 Prospective intervention Incentivised Ambulatory XDR-TB People receiving treatment for XDR-TB Similar to the ambulatory XDR-TB intervention, but incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $16,782.95 Prospective intervention (C ti d) Table 2. Interventions included in the Belarus analysis. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 10 / 24 PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Table 2. (Continued) Intervention Population(s) targeted Description Unit cost (US $, 2015) Spending (US$, 2015) Incentivised Ambulatory New Drugs XDR-TB People receiving treatment for XDR-TB Similar to the ambulatory XDR-TB intervention but with new and repurposed drugs, including bedaquiline and linezolid, added to the background regimen. Incorporates financial incentives (food packages, outcome-based financing) based on the Mogilev District pilot project $23,036.00 Prospective intervention Involuntary Isolation MDR-TB People receiving treatment for MDR-TB Treatment for people with MDR-TB with a history of adherence problems in a dedicated facility monitored by police $45,588.00 $10,895,532 Involuntary Isolation XDR-TB People receiving treatment for XDR-TB Treatment for people with XDR-TB with a history of adherence problems in a dedicated facility monitored by police $45,588.00 $5,698,500 Preventative interventions IPT for General Population All populations (contacts of people with active TB) Treatment of latent TB Infections with 6-months Isoniazid therapy in general population TB-contacts $11.52 $10,575 IPT for PLHIV PLHIV Treatment of latent TB Infections with 6-months Isoniazid in PLHIV $11.52 $2,477 BCG Vaccination 0–4 years Bacillus Calmette–Gue´rin (BCG) vaccination for newborns $1.32 $528,000 Interventions or activities not included in the optimisation analysis (spending fixed) Solid Culture People with MDR- or XDR-TB Cost of solid culture testing to identify and confirm resistance types of MDR-TB and XDR-TB $1.43 $362,108 Line Probe Assay People with MDR- or XDR-TB Cost of line probe assay (LPA) testing to identify and confirm resistance types of MDR-TB and XDR-TB $16.16 $78,938 Tuberculin Skin Test 0–4 years; PLHIV; contacts of people with active TB Cost of conducting a tuberculin skin test (TST) test to diagnose latent TB infections $4.32 $475,200 Palliative Care MDR/ XDR People receiving treatment for MDR- and XDR-TB Palliative care for MDR-TB and XDR-TB patients with a history of non-adherence, repeated treatment failure, and adverse reactions $5,108.00 $2,894,426 Alcohol Intervention People receiving treatment for active TB with problems of alcohol abuse Cost of alcohol programmes to support adherence to TB treatment regimens n/a $210,000 Management (Including HR) n/a Administrative costs n/a $892,061 Procurement Costs n/a As per TB sub-accounts of NHA n/a $649,349 Other Costs n/a As per TB sub-accounts of NHA n/a $255,055 d in the tool. of service delivery in addition to medicine, test kits, etc. All treatment unit costs are per DS-TB, MDR-TB or XDR-TB case. Note: All unit costs include the cost of service delivery in addition to medicine, test kits, etc. Optimised TB spending allocation in Belarus Optimised TB spending allocation in Belarus In 2015, an estimated US$61.8 million was spent on the TB programme and TB-related activi- ties in Belarus. The distribution of total TB spending across the various TB interventions in 2015 is shown in Fig 4. Annual population-wide mass-screening with chest X-rays accounted Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 11 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Table 3. Interventions with defined constraints in the optimisation analysis. Intervention name Minimum budget constraint relative to 2015 intervention spending Maximum budget constraint relative to 2015 intervention spending BCG vaccination 100% 100% Testing: TST1, LPA2, and solid culture testing 100% 100% Mass-screening (including X- ray) 50% - Active case finding for key populations 100% - Hospital-based treatments for DS-, MDR- and XDR-TB 30% - Palliative care 40% 40% Involuntary isolation for MDR- and XDR-TB 20% - 1 Tuberculin Skin Test: TST. 2 Line Probe Assay: LPA. https://doi.org/10.1371/journal.pcbi.1009255.t003 Table 3. Interventions with defined constraints in the optimisation analysis. entions with defined constraints in the optimisation analysis for one-sixth of total TB spending (US$9.8 million), with little investment in contact-tracing and other targeted active case finding interventions. Approximately 45% of total TB spending was invested in hospital-focused interventions (US$27.6 million), of which 74% was on DR-TB treatment (US$20.3 million). Another significant portion of total TB spending (approximately 25%) was on involuntary isolation facilities for drug-resistant treatment of patients at high-risk of loss-to-follow up, with a unit cost of US$45,588. The mathematically optimised allocation of TB spending that would simultaneously mini- mise active TB incidence and prevalence and TB-related mortality in Belarus according to our model is shown in Fig 2. Overall, recommendations from the analyses are that spending be shifted from hospital-focused interventions to outpatient treatment. Similarly, investment in annual mass-screening should be de-prioritised in order to prioritise targeted active case find- ing strategies. Spending on hospital focused treatment and involuntary isolation is reduced by 60% in the optimised budget compared with the 2015 spending allocation. In turn, spending on more cost-effective outpatient treatment interventions is increased to 40% of the optimised spending on treatment, equivalent to 20% of the total targeted TB budget. These reallocations could save around 30% of total treatment expenditure for re-investment in other interventions, while treatment coverage for people diagnosed with TB would increase from 81% to 90%. Optimised TB spending allocation in Belarus Sav- ings from the use of more cost effective outpatient treatment and reduced spending on mass screening, should ideally be reinvested to expand targeted screening and diagnosis. Rapid diag- nostic testing, targeted active case finding, and contact-tracing interventions are recom- mended for prioritisation, comprising 30% of total TB spending or 80% of spending on screening and diagnosis. Projected impact of optimised spending on the TB epidemic in Belarus Projected impact of 2015 and optimised allocations of national TB spending on key TB indicators among HIV-negative adults and people living with HIV from 2015 to 2035. Fig 5. Projected impact of 2015 and optimised allocations of national TB spending on key TB indicators among HIV-negative adults and people living with HIV from 2015 to 2035. https://doi.org/10.1371/journal.pcbi.1009255.g005 https://doi.org/10.1371/journal.pcbi.1009255.g005 Projected impact of optimised spending on the TB epidemic in Belarus Epidemiological projections are shown in Fig 5, where the grey lines assume that the amount and allocation of TB spending across interventions in 2015 is maintained until 2035. The prev- alence of TB is projected to decrease rapidly among HIV-negative populations aged 15–64 years, before stabilising around 2023. TB-related deaths are also estimated to decrease, although steadily. While there is a projected gradual decrease in the prevalence of TB among people living with HIV, the number of new TB infections and TB-related deaths are projected Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 12 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Fig 4. National TB spending in Belarus in 2015 compared with optimised annual budget to minimise active TB prevalence and incidence and TB-related mortality by 2035. https://doi.org/10.1371/journal.pcbi.1009255.g004 Fig 4. National TB spending in Belarus in 2015 compared with optimised annual budget to minimise active TB prevalence and incidence and TB-related mortality by 2035. https://doi.org/10.1371/journal.pcbi.1009255.g004 https://doi.org/10.1371/journal.pcbi.1009255.g004 to increase over this period—driven by a projected increase in the number of people living with HIV. In line with the projected overall decrease in TB infections, the number of people with DR-TB is estimated to decrease rapidly, reducing by 50% by 2035 compared with 2015 levels. An optimised allocation of national TB spending could yield significant improvements in key TB indicators, as shown in Fig 5. Among HIV-negative populations, the model estimates that an optimised allocation of spending could lead to a 45% reduction in adult TB prevalence by 2035 compared with the existing allocation. Similarly, an optimised allocation of spending is estimated to reduce TB-related deaths by 50% by 2035 compared with non-optimised spend- ing allocations, and by 70% compared with 2015 levels. In addition, an estimated 40% of all DR-TB infections in Belarus can be averted by 2035 through optimised reallocations in spend- ing. Among people living with HIV, between a 30% and 45% reduction in new infections, prevalence, and mortality could be realised by 2035, though the number people living with HIV with active TB is very small. 13 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Fig 5. Projected impact of 2015 and optimised allocations of national TB spending on key TB indicators among HIV-negative adults and people living with HIV from 2015 to 2035. https://doi.org/10.1371/journal.pcbi.1009255.g005 Fig 5. Discussion Optima TB is an open-access tool for allocative efficiency modelling, designed to inform prior- ity setting for TB responses. Grounded in available data, it enables different populations and co-morbidities to be defined to capture epidemic heterogeneities. This paper illustrates the potential of Optima TB analysis findings to inform NTP discussions on priority setting and TB response reform via a case study in Belarus. An optimised allocation of TB spending in Belarus was estimated to reduce TB prevalence and mortality by up to 50%, compared with prior pro- gramme approaches. The main factors behind the gains are shifts from annual population- wide mass-screening and inpatient focused care, to active case finding strategies and outpa- tient focused care. Savings from these reallocations could be reinvested in screening and diag- nosis, prioritising higher-yield interventions needed in Belarus such as contact tracing and rapid diagnostic testing [50, 59, 61, 73]. In addition, transitioning to cheaper outpatient care could reduce overall treatment spending by 30% for reallocations to other intervention, with no estimated reduction in the number of people on treatment. The average unit cost of an inpatient day is three or four times more than that of an ambulatory day for DS-TB or DR-TB care, respectively (see Table A in S2 File), driven by greater staff, overhead and capital costs. Shifts in spending from inpatient to outpatient focused care are therefore recommended, in line with findings from local literature and WHO guidelines [22, 74–76], which would offset reductions in total treatment spending and enable the NTP to cover approximately 90% of all people diagnosed with active TB. As well as reductions in prevalence and mortality, scaling up care for people with active TB through these reallocations may result in modest gains in inci- dence over time, as overall levels of infectiousness are reduced through increased diagnosis and treatment in the model. A central focus of the Belarus NTP is to provide appropriate care for people with DR-TB and to address the significant burden of drug-resistance in the country. Existing practices involve lengthy inpatient stays of 180 days (six months) on average before discharge following smear conversion. An optimised shift in spending from inpatient-focused care to outpatient DR-TB treatment, and to drug-regimens comprised of new and repurposed drugs such as bedaquiline and linezolid, is estimated to reduce the number of DR-TB infections by around 40%. Progress toward national and international TB targets The estimated progress of 2015 and optimised allocations of spending toward national and international targets is shown in Fig 6. The year 2015 is considered as the baseline at 100%, while different targets and milestones are indicated by the dashed lines. Under 2015 spending allocations, it is estimated that no milestones or targets will be met aside from the 2020 National Strategic Plan (NSP) target for incidence. The national NSP target of a 35% reduction in TB-related deaths by 2020 will likely be missed, with the necessary reduction achieved only by 2030. If programmatic spending is reprioritised optimally, projections suggest that 2020 NSP targets for both incidence and TB-related deaths could be met. While neither global mile- stones nor targets are met under an optimised combination of interventions with 2015 spend- ing levels, more significant progress will likely be made towards mortality targets relative to incidence. Fig 6. Progress toward national and global TB targets for (a) TB mortality and (b) new TB infections in the 15–64 age group, under 2015 and optimised allocations of national TB spending. https://doi.org/10.1371/journal.pcbi.1009255.g006 Fig 6. Progress toward national and global TB targets for (a) TB mortality and (b) new TB infections in the 15–64 age group, under 2015 and optimised allocations of national TB spending. https://doi.org/10.1371/journal.pcbi.1009255.g006 Fig 6. Progress toward national and global TB targets for (a) TB mortality and (b) new TB infections in the 15–64 age group, under 2015 and optimised allocations of national TB spending. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 14 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending Discussion Reduced prioritisation of inpatient care would enable the NTP to provide DR-TB services to a greater number of those in need, and free up funds that can be invested in newer drug reg- imens with proven efficacy [77]. Furthermore, it is important to note that the estimated gains in this analysis from prioritising outpatient care do not include considerations from the patient perspective. Many studies have highlighted the substantial indirect costs and loss of earnings experienced by patients due to hospitalisation [78, 79]. While it is estimated that 2020 NSP targets for incidence and mortality could be met under optimised allocations of spending,progress would still fall significantly short of meeting the global 2025 TB milestones and 2035 End TB Strategy targets. Other modelling studies have also estimated that countries may fall short of global milestones and targets [80]. To meet global targets in Belarus, as in other countries, new technologies and interventions will likely be required as well as substantial increases in cost-effective investment. Overall, the TB response in Belarus is largely focused on curative rather than preventative interventions (Table 2). Only the latter can reduce vulnerability to TB and the rate at which the large pool of people with latent TB progress to active TB each year through the pathways shown in Fig 2 [81, 82]. The insufficient progress towards global targets for reductions in TB incidence esti- mated in this analysis supports a large body of existing literature advocating for a more holistic response to the global burden of TB, which is based on an understanding that social determi- nants of disease such as living conditions and nutritional status significantly impact disease PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 15 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending progression to active TB [81, 83–87]. A recent modelling study suggests that the expansion of social protection programmes would yield significant accelerated progress toward global TB targets [88]. Overall, baseline projections in this analysis based on 2015 TB spending allocations appear to be largely in line with recent estimates of key TB indicators for Belarus. For example, the 3400 incident active cases projected in this analysis for 2018 (see Figs A-C in S1 File) falls within the active TB incidence estimates by the WHO for 2018 (between 2300 and 3700) [89]. Discussion However, while modelled annual TB-mortality at the time of analysis (1200) for 2014 was higher than the upper-bound of WHO estimates (870), [90] the latter have recently been revised and reduced by approximately a third for 2014 (580) [91]. Mortality projections from the analysis are therefore no longer in line with recent estimates. In addition, while this analysis was carried out before the publication of guidance for coun- try-level TB modelling [92], the analysis followed the principles listed in the guidance docu- ment (see S3 File for details). The timeliness of the Belarus analysis helped inform dialogue on national TB care, including a round-table consultation organised by the WHO country office in Minsk in 2017 and a regional reform meeting in Bishkek, during which cornerstones of reform were agreed [93]. This analysis has informed ongoing activities for TB financing and planning by the WHO Regional Office for Europe and Ministry of Health [94], and the find- ings have helped advocate for more and better quality ambulatory care. Recently, a national primary healthcare workers scheme has been introduced, which involves bonus payments for the provision of TB services (such as bonus payments of US $5 per day for visits made to TB patients’ homes by nurses for treatment observations [95]). That said, impact assessments are required to establish whether this Optima TB application influenced actual reallocations in spending and improved TB control because of any changes in decision making. Strengths and limitations A number of TB epidemiological modelling tools exist to support country-level decision-mak- ing, including TIME Impact, AuTuMN, SEARO, VI, and EMOD among others [12–14, 16, 96]. While most comprehensive models nowadays include the study of MDR-TB cases [12–14, 16, 96], only a few have an explicit structure able to capture co-morbidities or high-risk groups [12, 14, 96] and, of them, only Optima TB and AuTuMN [12] explicitly analyse XDR cases. More specifically, TIME and AuTuMN enable users to run a variety of epidemic scenarios, and TIME Impact can be combined with the OneHealth Tool to produce detailed costings of TB interventions and guide NTP implementation by assessing health system components such as infrastructure or human resource needs. In addition, linking to OneHealth allows for pri- vate sector considerations to be incorporated within a TIME model analysis, which is key in certain contexts, such as in India [97]. However, efforts to develop tools that can estimate a mathematically optimised allocation of spending across a number of interventions to maxi- mise a set of desired objectives have only recently begun [12]. The Optima TB tool draws on existing epidemiological tools and research in allocative efficiency. Other Optima tools have been applied in over 60 countries, and follow-up impact assessments have shown that Optima HIV studies have influenced actual allocations in HIV spending in some countries such as Sudan and led to improvements in disease control [10, 11, 98–100]. There are several additional characteristics particular to the Optima TB tool. First, the explicit care-cascade structure allows for the model to be directly initialised and parametrised using country-provided data such as notified cases, number of people initiated on treatment, and TB-related deaths without solely relying on incidence estimations. This means that the modelling output can more accurately represent the setting specific TB epidemic. Another PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 16 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending novel aspect that, to our knowledge, is unique in comparison to other TB tools, is a secondary latency pathway. This has multiple advantages as it not only allows individuals who had a pre- vious history of infection to be distinguished, it also allows to study different preventive strate- gies that confer partial protection against reactivation and disaggregate these individuals from the general population. Strengths and limitations Currently, the implementation of preventive strategies such as treat- ment of LTBI, is generally limited to only direct contacts of active cases and HIV positive indi- viduals, the model structure includes detailed capture of TB latency allowing for the testing of new strategies and their impact on the entire population. Key populations can also be flexibly defined and targeted in Optima TB. One of the strongest design goals of Optima TB has been to ensure that all available epi- demic data and estimates for TB for a given setting can be fully utilised by the model, while also maintaining an accessible interface that requires a minimum of data input. Ongoing work will continue to focus on updating default global values for model parameters to represent TB transmission, progression, and treatment efficacy, to incorporate future TB research findings and WHO recommendations. Future model development is ongoing and may also include an additional pre-reactivation compartment for latent TB if a setting is using or plans to imple- ment new diagnostic techniques capable of identifying people at higher risk of latent TB reacti- vation with a high degree of sensitivity [101]. As with any deterministic model, there are limitations to our approach. These are mainly related to the limited individual-level detail typical of population-level models and the homo- geneity between individuals belonging to the same sub-population. Moreover, as is the case with any modelling framework, the quality of the modelling output is tightly connected to the amount and quality of data informing it, particularly so here because of the use of country- informed data. Also, calibration plays a strong role in obviating the paucity of information on epidemic parameters related to infection progression. Several key limitations to the case study must be considered when interpreting the results. First, model projections are only as reliable as the data that informs them. For Belarus, as for other countries, there are gaps, errors and inconsistencies in and between different datasets, with diminishing quality when data are disaggregated by sub-population. Commonly missing values for TB prevalence or intervention spending means that these values must be estimated, and assumptions have to be made, as informed by local experts. In addition, determining a mathematically optimised allocation of spending is dependent on the availability of estimates on the effectiveness of individual interventions. Conclusions The Optima TB tool was applied in Belarus to determine an optimised allocation of national TB spending across TB interventions to best address defined objectives. A shift in program- matic priorities could reduce TB prevalence and mortality by 50% by 2035, compared with a continuation of the 2015 response. Similar reductions of around 40% in the number of people with drug-resistant TB could be achieved, which is a key priority of the national TB pro- gramme. These gains would be achieved through shifts from annual population-wide mass- screening and inpatient focused care, to active case finding strategies and outpatient focused care. Optima TB applications aim to use existing country data to help initiate or support dia- logue between national TB programme managers and Ministries of Health and Finance, to generate evidence-based findings to inform decisions and to reduce the burden of TB. Follow- up impact assessments will be required to determine whether this Optima TB application influenced future TB resource reallocations in Belarus, and whether there is evidence of improved TB control because of any changes in decision making. Strengths and limitations Optima TB aims to inform resource allocation across interventions based on the defined cost-coverage relationship for a given intervention. If a given intervention can be delivered more efficiently for example through lower costs for the same treatment regimen, this would affect the underlying ranking of the cost-effectiveness of the interventions and thus resource allocation decisions. Although some effectiveness parameters were informed by national data, others were sourced through a review of global lit- erature. Effectiveness can vary across different countries and settings and the latter, therefore, may not be contextually representative. Due to a lack of data, TB interventions for inmates are not included in estimates of national TB spending or within the optimisation analysis. At the time of this analysis, the capacity for comprehensive uncertainty analyses was not included in the Optima TB model. The updated Optima TB model allows uncertainty intervals to be specified for every input parameter, including key calibration parameters such as trans- mission and progression rates for a given setting in addition to epidemiological inputs. Uncer- tainty in model outputs is then generated by sampling from the distributions for each input parameter, resulting in a distribution of values for each output. In its most basic form, these can be used to produce confidence intervals for each output, but the full distributions allow covariance and higher order statistics to be analysed as well. 17 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending In addition, our assumption about the nature of the relationship between intervention spending and coverage in Belarus did not consider the possibility of diminishing marginal returns. Last, projections generated by the model use parameter values from the most recent year for which data are available. Intervention impacts were assumed not to change with time and simulations also did not assess varying allocations of resources over time. Despite this, the optimised allocation is based on the time horizon from 2017 to 2035 in order to ensure that immediate gains do not come at the expense of adverse impacts later on and to gauge progress toward international targets with optimised allocations. In practice, however, optimised alloca- tions should be revisited for timelines longer than three to five years. Supporting information S1 File. Transmission model and epidemiological rationale. Equations and table of parame- ters (Table A in S1 File) are included, as well as epidemiology background justifying the mod- el’s structure. Additional results are also included: (Fig A in S1 File) projections of new TB infections to 2035; (Fig B in S1 File) number of active TB infections with and without funding to current TB programs; and (Fig C in S1 File) projections f yearly TB deaths to 2035. (PDF) S2 File. Intervention cost and effect inputs, including. (Table A in S2 File) TB treatment and care cost assumptions; (Table B in S2 File) TB treatment intervention effectiveness inputs; and (Table C in S2 File) TB screening and diagnosis intervention inputs for yield. (DOC) Funding acquisition: Jolene Skordis. Investigation: Lara Gosce´, Gerard J. Abou Jaoude, Tom Palmer, Jasmina Panovska-Griffiths, Hassan Haghparast-Bidgoli. Methodology: Lara Gosce´, Gerard J. Abou Jaoude, David J. Kedziora, Clemens Benedikt, Azfar Hussain, Sarah Jarvis, Nicole Fraser-Hurt, Romesh Abeysuriya, Rowan Martin- Hughes, Sherrie L. Kelly, Robyn M. Stuart, Cliff C. Kerr, David P. Wilson, Hassan Haghpar- ast-Bidgoli, Jolene Skordis, Ibrahim Abubakar. Project administration: Gerard J. Abou Jaoude, Clemens Benedikt, Sarah Jarvis, Nejma Cheikh, Anna Roberts. Software: David J. Kedziora, Azfar Hussain, Sarah Jarvis, Romesh Abeysuriya, Rowan Martin- Hughes, Sherrie L. Kelly, Robyn M. Stuart, Cliff C. Kerr. Supervision: Feng Zhao, Marelize Gorgens, David J. Wilson, David P. Wilson, Hassan Hagh- parast-Bidgoli, Jolene Skordis, Ibrahim Abubakar. Validation: Lara Gosce´, Gerard J. Abou Jaoude, David J. Kedziora, Azfar Hussain, Sarah Jar- vis, Hassan Haghparast-Bidgoli, Ibrahim Abubakar. Visualization: Clemens Benedikt, Azfar Hussain, Sarah Jarvis. Visualization: Clemens Benedikt, Azfar Hussain, Sarah Jarvis. Writing – original draft: Lara Gosce´, Gerard J. Abou Jaoude. Writing – original draft: Lara Gosce´, Gerard J. Abou Jaoude. Writing – review & editing: Lara Gosce´, Gerard J. Abou Jaoude, David J. Kedziora, Clemens Benedikt, Azfar Hussain, Sarah Jarvis, Alena Skrahina, Dzmitry Klimuk, Henadz Hurevich, Nicole Fraser-Hurt, Nejma Cheikh, Romesh Abeysuriya, Rowan Martin-Hughes, Sherrie L. Kelly, Anna Roberts, Robyn M. Stuart, Tom Palmer, Jasmina Panovska-Griffiths, Cliff C. Kerr, David P. Wilson, Hassan Haghparast-Bidgoli, Jolene Skordis, Ibrahim Abubakar. Acknowledgments We thank the following people and organisations for their contributions to the study: Irina Oleinik, Hanna Shvanok from the World Bank; Inna Nekrasova, Marina Sachek, Vassily Aku- lov from the Republican Scientific and Practice Centre for Medical Technologies (RSPC MT); Alena Tkatcheva from the Ministry of Public Health of the Republic of Belarus; Viatcheslav Grankov, Valentin Rusovich from the World Health Organisation Belarus Country Office; PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 18 / 24 PLOS COMPUTATIONAL BIOLOGY Allocative Efficiency of TB Spending David Kokiashvili and George Sakvarelidze from The Global Fund to Fight AIDS, Tuberculo- sis and Malaria. David Kokiashvili and George Sakvarelidze from The Global Fund to Fight AIDS, Tuberculo- sis and Malaria. The findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the funding agency. Author Contributions Conceptualization: Lara Gosce´, Gerard J. Abou Jaoude, Clemens Benedikt, Alena Skrahina, Dzmitry Klimuk, Henadz Hurevich, Feng Zhao, Nicole Fraser-Hurt, Nejma Cheikh, Mare- lize Gorgens, David J. Wilson, David P. Wilson, Jolene Skordis, Ibrahim Abubakar. Data curation: Alena Skrahina, Dzmitry Klimuk, Henadz Hurevich. Formal analysis: Lara Gosce´, Gerard J. Abou Jaoude, David J. Kedziora, Clemens Benedikt, Azfar Hussain. Funding acquisition: Jolene Skordis. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009255 September 27, 2021 References 1. Centis R, D’Ambrosio L, Zumla A, Migliori GB. Shifting from tuberculosis control to elimination: Where are we? What are the variables and limitations? Is it achievable? International Journal of Infectious Diseases. 2017; 56:30–33. https://doi.org/10.1016/j.ijid.2016.11.416 PMID: 27916675 2. MacNeil A, Glaziou P, Sismanidis C, Maloney S, Floyd K. 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https://openalex.org/W2598651878	https://hal.science/hal-01521232/document	English	null	Transplanted lungs and the “white plague”	Medicine	2,017	cc-by	3,159	Transplanted lungs and the “white plague” A case-report and review of the literature Nadim Cassir, Robin Delacroix, Carine Gomez, Veronique Secq, Martine Reynaud-Gaubert, Pascal-Alexandre Thomas, Laurent Papazian, Michel Drancourt Nadim Cassir, Robin Delacroix, Carine Gomez, Veronique Secq, Martine Reynaud-Gaubert, Pascal-Alexandre Thomas, Laurent Papazian, Michel Drancourt To cite this version: Nadim Cassir, Robin Delacroix, Carine Gomez, Veronique Secq, Martine Reynaud-Gaubert, et al.. Transplanted lungs and the “white plague” A case-report and review of the literature. Medicine, 2017, 96 (13), pp.e6173. 10.1097/MD.0000000000006173. hal-01521232 Abstract Abstract Rationale: Solid organ transplant recipients, especially after lung transplantation, are at increased risk for Mycobacterium tuberculosis pulmonary tuberculosis due to lifelong immunosuppression. Patient concerns: A 41-year-old woman underwent a second bilateral lung transplantation that was complicated by fatal pulmonary tuberculosis. Diagnoses: Histological examination of a lung biopsy performed 6 weeks after retransplantation revealed a caseating granuloma and necrosis. Acid-fast bacilli were identiﬁed as rifampicin-susceptible M. tuberculosis by real-time polymerase chain reaction (PCR), conﬁrmed by culture 2 weeks later. Interventions: Our investigation led us to highly suspect that the transplanted lungs were the source of M. tuberculosis transmission. Lessons: In order to optimize diagnosis and treatment for lung recipients with latent or active tuberculosis, regular assessment of lower respiratory samples for M. tuberculosis, particularly during the 12-month period posttransplant should be implemented. Regarding donor-derived transmission, screening donor grafts with latent tuberculosis by M. tuberculosis real-time PCR in lymphoid and adipose tissues is an option that should be considered. Abbreviations: BAL = bronchoalveolar lavage, CT = chest-computerized tomography, DNA = deoxyribonucleic acid, IGRA = interferon-g release assay, LTBI = latent tuberculosis infection, PCR = polymerase chain reaction, SOT = solid organ transplant, TST = tuberculin skin test. Keywords: case-report, immunosuppression, lung, Mycobacterium tuberculosis, transplantation, tuberculosis Keywords: case-report, immunosuppression, lung, Mycobacterium tuberculosis, transplantation, t 1. Introduction tions complicate the management of tuberculosis, leading to a up to 30% mortality.[1] Posttransplantation tuberculosis may result from reactivating latent tuberculosis in the recipient or transmission of M. tuberculosis from a contagious person or from the transplant.[1] Risk for pulmonary tuberculosis is greater for lung transplant receivers compared with other SOT recipients.[3] In this population, the onset of pulmonary tuberculosis varies from 1 day to 12 months after lung transplantation.[4,5] Solid organ transplant (SOT) recipients are at increased risk for Mycobacterium tuberculosis pulmonary tuberculosis due to lifelong immunosuppression.[1] In low tuberculosis-prevalence regions, the frequency of pulmonary tuberculosis in SOT recipients varies from 1.2% to 6.5%.[2] In this population, diagnosis delay, treatment-related toxicities, and drug interac- In our hospital, diagnosing deadly pulmonary tuberculosis 8 weeks after bilateral lung transplantation led to investigate the source of M. tuberculosis. This case is reported anonymously in agreement with the advice n°2016–024 of the Méditerranée Infection Institute Ethics Committee. Editor: Duane R. Hospenthal. Editor: Duane R. Hospenthal. Funding: This study was supported by URMITE, IHU Méditerranée Infection, Marseille, France. The authors declare that they have no conﬂict of interest. a Aix Marseille Univ, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, IHU Méditerranée Infection, Marseille, b APHM, Service de Pneumologie, Equipe de Transplantation pulmonaire, c APHM, Service d’Anatomo-Pathologie, d APHM, Service de Chirurgie Thoracique, Equipe de Transplantation pulmonaire, e APHM, Service de Réanimation Détresses Respiratoires et Infections Sévères, Hôpital Nord, Marseille, France. a Aix Marseille Univ, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, IHU Méditerranée Infection, Marseille, b APHM, Service de Pneumologie, Equipe de Transplantation pulmonaire, c APHM, Service d’Anatomo-Pathologie, d APHM, HAL Id: hal-01521232 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. Medicine ® Medicine ® OPEN Clinical Case Report Received: 2 August 2016 / Received in ﬁnal form: 14 November 2016 / Accepted: 29 January 2017 Transplanted lungs and the “white plague” A case-report and review of the literature Nadim Cassir, MD, PhDa, Robin Delacroix, Pharm. Residenta, Carine Gomez, MDa,b, c a b Nadim Cassir, MD, PhDa, Robin Delacroix, Pharm. Residenta, Carine Gomez, MDa,b, Véronique Secq, MDc, Martine Reynaud-Gaubert, MD, PhDa,b, Pascal-Alexandre Thomas, MD, PhDa,d, Laurent Papazian, MD, PhDa,e, Michel Drancourt, MD, PhDa,∗ Véronique Secq, MDc, Martine Reynaud-Gaubert, MD, PhDa,b, Pascal-Alexandre Thomas, MD, PhDa,d, Laurent Papazian, MD, PhDa,e, Michel Drancourt, MD, PhDa,∗ http://dx.doi.org/10.1097/MD.0000000000006173 3. Discussion Acid-fast bacilli were identiﬁed as rifampicin-suscep- tible M. tuberculosis by real-time PCR. On postoperative day 65, the patient’s status worsened with severe hypoxemia, shock unresponsive to high dose cathecolamines, and multiorgan failure. The patient died on postoperative day 70, despite treatment combining isoniazid, rifampicin, ethambutol, and pyrazinamide. Retrospective real-time PCR testing of the explanted lung and BALs performed on postoperative days 1, 7, and 21 remained negative. apical posterior lobes and a bilateral pleural effusion (Fig. 1). The same day, a bronchoalveolar lavage (BAL) yielded a positive real-time PCR for rifampicin-susceptible M. tubercu- losis, conﬁrmed by culture on postoperative day 62. Tuberculin skin test (TST) or interferon-g release assay (IGRA) test were not performed. All the BALs performed on postoperative period yielded no other pathogen except for the one performed on day 60 that cultured Pseudomonas aeruginosa; the adjunctive antibiotic therapy was imipenem-cilastatin, 3g/d. Histological examination of a lung biopsy performed 6 weeks after retransplantation revealed a caseating granuloma and necrosis. Acid-fast bacilli were identiﬁed as rifampicin-suscep- tible M. tuberculosis by real-time PCR. On postoperative day 65, the patient’s status worsened with severe hypoxemia, shock unresponsive to high dose cathecolamines, and multiorgan failure. The patient died on postoperative day 70, despite treatment combining isoniazid, rifampicin, ethambutol, and pyrazinamide. Retrospective real-time PCR testing of the explanted lung and BALs performed on postoperative days 1, 7, and 21 remained negative. Current US and European guidelines recommend routine screening and treatment for latent tuberculosis infection (LTBI) in lung recipients but there is no controlled trial.[1,16] Assessing LTBI includes reviewing epidemiologic risk factors, chest radiography and a TST and/or IGRA. However, TSTs and IGRAs are less sensitive in immunosuppressed and/or critically ill patients than in the general population and they do not differentiate LTBI from active tuberculosis.[17] There is no controlled trial to support speciﬁc recommenda- tions regarding lung donors.[1] One option would be to screen lungs just before or at the time of transplantation as early antituberculous treatment and immunosuppression optimization are essential to successfully treat lung recipients with active tuberculosis.[15] In the case reported here, negative retrospective detection was obtained on ﬁxed rather than fresh biopsies. TST or IGRA testing in deceased donors is difﬁcult to perform and to interpret.[14] Because M. 3. Discussion Several lines of evidence indicate that the transplanted lungs were the source of fatal pulmonary tuberculosis in the patient who underwent a second bilateral lung transplantation. During her 9- year history of her ﬁrst bilateral lung transplant, the recipient had no known history of tuberculosis. In the month prior to second transplantation, she presented no clinical, CT-scan, or microbio- logical evidence of pulmonary tuberculosis. During regular monitoring, the ﬁrst positive respiratory sample tested positive for M. tuberculosis was obtained 42 days after the second transplantation, while immunosuppressive therapy had been administered for 9 years following the ﬁrst transplantation. Investigations found no evidence of a new infection posttrans- plant via healthcare-associated cross-transmission that could otherwise have explained this case. No case of active tuberculosis infection was diagnosed among her relatives, other patients or healthcare workers during the 3-month pretransplant period and the posttransplant stay in the thoracic surgery ward or intensive care unit. A donor-to-recipient lung transmission was suspected in 15 cases of pulmonary tuberculosis in lung transplant recipients since 1990 (Table 1).[5–15] It was conclusive in only 1 case reporting a 14-year-old girl with chronic bronchiectasis who was TST-negative before transplantation.[15] She received a bilateral lung transplant from a 51-year-old man born in the Philippines with a solitary pulmonary nodule that was found on perioperative palpation. Histologic analysis of this nodule indicated a caseating granuloma and necrosis with positive AFB staining and the recipient BAL performed on postoperative day 5 was positive for M. tuberculosis by PCR and culture. Early initiation of antituberculosis treatment and the omission of induction immunosuppressive therapy led to a favorable outcome. Figure 1. Chest computed-tomography obtained on day 68 after transplanta- tion. A: Bilateral lung parenchymal nodules with cavity in right lower lobe apical segment. B: Bilateral pleural effusion with nodule in posterior segment of right lower lobe. apical posterior lobes and a bilateral pleural effusion (Fig. 1). The same day, a bronchoalveolar lavage (BAL) yielded a positive real-time PCR for rifampicin-susceptible M. tubercu- losis, conﬁrmed by culture on postoperative day 62. Tuberculin skin test (TST) or interferon-g release assay (IGRA) test were not performed. All the BALs performed on postoperative period yielded no other pathogen except for the one performed on day 60 that cultured Pseudomonas aeruginosa; the adjunctive antibiotic therapy was imipenem-cilastatin, 3g/d. Histological examination of a lung biopsy performed 6 weeks after retransplantation revealed a caseating granuloma and necrosis. Medicine Cassir et al. Medicine (2017) 96:13 of per-transplantation right lung biopsy yielded Candida albicans. Retrospective M. tuberculosis real-time PCR yielded negative results on the left and right donor-lung biopsies. Both kidneys from the same donor were transplanted into 2 other recipients. Six months after transplantation, neither of the kidney recipients had developed any signs or symptoms suggestive of active tuberculosis. Figure 1. Chest computed-tomography obtained on day 68 after transplanta- tion. A: Bilateral lung parenchymal nodules with cavity in right lower lobe apica segment. B: Bilateral pleural effusion with nodule in posterior segment of right lower lobe. 2. Case report A 41-year-old Caucasian woman underwent a primary double lung transplantation for cystic ﬁbrosis in 2006. Her medical history was otherwise unremarkable and the patient had no known history of pulmonary tuberculosis or tuberculosis contact. On December 2015, she underwent retransplantation for chronic lung allograft dysfunction. During the month preceding retransplantation, 4 sputum specimens remained negative for acid-fast bacilli and speciﬁc M. tuberculosis culture and real-time polymerase chain reaction (PCR) testing. On postoperative day 42, deterioration of her respiratory status prompted a chest-computerized tomography (CT) scan reveal- ing sub-centimeter bilateral nodules primarily located in the ∗Correspondence: Michel Drancourt, Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, CNRS UMR 6020, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France (e-mail: Michel.drancourt@univ-amu.fr). Copyright © 2017 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.1097/MD.0000000000006173 1 1 Medicine 3. Discussion tuberculosis DNA is detected in lymphoid and adipose tissues surrounding the lungs in LTBI patients, the cost-effectiveness of rapid real-time PCR testing of the donor lungs has to be evaluated in various tuberculosis prevalence settings. The organ donor died of posttraumatic intracerebral hemor- rhage. He was a 47-year-old man with no history of lung disease or risk factors for tuberculosis other than chronic alcohol use and smoking. TST results were not available. During hospitalization, a lung CT-scan showed no signs of active or previous tuberculosis and no TST or IGRA test results were available. Routine cultures 2 Cassir et al. Medicine (2017) 96:13 www.md-journal.com Table 1 Cases of donor-derived pulmonary tuberculosis after lung transplantation since 1990. Recipient characteristics Donor characteristics Microbiological results in the recipient Delay from transplantation to diagnosis Treatment/Outcome Reference 23-y-old woman/heart-lung/pulmonary arterial hypertension/TST negative before transplant N.A. BAL samples showed AFB and culture yielded M. tuberculosis 4 mo N.A./Died Carlsen and Bergin[6] A 57-y-old woman/bilateral lung/chronic obstructive pulmonary disease/TST negative before transplant 45-y-old woman BAL samples showed AFB which were identiﬁed as M. tuberculosis by DNA probe and conﬁrmed by culture 3 mo Isoniazid, ethambutol and pyrazinamide/ died Miller et al[7] 42-y-old woman/single lung/end-stage chronic obstructive pulmonary disease Normal chest radiograph and no known prior history of M. tuberculosis BAL samples yielded M. tuberculosis—BAL showed AFB and cultures yielded M. tuberculosis 6 wk—idem Isoniazid, ethambutol, and pyrazinamide/ improved Ridgeway et al[8] 63-y-old woman/single lung/end-stage chronic obstructive pulmonary Infection DNA ﬁngerprints of both M. tuberculosis isolates were identical idem A 27-y-old man/bilateral lung/cystic ﬁbrosis 19-y-old New York City resident Lung biopsy specimens showed granulomatous inﬂammation with stainable AFB that yielded M. tuberculosis 3 mo—idem Isoniazid, ethambutol, pyrazinamide, and streptomycin during 3 mo, followed by 15 mo of isoniazid and ethambutol/improved Schulman et al[9] A 57-y-old man/bilateral lung/idiopathic bronchiectasis 25-y-old South American man who had immigrated to New York City 2 y earlier BAL samples showed AFB and culture yielded M. tuberculosis. Lung biopsy showed necrotizing granulomas idem 35-y-old woman/bilateral lung/end-stage pulmonary lymphangioleiomyomatosis/TST negative before transplant 51-y-old, non-smoking, recent immigrant from China Lung biopsy specimens showed granulomatous inﬂammation with stainable AFB that yielded XDR M. 3. Discussion tuberculosis 5 mo Isoniazid, rifampicin, pyrazinamide, and ethambutol replaced by levoﬂoxacin, prothionamide, and cycloserine together with para aminosalicylic acid (PAS)/ improved Lee (2003) 49-y-old woman/previous single lung transplant/ idiopathic pulmonary ﬁbrosis/end-stage bronchiolitis/bilateral sequential lung retransplantation Chest radiography with previously unnoticed pulmonary opacity BAL samples yielded M. tuberculosis 1 d Isoniazid, pyrazinamide, and levoﬂoxacin/ improved Winthrop et al[5] 28-y-old woman/heart-lung/pulmonary hypertension and restrictive cardiomyopathy 42-y-old man BAL samples showed AFB and culture yielded M. tuberculosis. Lung biopsy disclosed necrotizing granulomas positive for AFB 2.5 mo Isoniazid, rifampicin, pyrazinamide, and ethambutol/improved Place (2007) 16-y-old boy/heart-lung transplant/pulmonary arterial hypertension Contact with a patient with active tuberculosis for at least 1 y BAL samples culture yielded XDR M. tuberculosis 2.5 mo Ethambutol, cycloserine, ciproﬂoxacin, clarithromycin/improved Shitrit et al[12] 68-y-old man/single-lung/coal worker’s pneumoconiosis/TST negative before transplant 33-y-old man, emigrated from Peru 11 y before/TST positive at 24mm without LTBI prophylaxis Cultures from the pericardium and the BAL yielded M. tuberculosis 3 mo Ethambutol, isoniazid, pyrazinamide, and ciproﬂoxacin/died Boedefeld et al[13] 60-y-old woman/single lung/idiopathic pulmonary ﬁbrosis/TST negative before transplant 20-y-old man born in Mexico BAL samples showed AFB and culture yielded M. tuberculosis 5 mo Rifampin, isoniazid, pyrazinamide, and ethambutol during 3 mo followed by rifabutin and isoniazid/unrelated death Mortensen et al[14] Genotyping analysis of the strain revealed similarity with a cluster of patients from Mexico 3 3 3 Medicine Cassir et al. Medicine (2017) 96:13 (continued). Recipient characteristics Donor characteristics Microbiological results in the recipient Delay from transplantation to diagnosis Treatment/Outcome Reference 50-y-old woman/bilateral lung/TST negative before transplant 20-y-old man born in the USA, incarcerated 1 y before BAL samples showed AFB and culture yielded M. tuberculosis 2 mo Rifampin, isoniazid, ethambutol, and pyrazinamide/N.A. Mortensen et al[14] Genotyping analysis of the strain revealed similarity with a cluster of patients from a residential center near by the jail 50-y-old woman/bilateral lung/TST negative before transplant 20-y-old man born in the USA, traveled during 1 y just before donation in the Philippines BAL samples showed AFB and culture yielded M. tuberculosis 3 mo Rifampin, isoniazid, ethambutol, and pyrazinamide during 2 mo, followed by 7 mo of rifampin and isoniazid/N.A. 3. Discussion Mortensen et al[14] Genotyping analysis of the strain revealed similarity with a cluster of patients from the Philippines 14-y-old girl/bilateral lung/idiopathic bronchiectasis/TST negative before transplant 51-y-old man born in the Philippines/histologic analysis of a nodule from the donor graft indicated a granuloma with caseation and necrosis with positive AFB staining BAL samples were positive for M. tuberculosis by PCR and culture yielded M. tuberculosis 5 d Moxiﬂoxacin, isoniazid, ethambutol, and pyrazinamide during 2 mo, followed by 10 mo of moxiﬂoxacin, isoniazid, and ethambutol/improved Nizami et al[15] 41-y-old woman/previous bilateral lung transplant/ cystic ﬁbrosis/end-stage bronchiolitis obliterans/bilateral lung retransplantation 47-y-old, smocking, chronic alcohol user Lung biopsy yielded granuloma with caseation and necrosis. AFB were identiﬁed as rifampicin-susceptible M. tuberculosis by real-time PCR 8 wk Rifampin, isoniazid, ethambutol, and pyrazinamide/died This case AFB=acid fast bacilli, BAL=bronchoalveolar lavage, M. tuberculosis=Mycobacterium tuberculosis, NA=not available, PCR=polymerase chain reaction, TST=tuberculin skin test. 4 Cassir et al. Medicine (2017) 96:13 www.md-journal.com 4. Conclusion [6] Carlsen SE, Bergin CJ. Reactivation of tuberculosis in a donor lung after transplantation. AJR Am J Roentgenol 1990;154:495–7. Given the substantial morbidity and mortality associated with active tuberculosis in lung recipients, it is crucial to come up with an early diagnosis for those with latent or active tuberculosis in order to optimize their treatment. Regular assessment of lower respiratory samples for M. tuberculosis, particularly during the 12-month period posttransplant should be implemented. Regarding donor-derived transmission, screening donor grafts with LTBI by M. tuberculosis real-time PCR in lymphoid and adipose tissues is an option that should be considered. [7] Miller RA, Lanza LA, Kline JN, et al. Mycobacterium tuberculosis in lung transplant recipients. Am J Respir Crit Care Med 1995;152:374–6. [8] Ridgeway AL, Warner GS, Phillips P, et al. Transmission of Mycobacterium tuberculosis to recipients of single lung transplants from the same donor. Am J Respir Crit Care Med 1996;153:1166–8. [9] Schulman LL, Scully B, McGregor CC, et al. Pulmonary tuberculosis after lung transplantation. Chest 1997;111:1459–62. [10] Lee J, Yew WW, Wong CF, et al. Multidrug-resistant tuberculosis in a lung transplant recipient. J Heart Lung Transplant 2003;22:1168–73. [11] Place S, Knoop C, Remmelink M, et al. Paradoxical worsening of tuberculosis in a heart-lung transplant recipient. Transpl Infect Dis 2007;9:219–24. [12] Shitrit D, Bendayan D, Saute M, et al. Multidrug resistant tuberculosis following lung transplantation: treatment with pulmonary resection. Thorax 2004;59:79–80. References [13] Boedefeld RL, Eby J, Boedefeld WM, et al. Fatal Mycobacterium tuberculosis infection in a lung transplant recipient. J Heart Lung Transplant 2008;27:1176–8. [1] Horne DJ, Narita M, Spitters CL, et al. Challenging issues in tuberculosis in solid organ transplantation. Clin Infect Dis 2013;57:1473–82. [2] Aguado JM, Torre-Cisneros J, Fortun J, et al. Tuberculosis in solid-organ transplant recipients: consensus statement of the group for the study of infection in transplant recipients (GESITRA) of the Spanish Society of Infectious Diseases and Clinical Microbiology. Clin Infect Dis 2009;48:1276–84. [14] Mortensen E, Hellinger W, Keller C, et al. Three cases of donor-derived pulmonary tuberculosis in lung transplant recipients and review of 12 previously reported cases: opportunities for early diagnosis and prevention. Transpl Infect Dis 2014;16:67–75. [15] Nizami IY, Khan BJ, Saleh W, et al. Successful bilateral lung transplantation from a deceased donor with active Mycobacterium tuberculosis infection. Ann Thorac Surg 2014;97:e109–10. [3] Subramanian AK, Morris MI. AST infectious diseases community of practice. Mycobacterium tuberculosis infections in solid organ trans- plantation. Am J Transplant 2013;13:68–76. [16] Meije Y, Piersimoni C, Torre-Cisneros J, et al. ESCMID Study Group of infection in compromised hosts. Mycobacterial infections in solid organ transplant recipients. Clin Microbiol Infect 2014;20:89–101. [4] Singh N, Paterson DL. Mycobacterium tuberculosis infection in solid- organ transplant recipients: impact and implications for management. Clin Infect Dis 1998;27:1266–77. [17] Herrera V, Perry S, Parsonnet J, et al. Clinical application and limitations of interferon-gamma release assays for the diagnosis of latent tuberculosis infection. Clin Infect Dis 2011;52:1031–7. [5] Winthrop KL, Kubak BM, Pegues DA, et al. Transmission of Mycobacterium tuberculosis via lung transplantation. Am J Transplant 2004;4:1529–33. 5 5
https://openalex.org/W3111952697	https://europepmc.org/articles/pmc7752293?pdf=render	English	null	Time to Start Delivering Iron Chelation Therapy in Newly Diagnosed Severe β-Thalassemia	BioMed research international	2,020	cc-by	5,136	Susi Susanah ,1 Ponpon S. Idjradinata ,1 Nur M. Sari ,1 Lulu E. Rakhmilla ,2 Yunia Sribudiani ,3 Jessica O. Trisaputra ,4 and Octawyana Moestopo 4 Susi Susanah ,1 Ponpon S. Idjradinata ,1 Nur M. Sari ,1 Lulu E. Rakhmilla ,2 Yunia Sribudiani ,3 Jessica O. Trisaputra ,4 and Octawyana Moestopo 4 1Department of Child Health, Hematology-Oncology Division, Hasan Sadikin General Hospital/Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia 2Department of Public Health, Epidemiology and Biostatistic Division, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia Department of Biomedical Sciences, Division of Biochemistry and Molecular Biology, Faculty of Medicine, Universit Bandung 40161, Indonesia 4Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia 4Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia orrespondence should be addressed to Susi Susanah; susi_rshs@yahoo.co.id Correspondence should be addressed to Susi Susanah; susi_rshs@yahoo.co.id Received 7 June 2020; Revised 20 November 2020; Accepted 4 December 2020; Published 14 December 2020 Academic Editor: Ruxana Sadikot Copyright © 2020 Susi Susanah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Iron overload is still a major complication of severe β-thalassemia. Indication to start iron chelation therapy is based on serum ferritin (SF) or transferrin saturation (TS) level or the amount of transfusion. The goal of this study is to analyse the pattern of iron status, the amount of transfusion regarding the time to start iron chelator, and serum hepcidin levels in newly diagnosed severe β-thalassemia. Methods. A prospective cohort study was performed at Hasan Sadikin General Hospital on newly diagnosed severe β-thalassemia patients. Subjects had not received any blood transfusion with normal liver function test, CRP, and IL-6 levels who consumed normal diet according to age. The SF and TS levels indicate iron status, while hepcidin level indicates iron regulator status. Main indicator to start iron chelation therapy when SF level ≥1.000 ng/mL, TS level ≥70%, or after receiving transfusion at least 10 times. Statistical analysis used Mann–Whitney and Spearman. Results. Forty-two newly severe β-thalassemia, 30 (71.4%), were diagnosed before 1 year old, mean 9:9 ± 6:4 months, range 2–24 months. Range amount of transfusion until SF level reached ≥1,000 ng/mL were 4-12 times, mean 7 ± 2 times. Mean SF and TS level at diagnosis were 365:6 ± 194:9 ng/mL and 67:3 ± 22:5%, while hepcidin level was normal, mean 242:6 ± 58 ng/mL. 36/42 patients have reached SF >1000 ng/mL with amount of transfusion less than 10 times. Hindawi BioMed Research International Volume 2020, Article ID 8185016, 6 pages https://doi.org/10.1155/2020/8185016 Hindawi BioMed Research International Volume 2020, Article ID 8185016, 6 pages https://doi.org/10.1155/2020/8185016 Hindawi BioMed Research International Volume 2020, Article ID 8185016, 6 pages https://doi.org/10.1155/2020/8185016 Susi Susanah ,1 Ponpon S. Idjradinata ,1 Nur M. Sari ,1 Lulu E. Rakhmilla ,2 Yunia Sribudiani ,3 Jessica O. Trisaputra ,4 and Octawyana Moestopo 4 There was no signiﬁcant diﬀerence of SF, TS, and hepcidin levels when SF >1000 ng/mL in the group with amount of transfusion 7–12 and less than 7 (p = 0:454, p = 0:084, p = 0:765), respectively. A signiﬁcant positive correlation between SF and amount of transfusion was observed (p < 0:001; r = 0:781). Conclusion. Iron overload in severe β-thalassemia patients might occur earlier even before they received 10 times transfusion. Hepcidin serum level tends to increase when iron overload just started. 1. Introduction TS was measured by dividing serum iron by total iron binding capacity (TIBC). Iron status was assessed by measuring SF and TS using enzyme immu- noassays (Centaur XPT, Siemen). Serum hepcidin level was measured by human hepcidin, ELISA Kit (ELISA Reader, Rayto). Dietary intake was obtained by history taking regard- ing the subject’s food-consumption patterns involving exclu- sive breastfeeding, infant formula, and complementary foods. Hepcidin is suppressed by hypoxia and iron deﬁciency and upregulated by inﬂammation and iron loading [4, 5]. In condition of iron deﬁciency, low transferrin saturation (TS) suppressed hepcidin expression, but in IE as in severe β-thalassemia with high TS levels, hepcidin expression was also found to be suppressed [4]. Decreased hepcidin expres- sion as a result from iron overload in severe β-thalassemia is induced by erythropoiesis regulators as a response to IE [6, 7]. Due to the risk of those iron toxicities, it might be coun- tered as earlier as possible. The body iron status can be deter- mined by measuring serum ferritin (SF) and TS levels. Current practice to start iron chelation therapy based on Thalassemia International Federation (TIF) guidelines is when SF levels rise above 1,000 ng/mL or TS level ≥70% or after received of 10–20 times pack red cells (PRC) transfu- sions [2]. The study was approved by the ethical committee of Hasan Sadikin General Hospital, Faculty of Medicine, Uni- versitas Padjadjaran, Bandung, and was conducted according to the principles of the Declaration of Helsinki. Written informed consent for each patient was obtained from the parents prior to any study procedures being carried out. The period of recruitment was 16 months. Excess iron in β-thalassemia and severe HbE/β-thalasse- mia often develop early, even before the patient received any blood transfusion [4]. Evaluation of iron burden is essential for the determination of clinical outcomes, deciding when to commence chelation, selection of the regime that should be prescribed, the continuous monitoring of chelation eﬃ- cacy, and the ﬁne-tuning of the regime. Starting relative intensive chelation in younger children may prevent short stature and abnormal pubertal maturation as well as other iron-related morbidities, and signiﬁcantly improve survival [8–10]. Data were entered into Microsoft Excel, cross-checked, and analysed using SPSS version 25.0 (IBM Corp., Armonk, NY). Descriptive results of categorical variables were described as number and percentage, while continuous vari- ables were described as mean ± SD or median (IQR). 1. Introduction HbE/β-thalassemia generally demonstrate as transfusion- dependent thalassemia (TDT) cases causing iron overload which aggravated by chronic transfusion therapy [2]. Beta thalassemia is a group of hereditary disorders character- ized by reduction or absence of β-globin production resulting alpha globin chain accumulation, forming aggregate, which impairs red blood cell (RBC) production that leads to hemo- lytic anemia and ineﬀective erythropoiesis (IE) [1]. Ineﬀec- tive erythropoiesis leads to increased iron absorption in intestine [1, 2]. Beta thalassemia major and severe form of The main cause of severe β-thalassemia complications is iron overload. Excess accumulation of iron in organ (hemo- siderosis) leads to oxidative damage as a result of generation of reactive oxygen species (ROS). A remarkable variability of tissue iron distribution has been observed in severe β-thalas- semia: liver, heart, and endocrine glands are the organs which BioMed Research International 2 transfusion, (3) conﬁrmed transfusion-dependency and initi- ation of regular protocol of PRC transfusion to maintain hemoglobin level (Hb) >9 g/dL, and (4) iron chelation ther- apy were initiated when SF level >1000 ng/mL. The exclusion criteria were if they had any signs and symptoms of inﬂam- mation conﬁrmed by abnormal serum transaminase (AST, ALT), the level of CRP >5 mg/dL, and IL-6 >16.4 pg/mL. most severely aﬀected [1, 3]. Humans do not have any special mechanism that eﬀective to eliminate iron overload, so iron balance in the body was regulated by controlling its absorp- tion. When iron reservation is enough, the absorption is decreased on the contrary when iron reservation is low, the absorption will be increasing. Increased iron absorption is expected to be associated with hepcidin as a 25 amino acid peptide that regulates iron homeostasis. Hepcidin is the main regulator of iron homeostasis by controlling iron absorption in the small intestine and iron release from macrophages and liver [4]. ) g pg The patients were followed regarding to prove that all subjects clinically need regular transfusion, as TDT cases, and then, they will be monitored for the amount of transfu- sion as in 10–15 mL/kg of PRC when the Hb level was below 9 g/dL. Blood samples for the measurement of SF and Hb were collected and measured regularly concomitant with blood transfusion. Sampling was done in the morning, under fasting conditions. While hepcidin level and TS were mea- sured only at the ﬁrst diagnosis was conﬁrmed and as soon as SF level >1,000 ng/mL. 1. Introduction Spear- man’s correlation coeﬃcient was used to test the correlation between SF level and the amount of transfusion, while the Mann–Whitney test was used for the comparison between groups of amount of transfusion (7–12 and less than 7). A p value < 0.05 was considered as statistically signiﬁcant in all analyses. To date, the studies related to the severity of hemosidero- sis in severe β-thalassemia most reported patients who have had receiving multitransfusions, there was no study reported iron status and hepcidin level in newly diagnosed severe β- thalassemia patients. The purpose of this study was to analyse the pattern of the iron status (SF and TS levels) and hepcidin level in newly diagnosed severe β-thalassemia patients at ﬁrst diagnosis and when they reached iron overload, also regard- ing the time to initiate iron chelator. 3. Results There were 42 subjects who met the criteria as newly diag- nosed severe β-thalassemia. Most of them have β-thalasse- mia major (90%), and the rest were severe HbE/β- thalassemia (10%). When the diagnosis was conﬁrmed, the age of the subjects was less than 24 months, and 30/42 (71%) were under one year old with equal by gender. Most of the patients admitted to the hospital at ﬁrst diagnosis with severe anemia. Serum ferritin (SF) and TS level at diagnosis were already high, while hepcidin level was normal. 36/42 (85.7%) subjects already had a high level of SF based on age. At the time of diagnosis, serum iron level has already high while TIBC within normal limit, so most of the subjects 33/42 (78.5%) had high TS level based on age and increased to 39/42 (92.8%) when SF >1000 ng/mL. The high hepcidin 4. Discussion Most of the patients were admitted to the hospital with severe anemia similar with Trehan et al. reported that nearly 40% of subjects suﬀered from severe anemia, with hemoglobin <5.0 g/dL [11]. In β-thalassemia major and severe HbE/β-thalassemia, excess of unbound α-globin chains will aggregate and precip- itate adhering to the membrane erythroid precursors. This will cause cellular and membrane damage, apoptosis of ery- throid precursors in bone marrow leading to premature death and hence to IE [2, 12]. In β-thalassemia, combination of IE of developing erythroid precursor cell and increase hemolysis of mature RBC is the main causes of anemia [14]. Ineﬀective erythropoiesis in β-thalassemia caused increased iron absorption that is regulated by hepcidin mainly from dietary absorption in duodenum, recycled iron from macrophages, and released stored iron from hepato- cytes. When iron-deﬁcient hepatocyte produces less or no hepcidin, allowing iron to enter plasma and when iron is abundant, hepcidin will increase to limit further iron absorp- tion and release from stores. The suppressive eﬀect of eryth- ropoiesis on hepcidin in β-thalassemia was caused by IE. It is explained that in β-thalassemia with iron overload, the hep- cidin level was suppressed [15]. In this study, the hepcidin level at diagnosis was still normal (mean 242:6 ± 58:0) but tends to increase. However, only 6 out of 42 (14.3%) patients had high hepcidin level when SF >1000 ng/mL; this might due to the fact that the transfusion inhibits erythropoiesis that will increase hepcidin level [1, 15, 16]. The equal gender of the subjects was concordance with thalassemia characteristic as an inherited autosomal recessive disorder following Mendelian rule [2, 13]. Most of the patients were admitted to the hospital with severe anemia similar with Trehan et al. reported that nearly 40% of subjects suﬀered from severe anemia, with hemoglobin <5.0 g/dL [11]. In β-thalassemia major and severe HbE/β-thalassemia, excess of unbound α-globin chains will aggregate and precip- itate adhering to the membrane erythroid precursors. This will cause cellular and membrane damage, apoptosis of ery- throid precursors in bone marrow leading to premature death and hence to IE [2, 12]. In β-thalassemia, combination of IE of developing erythroid precursor cell and increase hemolysis of mature RBC is the main causes of anemia [14]. Normally, suﬃcient iron intake was needed for children’s physiological growth and development. On the contrary, children with TDT should be avoided from high iron intake including iron-fortiﬁed food. 2. Materials and Methods A prospective cohort study was carried out on 42 newly diag- nosed severe β-thalassemia patients at the Department of Child Health Hasan Sadikin General Hospital from October 2011 to March 2013, taken by consecutive sampling. The main inclusion criteria were (1) conﬁrmed diag- nosed of severe β-thalassemia, as determined by result of Hemoglobin (Hb) electrophoresis using high-performance liquid chromatography, (2) had never received any blood BioMed Research International 3 The diﬀerent response of hepcidin regulation in these two conditions (anemia and hypoxia) may be due to the diﬀer- ence of iron homeostasis regulation on responding to iron level. The existence of high iron circulation level at that time should be due to as a compensation mechanism to hypoxia and increased erythropoiesis. In the newly diagnosed severe β-thalassemia patients, the response of hepcidin should be still physiologic until in multitransfused severe β-thalassemia patients downregulation of hepcidin occur which causes anomaly. Apparently, hepcidin regulation by hypoxia was overlapped with iron status in the body. Most of iron excess disorder reﬂected dysregulation in iron status signal or ery- throid signal which cause expression of hepcidin inadequate to maintain normal iron homeostasis, and this condition is diﬀerent between newly diagnosed severe β-thalassemia and multitransfused β-thalassemia patients [2, 15]. level was found in 6/42 (14.2%) when SF >1000 ng/mL. In this study, 36/42 (85.7%) patients have reached SF >1000 ng/mL when the amount of transfusion less than 10 times (Table 1). The majority of our subjects (71.4%) were exclusively breastfed and continued until 2 years old with additional complementary food from 6 months old. y There was no signiﬁcant diﬀerence of SF, TS, and hepci- din levels when SF >1000 ng/mL in the group with amount of transfusion 7–12 and less than 7 (p = 0:454, p = 0:084, p = 0:765), respectively (Table 2). A signiﬁcant positive correlation between serum ferritin and amount of transfusion was observed in this study (p < 0:001; r = 0:781) (Figure 1). 4. Discussion Iron status can be seen from SF and TS level, even though liver iron concentration (LIC) is a gold standard to predict iron loading [17–19]. In this study, most of the subjects had already high SF and TS levels since diagnosis, while the mean of amount of transfusion was less than 10 times. Based on TIF guidelines, TS level ≥70% or SF level >1000 ng/mL for initiating iron chelation therapy, it means the severe β-thal- assemia patients should be initiated iron chelator earlier even before 10 transfusions. Coates et al. stated that the measure- ment of SF and TS can be used for iron overload screening. If TS >50% or SF >300 ng/mL on more than one occasion, it should be considered suspicions of iron overload [2, 20]. This study showed that most of the subjects were diagnosed under 2 years old; most of them were before 1 year old. This is in line with the characteristic of children with severe β- thalassemia who are generally diagnosed before the age of 2 years [2, 11]. Galanello and Origa also reported that severe β-thalassemia patients are generally diagnosed between 6 until 24 months [12]. Trehan et al. reported in their study that 52% patients with severe β-thalassemia were diagnosed before 1 year of age and one-third presented between 12 and 24 months of age [11]. It indicated that in severe β-thal- assemia, the genetic imbalance of globin chains causes severe hemolysis and anemia from an early age [2, 12]. In this present study, it was found that in the group amount of transfusion less than 7 had higher value of SF, TS, and hepcidin. It could be considered clinically that the subjects had already high SF and TS before and had reached SF >1000 ng/mL with less than 10 times transfusion. A signif- icant positive correlation between SF and amount of transfu- sion was observed in this study (p < 0:001; r = 0:781). Intensive transfusion for TDT patients leads to contributing factor for iron loading that proportional to the received blood. A unit 420 mL of PRC contains approximately 200 mg of iron or 0.47 mg/mL of whole blood donor [2, 21]. hemolysis and anemia from an early age [2, 12]. The equal gender of the subjects was concordance with thalassemia characteristic as an inherited autosomal recessive disorder following Mendelian rule [2, 13]. 4. Discussion The contribution of iron from diet is small compared to blood transfusion, only 1– 2 mg/day. Most of the subjects in this study receive exclusive breastfeeding and then added complementary foods at the age of 6 months above. It can be predicted that their esti- mated iron intake and its bioavailability only met their daily physiological requirement and will not contribute to iron overload in the body. This is in line with El Safy et al. study who stated there is no signiﬁcant diﬀerence of iron status in infants with β-thalassemia major who received exclusive breastfeeding, exclusive formula feeding, and combination of them. Iron concentration in human milk is lower com- pared to infant formula, and seemingly, it should not contrib- ute to iron burden in newly diagnosed TDT patients [2, 22]. Moreover, this study showed the newly diagnosed severe thalassemia baby has high iron body level since the begin- ning, at diagnosis mean SF level was 365:6 ± 194:9 with range y Ineﬀective erythropoiesis in β-thalassemia caused increased iron absorption that is regulated by hepcidin mainly from dietary absorption in duodenum, recycled iron from macrophages, and released stored iron from hepato- cytes. When iron-deﬁcient hepatocyte produces less or no hepcidin, allowing iron to enter plasma and when iron is abundant, hepcidin will increase to limit further iron absorp- tion and release from stores. The suppressive eﬀect of eryth- ropoiesis on hepcidin in β-thalassemia was caused by IE. It is explained that in β-thalassemia with iron overload, the hep- cidin level was suppressed [15]. In this study, the hepcidin level at diagnosis was still normal (mean 242:6 ± 58:0) but tends to increase. However, only 6 out of 42 (14.3%) patients had high hepcidin level when SF >1000 ng/mL; this might due to the fact that the transfusion inhibits erythropoiesis that will increase hepcidin level [1, 15, 16]. BioMed Research International 4 Table 1: The characteristic of subjects at diagnosis and at SF level >1000 ng/mL. Table 1: The characteristic of subjects at diagnosis and at SF level >1000 ng/mL. Table 1: The characteristic of subjects at diagnosis and at SF level >1000 ng/mL. 4. Discussion Characteristic At diagnosis (n = 42) At SF level >1000 ng/mL (n = 42) Iron status SF (ng/mL); mean ± SD; median (IQR) 365:6 ± 194:9; 319.5 (65.2– 768.3) 1,204:4 ± 141:7; 1,175.5 (1,002– 1,508) TS (%); mean ± SD; median (IQR) 67:3 ± 22:5; 70.0 (20.1–98.7) 78:5 ± 17:3; 82.6 (30.1–100.0) Hepcidin level (ng/mL); mean ± SD; median (IQR) 242:6 ± 58:0; 255.0 (114.5– 363.5) 402:7 ± 159:3; 366.6 (176.1– 876.0) Serum iron (μg/dL); mean ± SD; median (IQR) 177:7 ± 77:9; 173 (45–330) 182:4 ± 49:2; 189 (71–261) TIBC (μg/dL); mean ± SD; median (IQR) 258:7 ± 59:4; 265 (132–367) 233:0 ± 40:6; 230 (153–345) Amount of transfusion when SF level >1000 ng/mL; mean ± SD; median (IQR) 7:6 ± 2; 8 (4–12) Type of thalassemia; n (%) β-Thalassemia mayor 38 (90) Severe HbE/β-thalassemia 4 (10) Age (month); mean ± SD; median (IQR) 9:9 ± 6:4; 7.5(2–24) Gender; n (%) Male 22 (52) Female 20 (48) Hb (g/dL); mean ± SD; median (IQR) 5:1 ± 1:3; 5.2 (1.6–7.8) Dietary status; n (%) Exclusive breastfeeding Yes 42 (100%) No 0 (0%) Continued breastfeeding until 2 years old Yes 30 (71.4%) No 12 (38.6%) Complementary foods Yes 42 (100%) No 0 (0%) Table 2: Comparison between iron status and hepcidin at SF level >1000 ng/ml with group amount of transfusion (n = 42). Variable Amount of transfusion Mean ± SD Median (IQR) Mean diﬀerence 95% CI p value∗ Lower bound Upper bound SF level 1–6 1236:2 ± 155:5 1,214 (1,054– 1,491) 46.1 1,142.2 1,330.2 0.454 7–12 1190:1 ± 135:5 1162.0 (1002– 1508) 1,138.6 1,241.6 TS at SF >1000 ng/mL 1–6 84:1 ± 16:3 84.8 (36.2–100.0) 8.1 74.2 93.9 0.084 7–12 76:0 ± 17:5 81.8 (30.1–96.1) 69.3 82.6 Hepcidin at SF >1000 ng/mL 1–6 420:9 ± 193:0 367.9 (176.1– 876.0) 26.3 304.2 537.5 0.765 7–12 394:5 ± 144:7 362.6 (203.1– 785.6) 339.5 449.6 ∗Mann–Whitney test. parison between iron status and hepcidin at SF level >1000 ng/ml with group amount of transfusion (n = 42). Table 2: Comparison between iron status and hepcidin at SF level >1000 ng/ml with group amount of trans (65.2–768.3), while the mean TS level was 67:3 ± 22:5 with range (20.1–98.7) comparing normal SF and TS level in non- thalassemic children (SF 7–140 ng/mL; TS 6–38%), respec- tively. References [1] S. Gardenghi, R. W. Gray, and S. Rivella, “Anemia, ineﬀective erythropoiesis, and hepcidin: interacting factors in abnormal iron metabolism leading to iron overload in β-thalassemia,” Hematology/Oncology Clinics of North America, vol. 24, no. 6, pp. 1089–1107, 2010. [2] M. D. Cappellini, A. Cohen, J. Porter, A. Taher, and V. Viprakasit, Guidelines for the Management of Transfusion Dependent Thalassaemia (TDT), Thalassaemia International Federation, Nicosia, Cyprus, 3rd edition, 2014. Data Availability The data used to support the ﬁndings of this study are avail- able from the corresponding author upon request. [6] E. Nemeth, “Hepcidin in β-thalassemia,” Annals of the New York Academy of Sciences, vol. 1202, no. 1, pp. 31–35, 2010. [7] E. Nemeth, “Hepcidin and β-thalassemia major,” Blood, vol. 122, no. 1, pp. 3-4, 2013. 5. Conclusions [3] S. Gardenghi, P. Ramos, A. Follenzi et al., “Hepcidin and Hfe in iron overload in β-thalassemia,” Annals of the New York Academy of Sciences, vol. 1202, no. 1, pp. 221–225, 2010. Elevated iron status has been existed in newly diagnosed severe β-thalassemia, while hepcidin level still normal although the value tends to increase. It is suggested that iron chelator should be administered earlier. [4] A. El Beshlawy, I. Alaraby, M. S. A. Kader, D. H. Ahmed, and H. E. M. Adelrahman, “Study of serum hepcidin in hereditary hemolytic anemias,” Hemoglobin, vol. 36, no. 6, pp. 555–570, 2012. [5] S. Pasricha, D. M. Frazer, D. K. Bowden, and G. J. Anderson, “Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with β-thalassemia major: a longitudinal study,” Blood, vol. 122, no. 1, pp. 124–133, 2013. 4. Discussion It seemed that body iron load in newly diagnosed severe thalassemia is mostly due to increased iron absorption (65.2–768.3), while the mean TS level was 67:3 ± 22:5 with range (20.1–98.7) comparing normal SF and TS level in non- thalassemic children (SF 7–140 ng/mL; TS 6–38%), respec- tively. It seemed that body iron load in newly diagnosed severe thalassemia is mostly due to increased iron absorption related to IE and erythroid expansion mechanism which occurred since the early age [2, 13, 22, 23]. Apparently, iron chelator administration in severe β- thalassemia patients should not be only determined by the amount of transfusions. Further study was needed to assign 5 BioMed Research International 5 2500.0 2000.0 1500.0 Serum ferritin (ng/mL) 1000.0 R2 Linear = 0.587 p < 0.001 r = 0.781 500.0 4 6 8 Amount of transfusion 10 12 y = 3.32E2+1.1E2⁎x Figure 1: Correlation between serum ferritin level and amount of transfusion (Spearman’s correlation). A signiﬁcant positive correlation was observed in this study (p < 0:001, r = 0:781). 2500.0 2000.0 1500.0 Serum ferritin (ng/mL) 1000.0 R2 Linear = 0.587 p < 0.001 r = 0.781 500.0 4 6 8 Amount of transfusion 10 12 y = 3.32E2+1.1E2⁎x F 1 C l ti b t f iti l l d t f t f i (S ’ l ti ) A i iﬁ t iti l t Figure 1: Correlation between serum ferritin level and amount of transfusion (Spearman’s correlation). A signiﬁcant positive correlation was observed in this study (p < 0:001, r = 0:781). Acknowledgments time to initiate iron chelator administration in newly diag- nosed severe β-thalassemia patients, as nowadays, the medi- cation for them is available. Origa et al. also reported that early chelation therapy in children with TDT should be administered to prevent iron-related complication such as hypogonadism [24]. A randomized controlled trial by Elalfy et al. reported the safety and eﬃcacy of early start of iron chelation therapy in young children newly diag- nosed with TDT [25]. A multicentre study from Egypt, Indonesia, and Malaysia and also other studies of Makis et al. (Greece) and Chuansumrit et al. (Thailand) showed safety and eﬃcacy of oral iron chelator in TDT children 1–10 years old [26–28]. Detrimental eﬀects of iron over- load can be prevented with good adherences to iron chela- tion therapy [1, 9, 10]. time to initiate iron chelator administration in newly diag- nosed severe β-thalassemia patients, as nowadays, the medi- cation for them is available. Origa et al. also reported that early chelation therapy in children with TDT should be administered to prevent iron-related complication such as hypogonadism [24]. A randomized controlled trial by Elalfy et al. reported the safety and eﬃcacy of early start of iron chelation therapy in young children newly diag- nosed with TDT [25]. A multicentre study from Egypt, Indonesia, and Malaysia and also other studies of Makis et al. (Greece) and Chuansumrit et al. (Thailand) showed safety and eﬃcacy of oral iron chelator in TDT children 1–10 years old [26–28]. Detrimental eﬀects of iron over- load can be prevented with good adherences to iron chela- tion therapy [1, 9, 10]. We would like to thank the parents and children for partici- pating in this study. We also thank the doctors and nurses of Thalassemia Clinic for their dedicated care for our patients. BioMed Research International BioMed Research International 6 [24] R. Origa, F. Tatti, A. Zappu et al., “Earlier initiation of transfu- sional and iron chelation therapies in recently born children with transfusion-dependent thalassemia,” American Journal of Hematology, vol. 92, no. 11, pp. E627–E628, 2017. Sickle Cell Disease and β thalassemic patients,” European Jour- nal of Haematology, vol. 84, no. 1, pp. 72–78, 2010. [9] J. B. Porter, M. El-Alfy, V. Viprakasit et al., “Utility of labile plasma iron and transferrin saturation in addition to serum ferritin as iron overload markers in diﬀerent underlying ane- mias before and after deferasirox treatment,” European Jour- nal of Haematology, vol. 96, no. 1, pp. 19–26, 2016. [25] M. S. Elalfy, A. Adly, H. Awad, M. Tarif Salam, V. Berdoukas, and F. Tricta, “Safety and eﬃcacy of early start of iron chela- tion therapy with deferiprone in young children newly diag- nosed with transfusion-dependent thalassemia: a randomized controlled trial,” American Journal of Hematology, vol. 93, no. 2, pp. 262–268, 2018. [10] M. D. Cappellini, M. Bejaoui, L. Agaoglu et al., “Iron chelation with deferasirox in adult and pediatric patients with thalasse- mia major: eﬃcacy and safety during 5 years’ follow up,” Blood, vol. 118, no. 4, pp. 884–893, 2011. [26] M. S. Elalfy, T. T. Sari, C. L. Lee, F. Tricta, and A. El-Beshlawy, “The safety, tolerability, and eﬃcacy of a liquid formulation of deferiprone in young children with transfusional iron over- load,” Journal of Pediatric Hematology/Oncology, vol. 32, no. 8, pp. 601–605, 2010. [11] A. Trehan, N. Sharma, R. Das, D. Bansal, and R. K. Marwaha, “Clinicoinvestigational and demographic proﬁle of children with thalassemia major,” Indian Journal of Hematology and Blood Transfusion, vol. 31, no. 1, pp. 121–126, 2015. [27] A. Makis, N. Chaliasos, S. Alfantaki, P. Karagouni, and A. Siamopoulou, “Chelation Therapy with Oral Solution of Deferiprone in Transfusional Iron- Overloaded Children with Hemoglobinopathies,” Anemia, vol. 2013, Article ID 121762, 3 pages, 2013. [12] R. Galanello and R. Origa, “Beta-thalassemia,” Orphanet Jour- nal of Rare Disease, vol. 5, no. 1, 2010. [13] V. G. Sankaran, D. G. Nathan, and S. H. Orkin, “Thalasse- mias,” in Hematology and Oncology of Infancy and Childhood, S. H. Orkin, D. E. Fisher, D. Ginsburg, A. T. Look, S. E. Lux, and D. G. Nathan, Eds., Elsevier Saunders, Philadelphia, USA, 8th edition, 2015. [28] A. Chuansumrit, D. Songdej, N. Sirachainan, P. Wongwerawattanakoon, P. Kadegasem, and W. Conflicts of Interest [8] A. Koren, D. Fink, O. Admoni, Y. Tennenbaum-Rakover, and C. Levin, “Non-transferrin bound labile plasma iron and iron overload in Sickle Cell Disease: a comparative study between The authors declare that there is no conﬂict of interests regarding the publication of this paper. BioMed Research International Sasanakul, “Safety proﬁle of a liquid formulation of deferiprone in young children with transfusion-induced iron overload: a 1-year experience,” Paediatric and International Child Health, vol. 36, no. 3, pp. 209–213, 2016. [14] K. Leecharoenkiat, P. Lithanatudom, W. Sornjai, and D. R. Smith, “Iron dysregulation in beta-thalassemia,” Asian Paciﬁc Journal of Tropical Medicine, vol. 9, no. 11, pp. 1035–1043, 2016. [15] T. Ganz and E. 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Kompani, “Serum ferritin levels and irregular use of iron chelators predict liver iron load in patients with major beta thalassemia: a cross-sectional study,” Croatian Medical Jour- nal, vol. 60, no. 5, pp. 405–413, 2019. [20] T. D. Coates, S. Carson, J. C. Wood, and V. Berdoukas, “Man- agement of iron overload in hemoglobinopathies: what is the appropriate target iron level?,” Annals of the New York Acad- emy of Sciences, vol. 1368, no. 1, pp. 95–106, 2016. [21] F. Danjou, Z. I. Cabantchik, R. Origa et al., “A decisional algo- rithm to start iron chelation in patients with beta thalassemia,” Haematologica, vol. 99, no. 3, pp. e38–e40, 2014. [22] U. R. El Safy, M. M. Fathy, T. H. Hassan et al., “Eﬀect of breast- feeding versus infant formula on iron status of infants with beta thalassemia major,” International breastfeeding journal, vol. 12, no. 1, 2017. [23] P. Lanzkowsky, J. M. Lipton, and J. D. 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https://openalex.org/W2303233021	https://hal.archives-ouvertes.fr/hal-01338300/file/0026026-03.pdf	English	null	Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans	AMB express	2,016	cc-by	8,338	To cite this version: Eve Dondelinger, Nathalie Aubry, Fadhel Ben Chaabane, Celine Cohen, Jean Tayeb, et al.. Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hy- drolysis of cellulose in presence of xylans. AMB Express, 2016, 6, pp.24. 10.1186/s13568-016-0196-x. hal-01338300 Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans ndelinger, Nathalie Aubry, Fadhel Ben Chaabane, Celine Cohen, Jean Tayeb, Caroline Rémond Tayeb, Caroline Rémond Tayeb, Caroline Rémond © 2016 Dondelinger et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemi- cellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes devel- oped in the context of lignocellulosic biorefinery. Keywords: Cellulase, β-Glucosidase, Xylanase, β-xylosidase, Ethanol, Biorefinery HAL Id: hal-01338300 https://hal.science/hal-01338300v1 Submitted on 28 Jun 2016 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. Dondelinger et al. AMB Expr (2016) 6:24 DOI 10.1186/s13568-016-0196-x Open Access Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans Eve Dondelinger1,2, Nathalie Aubry1,2, Fadhel Ben Chaabane3, Céline Cohen3, Jean Tayeb1,2 and Caroline Rémond1,2* Eve Dondelinger1,2, Nathalie Aubry1,2, Fadhel Ben Chaabane3, Céline Cohen3, Jean Tayeb1,2 and Caroline Rémond1,2* Introduction composition of biomass to hydrolyse. Depending on lig- nocellulosic biomass and on the severity of the pretreat- ment technology, xylose and xylo-oligosaccharides (XOs) are released to various extents during the pretreatment step (Chandra et al. 2007). Different approaches can be developed to improve enzymatic fractionation of lig- nocellulose. Pretreatment of wheat straw at high solid loading (20 % DM) in presence of xylanase conducted to an increase of glucan hydrolysis by Celluclast 1.5 L by a factor 2.1 (Remond et al. 2010). Supplementation of cel- lulosic cocktails with hemicellulases allowed improv- ing hydrolysis yields of cellulose and hemicelluloses: while increasing the hydrolysis of cellulose and xylan A challenge for producing glucose from enzymatic hydrolysis of lignocellulosic biomass while limiting the process cost is to perform enzymatic hydrolysis at high solid concentration and to use low enzyme loadings. In this context, the development of optimized enzyme mixtures is of interest. Enzymatic cocktails have to be adapted to lignocellulosic biomass as well as to pre- treatment technology which impact largely chemical Correspondence: caroline.remond@univ‑reims.fr 1 Université de Reims Champagne-Ardenne, UMR614 Fractionnement des AgroRessources et Environnement, 51100 Reims, France Full list of author information is available at the end of the article Correspondence: caroline.remond@univ‑reims.fr 1 Université de Reims Champagne-Ardenne, UMR614 Fractionnement des AgroRessources et Environnement, 51100 Reims, France Full list of author information is available at the end of the article Enzymes production in bioreactors Two strains from T. reesei (CL847 and TR3002) were used to produce the different enzymatic cocktails used in this study. Trichoderma reesei CL847 is a cellulase hyperproducer strain obtained from NG14 Rut-C30 strain by several steps of mutagenesis and selection, from Cayla Company, Toulouse, France (Portnoy et al. 2011). The strain TR3002 was obtained from the CL847 after introduction of an improved β-glucosidase gene (Ayrin- hac et al. 2011). Spores were conserved in cryotubes at −80 °C with 50 % glycerol. The protocol used to produce the enzymatic cocktails consists in two phases as described previously (Jourdier et al. 2013). Bioreactor cultivations were carried out in Dasgip fedbatch-pro bioreactors with an initial working volume of 750 mL. For each bioreactor, a preculture was performed in a Fernbach flask with 250 mL flask medium culture, inoculated with 106 spores, incubated 72 h at 150 rpm and 30 °C in an Infors rotary shaker. Seventy- five milli-liters were then used to inoculate the bioreac- tor. Growth phase in batch was performed on 15 g/L glucose at pH 4.8 and 27 °C for 24 h. Then fed-batch was performed at pH 4.0 and 25 °C with feeding at 2 mL/h by a 250 g/L mixed sugars solution (Table 1). The pH was automatically adjusted with 5.5 N NH3 solution. Aeration rate was fixed at 30 sL/h and agitation was regulated to maintain at least 40 % dissolved oxygen of its saturation. The strategy developed in the present study was based on the use of enzymatic cocktails obtained from T. ree- sei. In this context, various enzymatic cocktails were pre- pared from T. reesei after cultivation with different sugars sources and ratios. This allowed obtaining cocktails con- taining various levels of cellulases and hemicellulases activities. These cocktails were tested for hydrolysis of Avicel. The effect of the presence of added xylans dur- ing the hydrolysis of Avicel was also investigated. Experi- ments were conducted at high substrate loading and with low enzymes loading in order to mimic the conditions of an industrial process. The experiments for TR2 and TR4 production were performed with the strain CL847 while the experiments concerning TR1, TR3 and TR5 were performed with the strain TR3002. The enzymatic cocktails were chosen in order to have contrasted enzymatic activities. Dondelinger et al. AMB Expr (2016) 6:24 Page 2 of 9 obtained from Cascade Analytical Reagents & Biochemi- cals (Corvallis, Oregon, USA). XOs contained DP2–5 oligosaccharides. from steam-explosed corn stover, the supplementation by GH11 xylanases of commercial cellulase (Celluclast 1.5 L) allows reducing the cellulase loading by a factor 7 (Hu et al. 2011). This was attributed to the removal of xylans and to the increase of cellulose accessibility by enhancing fiber porosity and swelling (Hu et al. 2011). Another strategy is to develop enzymatic cocktails pos- sessing both cellulases and hemicellulases activities by growing micro-organisms onto various simple or com- plex substrates. In this way, improved cellulases cocktails contain high hemicellulases level. Recently, Trichoderma reesei was cultivated in presence of various commercial sugars to evaluate the impact of these carbon sources onto enzymes produced (Jourdier et al. 2013). In pres- ence of high xylose concentration, cellulases activities (endoglucanase, cellobiohydrolase and β-glucosidase) decreased whereas xylanase activity was more important compared to culture without xylose (Jourdier et al. 2013). Even if significant progress to obtain efficient enzymatic cocktails has already been achieved, their improvement remains a challenge in case of lignocellulosic biomass hydrolysis. To the best of our knowledge, no study con- cerns the production of enzymes by T. reesei growing on hemicellulolytic hydrolysates. Enzymes production in bioreactors The objective of this work was to highlight the impact of the cellulase and some hemicellulases activities in vari- ous contrasted enzymatic cocktails during hydrolysis of Avicel, of xylans, and simultaneous hydrolysis of both substrates. Furthermore, combinations of different enzy- matic cocktails were evaluated in order to improve the global efficiency of cocktails for cellulose hydrolysis. All cocktails tested in our study were complete cocktails pro- duced by T. reesei onto various carbon sources more or less enriched in C5 and C6 sugars. Hemicellulosic hydrolysates referred to pentose (C5) extracts obtained after steam explosion of wheat straw under acidic conditions (H2SO4 presoaking), followed by washing with water and further concentration by evap- oration as described by Warzywoda et al. (1992). The analytical composition of the hemicellulosic hydrolyzate Table 1 Sugar composition used during the fed-batch mode. C5 refers to hemicellulosic hydrolysate Experiment Carbon source Strain TR1 20 % xylose/25 % lactose/55 % glucose TR3002 TR2 100 % C5 CL847 TR3 10 % C5/25 % Lactose/65 % glucose TR3002 TR4 100 % Lactose CL847 TR5 75 % C5/25 % Lactose TR3002 Table 1 Sugar composition used during the fed-batch mode. C5 refers to hemicellulosic hydrolysate Experiment Carbon source Strain TR1 20 % xylose/25 % lactose/55 % glucose TR3002 TR2 100 % C5 CL847 TR3 10 % C5/25 % Lactose/65 % glucose TR3002 TR4 100 % Lactose CL847 TR5 75 % C5/25 % Lactose TR3002 Table 1 Sugar composition used during the fed-batch mode. C5 refers to hemicellulosic hydrolysate Experiment Carbon source Strain TR1 20 % xylose/25 % lactose/55 % glucose TR3002 TR2 100 % C5 CL847 TR3 10 % C5/25 % Lactose/65 % glucose TR3002 TR4 100 % Lactose CL847 TR5 75 % C5/25 % Lactose TR3002 Table 1 Sugar composition used during the fed-batch mode. C5 refers to hemicellulosic hydrolysate Results Proteins were measured with the Lowry method (Lowry et al. 1951). Prior to quantification, samples were washed with 10 % trichloroacetic acid during 30 min at 4 °C. Supernatants were thrown after 5 min of centrifuga- tion at 13,000 rpm. Pellets were dried 5 min in a speed- vac and the precipitates were dissolved with 0.08 % sodium hydroxide and 0.4 % sodium carbonate. Proteins Materials and methods Materials Microcrystalline cellulose (Avicel PH-101) and xylose were purchased from Sigma–Aldrich® (St Louis, MO, USA), cellulose content >97 %. Beechwood xylan was supplied by Carl Roth® (Karlsruhe, Germany). XOs were Dondelinger et al. AMB Expr (2016) 6:24 Page 3 of 9 concentration was measured in supernatants against BSA standards (0–500 µg/mL). used in this study was: 174 g/L of xylose, 22.5 g/L of arab- inose, 27.5 g/L of glucose, 21 g/L of oligomers. For the preculture before bioreactor cultivations, the medium composition was: cornsteep solid 1.5 g/L; dipotassium phtalate 6 g/L; H3PO4 85 % 0.8 mL/L; (NH4)2SO4 4.2 g/L; MgSO4,7H2O 0.3 g/L; CaCl2,2H2O 0.15 g/L; FeSO4–7H2O 30 mg/L; MnSO4,H2O 6 mg/L; ZnSO4,7H2O 8 mg/L; CoNO3,6H2O 9 mg/L; H3BO3 1 mg/L. pH was adjusted to 6.0 with NaOH 30 %. Carbohydrates quantificationh The glucose concentration was assessed by a glucose oxidase assay with an Analox GL6 glucose analyzer (Imlab, Lille France) and with a standard glucose solu- tion (144 mg/dL, Imlab, Lille France). Quantification of xylose and XOs was performed by HPAEC-PAD (Dionex, Thermo Scientific, Courtaboeuf, France). Before analysis, all samples were filtered (PTFE, 0.22 µm) before injec- tion on a CarboPac PA-1 column (4 × 250 mm, Dionex). Xylose was eluted as previously described (Remond et al. 2010) with fucose as internal standard. XOs (DP2–DP6) were eluted with a 100 mM NaOH and 300 mM sodium acetate gradient with a flow rate of 1 mL/min. Detection was carried out by pulsed amperometry (ED 40, Dionex) and signal sensitivity was increased with a post-column module delivering 300 mM NaOH. i Endo-β-1,4-xylanase activity was determined by measuring the reducing sugars liberated from beech- wood xylan as previously described (Rakotoarivonina et al. 2012). Reaction mixture contained 0.9 mL 0.5 % xylan (w/v) in 50 mM citrate phosphate buffer pH 4.8 and 0.1 mL enzyme solution. Reactions were conducted at 50 °C for 10 min. One unit (IU) was defined as the quantity of enzyme required to liberate 1 µmol of xylose equivalent per minute at 50 °C. Filter paper activity (FPU) describing the global cel- lulolytic activity was assayed according to the IUPAC standard Filter Paper Assay (Ghose 1987). The amount of released sugars was quantified from filter paper strip (Whatman no.1, 1 × 6 cm) and reducing sugars were estimated by the DNS method (Miller 1959). One unit of enzyme activity corresponds to the amount of enzyme required to release 1 µmol of glucose equivalent per min- ute under the assay conditions. Yields of glucose and xylose released were calculated according to their quantity introduced during the reac- tions by taking into account their conversion from cel- lulose and xylans (anhydro correction of 0.9 and 0.88 for glucose and xylose respectively). Yields of XOs released were expressed on the basis of xylose initially present in reaction. Enzyme assays β-xylosidase and β-glucosidase activities were deter- mined by incubating 0.1 mL of enzymatic cocktail with 0.9 mL ρ-nitrophenyl-β-d-xyloside or ρ-nitrophenyl-β- d-glucoside as substrates at 5 mM. Reactions were per- formed during 10 min in 50 mM citrate phosphate buffer, pH 4.8 with appropriate dilute enzyme solutions at 50 °C. Release of ρ-nitrophenol (ρNP) was measured by con- tinuous monitoring at 401 nm. One unit of β-xylosidase or β-glucosidase activities was defined as the amount of enzyme releasing 1 µmol of ρNP per minute using the defined conditions. Hydrolysis samples were taken after 24, 48 and 72 h of hydrolysis and were boiled for 10 min to terminate the reaction and stored at −20 °C until carbohydrates analy- sis. All assays were performed in triplicate. Enzymatic hydrolysis Hydrolysis of 10 % (w/v) Avicel and of 1.5 % (w/v) xylans was performed with enzymes cocktails with a loading of 10 mg proteins/g Avicel or xylans. Reactions were car- ried out in 50 mM citrate phosphate buffer (pH 4.8) with chloramphenicol (100 ppm) in a thermostatically con- trolled system Tornado Radleys® (Interchim, Montluçon, France) at 45 °C under agitation at 150 rpm. For some Avicel hydrolysis experiments, 1.5 % (w/v) beechwood xylan, xylose or XOs were added. Experiments conducted with mixtures of cocktails were performed with TR1 sup- plemented with TR5 with two different ratios of proteins quantities (1/1 and 4/1) with a total protein loading cor- responding to 10 mg/g Avicel. For bioreactor cultivations, the medium composi- tion was: cornsteep solid 1.5 g/L; KOH 1.66 g/L; H3PO4 85 % 2.5 mL/L; (NH4)2SO4 2.8 g/L; MgSO4,7H2O 0.6 g/L; CaCl2,2H2O 0.6 g/L; FeSO4–7H2O 60 mg/L; MnSO4,H2O 12 mg/L; ZnSO4,7H2O 16 mg/L; CoNO3,6H2O 18 mg/L; H3BO3 2 mg/L. pH was adjusted to 4.8 with NH3 20 %. Production and characterization of various enzymatic cocktails This activity was rather abundant in cocktail TR3 (10.5 IU/mg) and was intermediary for the cocktail TR1 (6.1 IU/mg). Concerning hemicellulases activities, xyla- nase and β-xylosidase activities were measured. Cock- tails TR2 and TR1 contained high level of xylanase activity (59.1 IU/mg and 53.5 IU/mg respectively). Xyla- nase activity was lowest for cocktail TR4 (11.9 IU/mg) and was intermediary for cocktails TR3 and TR5 (26.7 and 37.8 IU/mg respectively). Cocktail TR2 which was rich in xylanase activity possessed also high levels of β-xylosidase activity (0.3 IU/mg). However one could observe that cocktail TR1 which presented an important xylanase activity (53.5 IU/mg) did not possess impor- tant level of xylosidase activity (0.1 IU/mg). The higher β-xylosidase (0.5 IU/mg) activity was for cocktail TR5 whereas this activity was low for cocktail TR4 (0.03 IU/ mg). Finally cocktail TR3 contained intermediary level of β-xylosidase (0.3 IU/mg). TR3 and TR4 presented similar FPU activity (0.5 FPU/ mg) and cocktail TR2 possessed a rather important FPU activity (0.6 FPU/mg). The β-glucosidase activity was the most important in cocktail TR5 (13.1 IU/mg) and decreased by a factor 9 in cocktails TR2 and TR4 (1.4 IU/ mg). This activity was rather abundant in cocktail TR3 (10.5 IU/mg) and was intermediary for the cocktail TR1 (6.1 IU/mg). Concerning hemicellulases activities, xyla- nase and β-xylosidase activities were measured. Cock- tails TR2 and TR1 contained high level of xylanase activity (59.1 IU/mg and 53.5 IU/mg respectively). Xyla- nase activity was lowest for cocktail TR4 (11.9 IU/mg) and was intermediary for cocktails TR3 and TR5 (26.7 and 37.8 IU/mg respectively). Cocktail TR2 which was rich in xylanase activity possessed also high levels of β-xylosidase activity (0.3 IU/mg). However one could observe that cocktail TR1 which presented an important xylanase activity (53.5 IU/mg) did not possess impor- tant level of xylosidase activity (0.1 IU/mg). The higher β-xylosidase (0.5 IU/mg) activity was for cocktail TR5 whereas this activity was low for cocktail TR4 (0.03 IU/ mg). Finally cocktail TR3 contained intermediary level of β-xylosidase (0.3 IU/mg). Fig. 1 Enzymatic hydrolysis of Avicel 10 % (w/v) by different enzy- matic cocktails at 10 mg proteins/g Avicel without xylans (a) or in presence of xylans 15 g/L (b). Mean values and standard deviations of triplicates are presented similar kinetic for glucose release which attained respec- tively 34.6 ± 2.2 % and 33.7 ± 2.2 % after 72 h. Production and characterization of various enzymatic cocktails Glucose yields were lowest in case of cocktail TR2 and reached 25.5 ± 2.7 % at 72 h. Production and characterization of various enzymatic cocktails Enzymatic cocktails presented various enzymatic activi- ties ratios (Table 2). Cocktail TR1 was the most rich in FPU activity (0.7 FPU/mg) whereas cocktail TR5 con- tained the lowest FPU activity (0.4 FPU/mg). Cocktails Dondelinger et al. AMB Expr (2016) 6:24 Page 4 of 9 TR3 and TR4 presented similar FPU activity (0.5 FPU/ mg) and cocktail TR2 possessed a rather important FPU activity (0.6 FPU/mg). The β-glucosidase activity was the most important in cocktail TR5 (13.1 IU/mg) and decreased by a factor 9 in cocktails TR2 and TR4 (1.4 IU/ mg). This activity was rather abundant in cocktail TR3 (10.5 IU/mg) and was intermediary for the cocktail TR1 (6.1 IU/mg). Concerning hemicellulases activities, xyla- nase and β-xylosidase activities were measured. Cock- tails TR2 and TR1 contained high level of xylanase activity (59.1 IU/mg and 53.5 IU/mg respectively). Xyla- nase activity was lowest for cocktail TR4 (11.9 IU/mg) and was intermediary for cocktails TR3 and TR5 (26.7 and 37.8 IU/mg respectively). Cocktail TR2 which was rich in xylanase activity possessed also high levels of β-xylosidase activity (0.3 IU/mg). However one could observe that cocktail TR1 which presented an important xylanase activity (53.5 IU/mg) did not possess impor- tant level of xylosidase activity (0.1 IU/mg). The higher β-xylosidase (0.5 IU/mg) activity was for cocktail TR5 whereas this activity was low for cocktail TR4 (0.03 IU/ mg). Finally cocktail TR3 contained intermediary level of β-xylosidase (0.3 IU/mg). Table 2 Proteins concentrations and enzymatic activities (FPU, β-glucosidase, xylanase, β-xylosidase) present in the enzymatic cocktails TR1 TR2 TR3 TR4 TR5 Proteins (g/L) 154.0 32.0 58.5 68.0 26.0 FPU (IU/mg) 0.7 0.6 0.5 0.5 0.4 β-Glucosidase (IU/mg) 6.1 1.4 10.5 1.4 13.1 Xylanase (IU/mg) 53.5 59.1 26.7 11.9 37.8 β-Xylosidase (IU/mg) 0.1 0.3 0.3 0.03 0.5 Table 2 Proteins concentrations and enzymatic activities (FPU, β-glucosidase, xylanase, β-xylosidase) present in the enzymatic cocktails Fig. 1 Enzymatic hydrolysis of Avicel 10 % (w/v) by different enzy- matic cocktails at 10 mg proteins/g Avicel without xylans (a) or in presence of xylans 15 g/L (b). Mean values and standard deviations of triplicates are presented TR3 and TR4 presented similar FPU activity (0.5 FPU/ mg) and cocktail TR2 possessed a rather important FPU activity (0.6 FPU/mg). The β-glucosidase activity was the most important in cocktail TR5 (13.1 IU/mg) and decreased by a factor 9 in cocktails TR2 and TR4 (1.4 IU/ mg). Enzymatic hydrolysis of Avicel with various contrasted enzymatic cocktailsh 2 Yields of xylose and xylo-oligosaccharides (XOs) released from xylans (1.5 %, w/v) at 72 h with enzymatic cocktails at 10 mg proteins/g xylans. Mean values and standard deviations of triplicates are presented Fig. 2 Yields of xylose and xylo-oligosaccharides (XOs) released from xylans (1.5 %, w/v) at 72 h with enzymatic cocktails at 10 mg proteins/g xylans. Mean values and standard deviations of triplicates are presented represented 5.80 ± 0.13 % and 5.13 ± 0.15 % respec- tively for cocktails TR1 and TR4. Enzymatic hydrolysis of Avicel in presence of xylans with various contrasted enzymatic cocktails However, an important observation is that for all enzymatic cocktails and during the entire reactions, yields of glucose were lower than those obtained when catalysis was performed in absence of xylans. After 72 h, yields were decreased by factor 1.33 and 1.32 for TR1 and TR5 whereas the decrease was 1.24 and 1.20 for TR2 and TR4. The yield was less affected for cocktail TR3 as it decreased 1.1-fold.i Fig. 3 Yields of xylose and xylo-oligosaccharides (XOs) released in presence of cellulose Avicel (10 %, w/v) and xylans (1.5 %, w/v) at 72 h with enzymatic cocktails at 10 mg proteins/g Avicel. Mean values and standard deviations of triplicates are presented with the level of xylosidase activity poorly present in TR4 and TR1 cocktails. In the present study, hydrolysis of Avicel was tested with TR1 cocktail in presence of xylose or in presence of XOs (DP 2–5) (Fig. 4). Whereas xylose did not lead to any significant reduction in glucose yields, presence of XOs from DP2 to 5 induced a lower glucose release from Avicel notably at 48 and 72 h. Glucose yields were respectively 35.8 and 42.1 % after 48 and 72 h without XOs and decreased to 29.1 and 36 % after 48 and 72 h in presence of XOs which represents a decrease by 1.23-fold and 1.17-fold respectively. Similar reduction of glucose release was obtained for other enzymatic cocktails (data not shown). Quantification of xylose and XOs present in solutions at 72 h is presented in Fig. 3. As observed for hydroly- sis reactions of xylans, the various enzymatic cocktails did not present the same efficiency for xylans hydroly- sis. Cocktails TR5 and TR2 were the most efficient and liberated xylose with yields respectively 90.7 ± 6.7 % and 87.3 ± 1.2 %. XOs concentrations were respec- tively 4.7 ± 0.2 % and 3.7 ± 0.7 % for TR5 and TR2. Yield of xylose released was respectively 81.5 ± 2.7 %, 74.2 ± 6.3 % and 72.4 ± 4.5 % for cocktails TR3, TR1 and TR4 respectively. XOs concentrations reached 3.7 ± 0.6 %, 11.8 ± 2.2 % and 14.4 ± 0.7 % respectively for TR3, TR4 and TR1. These results are in accordance Enzymatic hydrolysis of Avicel in presence of xylans with various contrasted enzymatic cocktails Hydrolysis experiments of cellulose were conducted in presence of xylans with the different cocktails. Figure 1b represents the kinetic of glucose released during 72 h of reaction. After 72 h of reactions, yields were 20.5 ± 2.8 %, 26.3 ± 1.2 %, 28.1 ± 2.8 %, 31.7 ± 1.4 % and 36.0 ± 1.4 % respectively for cocktails TR2, TR5, TR4, TR1 and TR3. This classification of increased efficiency displayed according to cocktails was similar to the one obtained for reactions catalyzed without xylans (Fig. 1a). However, an important observation is that for all enzymatic cocktails and during the entire reactions, yields of glucose were lower than those obtained when catalysis was performed in absence of xylans. After 72 h, yields were decreased by factor 1.33 and 1.32 for TR1 and TR5 whereas the decrease was 1.24 and 1.20 for TR2 and TR4. The yield was less affected for cocktail TR3 as it decreased 1.1-fold. Quantification of xylose and XOs present in solutions at 72 h is presented in Fig. 3. As observed for hydroly- sis reactions of xylans, the various enzymatic cocktails did not present the same efficiency for xylans hydroly- sis. Cocktails TR5 and TR2 were the most efficient and liberated xylose with yields respectively 90.7 ± 6.7 % and 87.3 ± 1.2 %. XOs concentrations were respec- tively 4.7 ± 0.2 % and 3.7 ± 0.7 % for TR5 and TR2. Yield of xylose released was respectively 81.5 ± 2.7 %, 74.2 ± 6.3 % and 72.4 ± 4.5 % for cocktails TR3, TR1 and TR4 respectively. XOs concentrations reached 3.7 ± 0.6 %, 11.8 ± 2.2 % and 14.4 ± 0.7 % respectively for TR3, TR4 and TR1. These results are in accordance Hydrolysis experiments of cellulose were conducted in presence of xylans with the different cocktails. Figure 1b represents the kinetic of glucose released during 72 h of reaction. After 72 h of reactions, yields were 20.5 ± 2.8 %, 26.3 ± 1.2 %, 28.1 ± 2.8 %, 31.7 ± 1.4 % and 36.0 ± 1.4 % respectively for cocktails TR2, TR5, TR4, TR1 and TR3. This classification of increased efficiency displayed according to cocktails was similar to the one obtained for reactions catalyzed without xylans (Fig. 1a). Enzymatic hydrolysis of Avicel with various contrasted enzymatic cocktailsh Xylan conversion was studied with the various enzy- matic cocktails. Yields of xylose and XOs from DP2 to 5 were quantified after 72 h of catalysis (Fig. 2). In case of cocktails TR2 and TR5, xylose release was respectively 77.9 ± 2.2 % and 75.9 ± 0.6 % of total xylose calculated from xylans content. In parallel, XOs content (calcu- lated as % of total xylose from xylans) were low for reac- tions performed with these both cocktails and reached respectively 0.25 ± 0.05 % and 0.76 ± 0.02 % respec- tively for TR2 and TR5. Xylose production was less important for cocktail TR3 and reached 68.8 ± 5.1 %. In this case, XOs content represented 2.44 ± 0.15 %. Cocktails TR1 and TR4 were less efficient for xylose release which attained respectively 48.7 ± 3.2 % and 46.3 ± 0.3 % respectively. These both cocktails were also less efficient for XOs hydrolysis as XOs content The various enzymatic cocktails were evaluated for their hydrolysis efficiency on cellulose Avicel. In order to reveal subtle differences between all cocktails and to favor low protein concentration use as it should be the case for industrial processes, catalysis was performed with 10 mg proteins/g cellulose. Furthermore, cellu- lose loading was high as reactions were conducted with 10 % (w/v) of cellulose. The glucose yields are presented in Fig. 1a. For all cocktails, reactions were not finished after 72 h of reaction. This was not surprising as protein concentration was low during hydrolysis experiments. Cocktails TR1 and TR3 were the most efficient for Avicel hydrolysis. Glucose yields were respectively 42.1 ± 0.8 % and 39.5 ± 1.6 % respectively for both these cocktails after 72 h of reaction. Cocktails TR5 and TR4 presented Dondelinger et al. AMB Expr (2016) 6:24 Page 5 of 9 Fig. 3 Yields of xylose and xylo-oligosaccharides (XOs) released in presence of cellulose Avicel (10 %, w/v) and xylans (1.5 %, w/v) at 72 h with enzymatic cocktails at 10 mg proteins/g Avicel. Mean values and standard deviations of triplicates are presented Fig. 2 Yields of xylose and xylo-oligosaccharides (XOs) released from xylans (1.5 %, w/v) at 72 h with enzymatic cocktails at 10 mg proteins/g xylans. Mean values and standard deviations of triplicates are presented Fig. Effect of cocktails mixture for hydrolysis efficiency Hydrolysis experiments were performed wi these cocktails mixtures in same conditions as for pr vious experiments: 10 % Avicel in presence of xylan 15 g/L with a total loading of 10 mg proteins/g Avic For both ratios tested, release of glucose reached sim lar yields (33.1 ± 1.2 % and 33.5 ± 1.9 % respectively f ratios 1/1 and 4/1 after 72 h) as obtained for TR1 alon (31.7 ± 1.4 % after 72 h) (Fig. 5). Compared to TR5 alon (26.3 ± 1.2 %), both mixtures led to increased gluco yields which were 1.27-fold higher at 72 h indicating th the more important cellulase activity in cocktail mixtur Table 3 Proteins concentrations and enzymatic activiti measured from cocktails mixtures (FPU, β-glucosidas xylanase, β-xylosidase) present in the cocktails mixtures TR1/TR5 1/1 TR1/TR5 4 Proteins (g/L) 89.4 125.0 FPU (IU/mg) 0.5 0.6 β-Glucosidase (IU/mg) 10.2 7.0 Xylanase (IU/mg) 45.6 50.0 β-Xylosidase (IU/mg) 0.3 0.2 In that way, mixtures were prepared with TR1 supple- mented with TR5 with two different ratios of proteins quantities (1/1 and 4/1) with a total protein loading corresponding to 10 mg/g Avicel (Table 3). These com- binations were chosen in order to obtain cocktails pre- senting high levels of FPU, β-glucosidase, xylanase and β-xylosidase activities. For both ratios tested, mixing TR1 with TR5 induced a decrease of FPU and xylanase activities whereas β-glucosidase and β-xylosidase activi- ties were increased compared to TR1. In contrary, in case of TR5, complementation with TR1 with both ratios induced more important FPU and xylanase activities and less high levels of β-glucosidase and β-xylosidase activities. Hydrolysis experiments were performed with these cocktails mixtures in same conditions as for pre- vious experiments: 10 % Avicel in presence of xylans 15 g/L with a total loading of 10 mg proteins/g Avicel. For both ratios tested, release of glucose reached simi- lar yields (33.1 ± 1.2 % and 33.5 ± 1.9 % respectively for ratios 1/1 and 4/1 after 72 h) as obtained for TR1 alone (31.7 ± 1.4 % after 72 h) (Fig. 5). Compared to TR5 alone (26.3 ± 1.2 %), both mixtures led to increased glucose yields which were 1.27-fold higher at 72 h indicating that the more important cellulase activity in cocktail mixtures could probably be responsible for these higher yields. Xylose and XOs present at 72 h were quantified (Fig. 6). Effect of cocktails mixture for hydrolysis efficiency Xylose release was higher for ratio 4/1 (95.2 ± 1.9 %) compared to ratio 1/1 (87 ± 3.4 %). The xylose release is more important with TR1/TR5: 1/4 mixture compared to TR5 alone (90.7 %). XOs were detected with concentra- tions reaching 4.3 ± 0.3 % and 5.5 ± 0.4 % respectively for TR1/TR5 ratios 1/4 and 1/1. Effect of cocktails mixture for hydrolysis efficiency In order to investigate the impact of enzymatic activi- ties enrichment, experiments were conducted with some cocktails mixtures. The objective was to test their effect on hydrolysis of cellulose in presence of xylans. Dondelinger et al. AMB Expr (2016) 6:24 Page 6 of 9 Fig. 4 Yields of glucose released from Avicel (10 %, w/v) in absence or in presence of xylose and XOs (DP 2–5) with TR1 cocktail at 10 mg proteins/g Avicel. Mean values and standard deviations of triplicates are presented I th t i t d ith TR1 l Fig. 4 Yields of glucose released from Avicel (10 %, w/v) in absence or in presence of xylose and XOs (DP 2–5) with TR1 cocktail at 10 mg proteins/g Avicel. Mean values and standard deviations of triplicates are presented Fig. 5 Enzymatic hydrolysis of Avicel 10 % (w/v) in presence of xylans 15 g/L with different enzymatic cocktails and mixtures of cocktails with 10 mg total proteins/g Avicel. Mean values and standard devia- tions of triplicates are presented Fig. 5 Enzymatic hydrolysis of Avicel 10 % (w/v) in presence of xylans 15 g/L with different enzymatic cocktails and mixtures of cocktails with 10 mg total proteins/g Avicel. Mean values and standard devia- tions of triplicates are presented Fig. 4 Yields of glucose released from Avicel (10 %, w/v) in absence or in presence of xylose and XOs (DP 2–5) with TR1 cocktail at 10 mg proteins/g Avicel. Mean values and standard deviations of triplicates are presented Fig. 5 Enzymatic hydrolysis of Avicel 10 % (w/v) in presence of xylans 15 g/L with different enzymatic cocktails and mixtures of cocktails with 10 mg total proteins/g Avicel. Mean values and standard devia- tions of triplicates are presented In that way, mixtures were prepared with TR1 suppl mented with TR5 with two different ratios of protein quantities (1/1 and 4/1) with a total protein loadin corresponding to 10 mg/g Avicel (Table 3). These com binations were chosen in order to obtain cocktails pr senting high levels of FPU, β-glucosidase, xylanase an β-xylosidase activities. For both ratios tested, mixin TR1 with TR5 induced a decrease of FPU and xylana activities whereas β-glucosidase and β-xylosidase activ ties were increased compared to TR1. In contrary, case of TR5, complementation with TR1 with both rati induced more important FPU and xylanase activiti and less high levels of β-glucosidase and β-xylosida activities. Discussion d As beechwood xylans used for enzymatic hydrolysis experiments con- tain few arabinose (<1 % DM) and no esterified groups (acetyl, feruloyl), arabinosidase and esterases activities were not quantified. qi In regards of enzymatic activities present in the dif- ferent cocktails, efficiency of cocktails for cellulose hydrolysis could be explained by FPU and β-glucosidase activities levels. High FPU and β-glucosidase activi- ties considered as dissociated do not allow explaining the various glucose yields obtained with the different cocktails. Indeed, TR2 and TR5 cocktails which contain respectively high FPU and β-glucosidase activities are not those generating maximal glucose release. Cocktails TR1 and TR3 giving rise to the most important glucose yields were characterized by high levels of FPU activities as well as by low ratios between FPU and β-glucosidase activi- ties (respectively 0.11 and 0.05). In comparison, lower efficiency of cocktails TR5 and TR4 for cellulose hydroly- sis could be explained by a less important FPU activity for TR5 (in spite of a low ratio FPU/β-glucosidase: 0.03) and by a higher ratio FPU/β-glucosidase (0.36) for TR4. In case of cocktail TR2, FPU activity was as important as for cocktail TR4 and higher compared to FPU activity of TR5, however the high ratio FPU/β-glucosidase (0.43) was probably responsible for the limited glucose release. β-Glucosidase activity represents an essential factor for the design of cellulase cocktails. Indeed β-glucosidases are responsible for glucose release from cellobiose pro- duced synergistically by endoglucanases and CBH dur- ing cellulose hydrolysis. Furthermore β-glucosidases decrease the accumulation of cellobiose during cataly- sis and thus limit CBH inhibition by this disaccharide (Holtzapple et al. 1990). In that way, recent commer- cial cellulase cocktails have been supplemented with β-glucosidase activity. Fig. 6 Yields of xylose and xylo-oligosaccharides (XOs) released in presence of cellulose Avicel (10 %, w/v) and xylans (1.5 %, w/v) at 72 h with TR1 and TR5 alone or with mixtures of TR1/TR5. For all condi- tions, the enzyme loading was 10 mg total proteins/g Avicel. Mean values and standard deviations of triplicates are presented source in the fed-batch solution. TR4 corresponds to an experiment where only the lactose was used in the fed- batch solution. Ten percent of hemicellulosic hydrolysate was used as carbon source during the culture experiment of TR3 and 75 % during the culture experiment of TR5. Finally, the experiment TR1 was carried out using 20 % xylose instead of the hemicellulosic hydrolysate. Discussion d Contrasted enzymatic activities ratios characterizing the obtained cocktails could be related to the strain and to the substrates used during the culture of T. reesei for enzymes production. The experiments for TR2 and TR4 production were performed with the strain CL847 while the experiments concerning TR1, TR3 and TR5 were performed with the strain TR3002 which have an improved β-glucosidase expression capacity explain- ing why β-glucosidase activity is higher in case of these three cocktails. For all the experiments, enzymes pro- duction was carried out in carbon-limited fed-batch mode with lactose, hemicellulosic hydrolysate (C5) and mix of lactose, glucose xylose and C5 with different pro- portions. The hemicellulosic hydrolysate corresponds to the water extracts of steam-exploded biomass. It is mainly composed of monomeric pentoses (xylose, arab- inose) and oligomeric pentoses both resulting from the thermo-chemical hydrolysis. Before being used for cel- lulase biosynthesis, the hemicellulosic hydrolysate was mixed with lactose and eventually glucose in the feed- ing solution as described previously (Ben Chaabane and Marchal 2013). TR2 corresponds to an experiment where only the hemicellulosic hydrolysate was used as carbon Table 3 Proteins concentrations and enzymatic activities measured from cocktails mixtures (FPU, β-glucosidase, xylanase, β-xylosidase) present in the cocktails mixtures TR1/TR5 1/1 TR1/TR5 4/1 Proteins (g/L) 89.4 125.0 FPU (IU/mg) 0.5 0.6 β-Glucosidase (IU/mg) 10.2 7.0 Xylanase (IU/mg) 45.6 50.0 β-Xylosidase (IU/mg) 0.3 0.2 Table 3 Proteins concentrations and enzymatic activities measured from cocktails mixtures (FPU, β-glucosidase, xylanase, β-xylosidase) present in the cocktails mixtures Page 7 of 9 Dondelinger et al. AMB Expr (2016) 6:24 Fig. 6 Yields of xylose and xylo-oligosaccharides (XOs) released in presence of cellulose Avicel (10 %, w/v) and xylans (1.5 %, w/v) at 72 h with TR1 and TR5 alone or with mixtures of TR1/TR5. For all condi- tions, the enzyme loading was 10 mg total proteins/g Avicel. Mean values and standard deviations of triplicates are presented on carbon sources (Juhász et al. 2005). One could sup- pose that using various C5 and C6 carbon sources led to modulations of secreted enzymes by CL847 and TR3002 strains used in our study. The objective of our study was to relate the enzymatic activities to the yields of cel- lulose and xylans hydrolysis. In this context, the char- acterization of enzymatic cocktails was based on the measurement of enzymatic activities (FPU, β-glucosidase, xylanase, β-xylosidase) supposed to play an essential role during cellulose and xylans hydrolysis. Discussion d Results indicate that xylose induces high xylanase activity but lowers β-xylosidase activity compared to hemicellulosic hydrolysates. Various enzymes, expressed by T. reesei, are involved in lignocellulosic biomass fractionation. In a previous study concerning the analysis of the secretome of T. ree- sei CL847 growing on lactose-based media, 22 biomass- degrading enzymes were identified and represented 93 % of the secretome (Herpoel-Gimbert et al. 2008). These enzymes correspond notably to 2 cellobiohydrolases (CBH), 4 endoglucanases, 1 β-glucosidase, 3 xylanases, 1 β-xylosidase, 1 mannanase. Cellulases and hemicellu- lases production by T. reesei is known to be dependent TR2 and TR5 cocktails, which were the most efficient for xylose production from xylans and for which XOs content were the less abundant, possessed both high xylanase and β-xylosidase activities. Xylanase activ- ity was less important within cocktail TR3 which could probably explain the lower yield of xylans conversion into xylose compared to TR2 and TR5 cocktails. The low β-xylosidase activity level within TR1 and TR4 Dondelinger et al. AMB Expr (2016) 6:24 Page 8 of 9 of TR1/TR5 mixtures was most important for xylose release but no gain was obtained for glucose production. In case of complex enzymatic mixtures, as it is the case in our study, correlating yields of products to levels of enzy- matic activities is not an easy task. Adding pure enzymes to complex enzymatic cocktails represents a simplest approach. In this way, Gao et al. (2011) tailored optimal enzymatic cocktails including cellulases (endoglucanase, cellobiohydrolase and β-glucanase activities) and hemi- cellulases (xylanase, β-xylosidase, α-arabinosidase and α-glucuronidase activities) for the hydrolysis of AFEX pretreated corn stover (Gao et al. 2011). This allowed recovering high yields of glucose (80 %) and xylose (70 %) with a reasonable protein loading (20 mg/g glucan). In a same way, on steam exploded wheat straw the supple- mentation of commercial cellulases with a xylanase and an arabinosidase gave rise to 10 % higher glucose yield (Alvira et al. 2011). Discussion d Another previous study showed that improvement of enzymatic cocktails largely depends on the substrate used for hydrolysis: the supplementation of a cellulase cocktail with xylanase and β-xylosidase activi- ties improved glucan conversion from corn stover pre- treated with AFEX and dilute acid (increase of 27 and 8 % respectively); furthermore the addition of these both hemicellulases gave more benefic impact when adding them several hours before the addition of cellulase com- pared to a latter addition (Qing and Wyman 2011). cocktails could be responsible of the low xylose produc- tion observed with these cocktails in spite of a high xyla- nase activity level for TR1 cocktail. In comparison with hydrolysis experiments performed on xylans, total yields of xylose release were more impor- tant for hydrolysis of xylans in presence of Avicel. This could be explained by a loading of enzymatic proteins 6.6-folds more important for hydrolysis of Avicel in pres- ence of xylans compared to enzymes loading for xylans hydrolysis. Globally, when hydrolysis was conducted simultaneously onto cellulose and xylans, release of glu- cose was decreased compared to action onto separated Avicel. This indicates a lesser efficiency of cellulases in this case. Recent studies revealed that xylose, XOs and xylans have a negative impact during hydrolysis of cellu- lose with cellulases. For XOs, their negative impact dur- ing cellulose hydrolysis with cellulases was reported in numerous studies (Hu et al. 2013; Shi et al. 2011). XOs inhibitory effect is higher than the one obtained in pres- ence of xylose (Qing et al. 2010). A mixture of XOs from DP7 to 16 was recovered from hydrothermally pretreated wheat straw (Kont et al. 2013) and these oligosaccharides induced an inhibitory effect 100-fold more important on CBH from T. reesei than cellobiose. By mimicking the structure of cellulose chain, these oligosaccharides bind the active site of CBHs (Kont et al. 2013). Competitive inhibition seems to be partly responsible of the negative impact of XOs on cellulases efficiency (Qing et al. 2010) notably on CBHI (Zhang and Viikari 2012). Structural resolution of the CBH Cel7A from Hypocrea jecorina complexed with XOs indicated that xylotriose, xylotetra- ose and xylopentaose bind predominantly to the entrance of the substrate-binding tunnel of the enzyme and that an second alternative binding mode occurs near the catalytic center of the enzyme (Momeni et al. 2015). Authors’ contributions C d d h FBC designed the experiments for the production of the various enzymatic cocktails. CC produced the enzymatic cocktails in bioreactors. CR and JT designed the enzymatic hydrolysis experiments. NA carried out the characteri- zation of the enzymatic cocktails (proteins and activities quantifications). ED performed the enzymatic hydrolysis experiments and quantified the sugars produced. FBC, JT and CR analyzed all results. CR drafted the manuscript. All authors read and approved the final manuscript. Results obtained with cocktails mixtures indicate that TR1/TR5 with both ratios represented improved enzy- matic cocktails for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Mix- ture 4/1 was also more effective for xylose release com- pared to TR5 alone. In comparison to TR1, the efficiency Discussion d The data obtained during hydrolysis of Avicel in presence of xylans, indicate that a larger proportion of residual cel- lulose and a lesser extent part of xylans could remain in reactional media. Previous experiments demonstrated that presence of xylans was a factor decreasing cellu- lases efficiency notably by limiting cellulose accessibility (Penttilä et al. 2013; Zhang et al. 2012; Zhang and Vii- kari 2014). This could be attributed to the adsorption of xylans chains onto cellulose surface (Kohnke et al. 2008, 2011). One could not exclude that binding of xylans chains into the active site of cellulases occurs leading to their inhibition (Zhang et al. 2012). Results obtained with cocktails mixtures indicate that p g y In our study, T. reesei strains modify their enzymatic activities levels produced according to the sugar nature present as carbon sources. Hydrolysis of Avicel with the various cocktails was more important when cock- tails were rich in FPU activity and when ratio FPU/β- glucosidase was low. The presence of xylans during Avicel hydrolysis impacted negatively the efficiency of cellulases for glucose release. 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https://openalex.org/W1528677578	http://diposit.ub.edu/dspace/bitstream/2445/131543/1/577693.pdf	English	null	Searches for the decays<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msup><mml:mi>B</mml:mi><mml:mn>0</mml:mn></mml:msup><mml:mo>→</mml:mo><mml:msup><mml:mi>l</mml:mi><mml:mo>±</mml:mo></mml:msup><mml:msup><mml:mi>τ</mml:mi><mml:mo>∓</mml:mo></mml:msup></mml:math>and<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msup><mml:mi>B</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mo>→</mml:mo><mml:msup><mml:mi>l</mml:mi><mml:mo>+</mml:mo></…	Physical review. D. Particles, fields, gravitation, and cosmology/Physical review. D, Particles, fields, gravitation, and cosmology	2,008	public-domain	8,976	Searches for the decays B0 ! l and Bþ ! lþ (l ¼ e, ) using hadron Searches for the decays B0 ! l and Bþ ! lþ (l ¼ e, ) using hadronic tag reconstruction d Bþ ! lþ (l ¼ e, ) using hadronic tag reconstruction B. Aubert,1 M. Bona,1 Y. Karyotakis,1 J. P. Lees,1 V. Poireau,1 X. Prudent,1 V. Tisserand,1 A. Zghiche,1 J. Garra Tico,2 E. Grauges,2 L. Lopez,3 A. Palano,3 M. Pappagallo,3 G. Eigen,4 B. Stugu,4 L. Sun,4 G. S. Abrams,5 M. Battaglia,5 D. N. Brown,5 J. Button-Shafer,5 R. N. Cahn,5 R. G. Jacobsen,5 J. A. Kadyk,5 L. T. Kerth,5 Yu. G. Kolomensky,5 G. Kukartsev,5 D. Lopes Pegna,5 G. Lynch,5 I. L. Osipenkov,5 M. T. Ronan,5,* K. Tackmann,5 T. Tanabe,5 W. A. Wenzel,5 P. del Amo Sanchez,6 C. M. Hawkes,6 N. Soni,6 A. T. Watson,6 H. Koch,7 T. Schroeder,7 D. Walker,8 D. J. Asgeirsson,9 T. Cuhadar-Donszelmann,9 B. G. Fulsom,9 C. Hearty,9 T. S. Mattison,9 J. A. McKenna,9 M. Barrett,10 A. Khan,10 M Saleem 10 L Teodorescu 10 V E Blinov 11 A D Bukin 11 A R Buzykaev 11 V P Druzhinin 11 V B Golubev 11 , , , , , , , g , T. Cuhadar-Donszelmann,9 B. G. Fulsom,9 C. Hearty,9 T. S. Mattison,9 J. A. McKenna,9 M. Barrett,10 A. Khan,10 M. Saleem,10 L. Teodorescu,10 V. E. Blinov,11 A. D. Bukin,11 A. R. Buzykaev,11 V. P. Druzhinin,11 V. B. Golubev,11 T. Cuhadar-Donszelmann, B. G. Fulsom, C. Hearty, T. S. Mattison, J. A. McKenna, M. Barrett, A. Khan, M. Saleem,10 L. Teodorescu,10 V. E. Blinov,11 A. D. Bukin,11 A. R. Buzykaev,11 V. P. Druzhinin,11 V. B. Golubev,11 y p y y I. Eschrich,12 D. Kirkby,12 A. J. Lankford,12 P. Lund,12 M. Mandelkern,12 E. C. Martin,12 D. P. Stoker,12 S. Abachi,13 13 14 14 14 14 14 14 15 15 C. Buchanan,13 J. W. Gary,14 F. Liu,14 O. Long,14 B. C. Shen,14,* G. M. Vitug,14 L. Zhang,14 H. P. Paar,15 S. Rahatlou,15 V. Sharma,15 C. Campagnari,16 T. M. Hong,16 D. Kovalskyi,16 J. D. Richman,16 T. W. Beck,17 A. M. Eisner,17 C. J. Flacco,17 C. A. Heusch,17 J. Kroseberg,17 W. S. Lockman,17 T. Schalk,17 B. A. Schumm,17 A. Seiden,17 M. G. Wilson,17 L. O. Winstrom,17 E. Chen,18 C. H. Cheng,18 D. A. Doll,18 B. Echenard,18 F. Fang,18 D. G. Hitlin,18 I. Narsky,18 T. Piatenko,18 F. C. Porter,18 R. Andreassen,19 G. Mancinelli,19 B. T. Meadows,19 K. Mishra,19 M. D. Sokoloff, F. Blanc, P. C. Bloom, W. T. Ford, J. F. Hirschauer, A. Kreisel, M. Nagel, U. Nauenberg, A. Olivas,20 J. G. Smith,20 K. A. Ulmer,20 S. R. Wagner,20 R. Ayad,21,+ A. M. Gabareen,21 A. Soffer,21,‡ W. H. Toki,21 R. J. Wilson,21 D. D. Altenburg,22 E. RAPID COMMUNICATIONS RAPID COMMUNICATIONS RAPID COMMUNICATIONS PHYSICAL REVIEW D 77, 091104(R) (2008) d Bþ ! lþ (l ¼ e, ) using hadronic tag reconstruction Feltresi,22 A. Hauke,22 H. Jasper,22 J. Merkel,22 A. Petzold,22 B. Spaan,22 K. Wacker,22 V. Klose,23 M. J. Kobel,23 H. M. Lacker,23 W. F. Mader,23 R. Nogowski,23 J. Schubert,23 K. R. Schubert,23 R. Schwierz,23 J. E. Sundermann,23 A. Volk,23 D. Bernard,24 G. R. Bonneaud,24 E. Latour,24 V. Lombardo,24 Ch. Thiebaux,24 M. Verderi,24 P. J. Clark,25 W. Gradl,25 S. Playfer,25 A. I. Robertson,25 J. E. Watson,25 M. Andreotti,26 D. Bettoni,26 C. Bozzi,26 R. Calabrese,26 A. Cecchi,26 G. Cibinetto,26 P. Franchini,26 E. Luppi,26 M. Negrini,26 A. Petrella,26 L. Piemontese,26 E. Prencipe,26 V. Santoro,26 F. Anulli,27 R. Baldini-Ferroli,27 A. 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(BABAR Collaboration) 1Laboratoire de Physique des Particules, IN2P3/CNRS et Universite´ de Savoie, F-74941 Annecy-Le-Vieux, France 2Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain 3Universita` di Bari, Dipartimento di Fisica and INFN, I-70126 Bari, Italy 4University of Bergen, Institute of Physics, N-5007 Bergen, Norway 5Lawrence Berkeley National Laboratory and University of California, Berkeley, California 94720, USA 6University of Birmingham, Birmingham, B15 2TT, United Kingdom 7Ruhr Universita¨t Bochum, Institut fu¨r Experimentalphysik 1, D-44780 Bochum, Germany 8University of Bristol, Bristol BS8 1TL, United Kingdom 9University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 10Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom 11Budker Institute of Nuclear Physics, Novosibirsk 630090, Russia 12University of California at Irvine, Irvine, California 92697, USA 13University of California at Los Angeles, Los Angeles, California 90024, USA 14University of California at Riverside, Riverside, California 92521, USA 15University of California at San Diego, La Jolla, California 92093, USA 16University of California at Santa Barbara, Santa Barbara, California 93106, USA 17University of California at Santa Cruz, Institute for Particle Physics, Santa Cruz, California 95064, USA 18California Institute of Technology, Pasadena, California 91125, USA 19University of Cincinnati, Cincinnati, Ohio 45221, USA 20University of Colorado, Boulder, Colorado 80309, USA 21Colorado State University, Fort Collins, Colorado 80523, USA 22Universita¨t Dortmund, Institut fu¨r Physik, D-44221 Dortmund, Germany 23Technische Universita¨t Dresden, Institut fu¨r Kern- und Teilchenphysik, D-01062 Dresden, Germany 24Laboratoire Leprince-Ringuet, CNRS/IN2P3, Ecole Polytechnique, F-91128 Palaiseau, France 25University of Edinburgh, Edinburgh EH9 3JZ, United Kingdom 26Universita` di Ferrara, Dipartimento di Fisica and INFN, I-44100 Ferrara, Italy 27Laboratori Nazionali di Frascati dell’INFN, I-00044 Frascati, Italy 28Universita` di Genova, Dipartimento di Fisica and INFN, I-16146 Genova, Italy 29Harvard University, Cambridge, Massachusetts 02138, USA 30Universita¨t Heidelberg, Physikalisches Institut, Philosophenweg 12, D-69120 Heidelberg, Germany 31Imperial College London, London, SW7 2AZ, United Kingdom 32University of Iowa, Iowa City, Iowa 52242, USA 1Laboratoire de Physique des Particules, IN2P3/CNRS et Universite´ de Savoie, F-74941 Annecy-Le-Vieux, France 2Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain 3Universita` di Bari, Dipartimento di Fisica and INFN, I-70126 Bari, Italy 4University of Bergen, Institute of Physics, N-5007 Bergen, Norway 5Lawrence Berkeley National Laboratory and University of California, Berkeley, California 94720, USA 6University of Birmingham, Birmingham, B15 2TT, United Kingdom 7Ruhr Universita¨t Bochum, Institut fu¨r Experimentalphysik 1, D-44780 Bochum, Germany 8University of Bristol, Bristol BS8 1TL, United Kingdom 9University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 10Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom 11Budker Institute of Nuclear Physics, Novosibirsk 630090, Russia 12University of California at Irvine, Irvine, California 92697, USA 13University of California at Los Angeles, Los Angeles, California 90024, USA 14University of California at Riverside, Riverside, California 92521, USA 15University of California at San Diego, La Jolla, California 92093, USA 16University of California at Santa Barbara, Santa Barbara, California 93106, USA 17University of California at Santa Cruz, Institute for Particle Physics, Santa Cruz, California 95064, USA 18California Institute of Technology, Pasadena, California 91125, USA 19University of Cincinnati, Cincinnati, Ohio 45221, USA 20University of Colorado, Boulder, Colorado 80309, USA 21Colorado State University, Fort Collins, Colorado 80523, USA 22Universita¨t Dortmund, Institut fu¨r Physik, D-44221 Dortmund, Germany 23Technische Universita¨t Dresden, Institut fu¨r Kern- und Teilchenphysik, D-01062 Dresden, Germany 24Laboratoire Leprince-Ringuet, CNRS/IN2P3, Ecole Polytechnique, F-91128 Palaiseau, France 25University of Edinburgh, Edinburgh EH9 3JZ, United Kingdom 26Universita` di Ferrara, Dipartimento di Fisica and INFN, I-44100 Ferrara, Italy 27Laboratori Nazionali di Frascati dell’INFN, I-00044 Frascati, Italy 28Universita` di Genova, Dipartimento di Fisica and INFN, I-16146 Genova, Italy 29Harvard University, Cambridge, Massachusetts 02138, USA 30Universita¨t Heidelberg, Physikalisches Institut, Philosophenweg 12, D-69120 Heidelberg, Germany 31Imperial College London, London, SW7 2AZ, United Kingdom 32University of Iowa, Iowa City, Iowa 52242, USA y g Heidelberg, Physikalisches Institut, Philosophenweg 12, D-69120 Heidelberg, Germany 31Imperial College London, London, SW7 2AZ, United Kingdom 32University of Iowa, Iowa City, Iowa 52242, USA PHYSICAL REVIEW D 77, 091104(R) (2008) J. Olsen,61 A. J. S. Smith,61 A. V. Telnov,61 E. Baracchini,62 G. Cavoto,62 D. del Re,62 E. Di Marco,62 R. Faccini,62 F. Ferrarotto,62 F. Ferroni,62 M. Gaspero,62 P. D. Jackson,62 M. A. Mazzoni,62 S. Morganti,62 G. Piredda,62 F. Polci,62 F. Renga,62 C. Voena,62 M. Ebert,63 T. Hartmann,63 H. Schro¨der,63 R. Waldi,63 T. Adye,64 B. Franek,64 E. O. Olaiya,64 W. Roethel,64 F. F. Wilson,64 S. Emery,65 M. Escalier,65 A. Gaidot,65 S. F. Ganzhur,65 G. Hamel de Monchenault,65 W. Kozanecki,65 G. Vasseur,65 Ch. Ye`che,65 M. Zito,65 X. R. Chen,66 H. Liu,66 W. Park,66 M. V. Purohit,66 R. M. White,66 J. R. Wilson,66 M. T. Allen,67 D. Aston,67 R. Bartoldus,67 P. Bechtle,67 R. Claus,67 J. P. Coleman,67 M. R. Convery,67 J. C. Dingfelder,67 J. Dorfan,67 G. P. Dubois-Felsmann,67 W. Dunwoodie,67 R. C. Field,67 T. Glanzman,67 S. J. Gowdy,67 M. T. Graham,67 P. Grenier,67 C. Hast,67 W. R. Innes,67 J. Kaminski,67 M. H. Kelsey,67 H. Kim,67 P. Kim,67 M. L. Kocian,67 D. W. G. S. Leith,67 S. Li,67 S. Luitz,67 V. Luth,67 H. L. Lynch,67 D. B. MacFarlane,67 H. Marsiske,67 R. Messner,67 D. R. Muller,67 S. Nelson,67 C. P. O’Grady,67 I. Ofte,67 A. Perazzo,67 M. Perl,67 B. N. Ratcliff,67 A. Roodman,67 A. A. Salnikov,67 R. H. Schindler,67 J. Schwiening,67 A. Snyder,67 D. Su,67 M. K. Sullivan,67 K. Suzuki,67 S. K. Swain,67 J. M. Thompson,67 J. Va’vra,67 A. P. Wagner,67 M. Weaver,67 W. J. Wisniewski,67 M. Wittgen,67 D. H. Wright,67 H. W. Wulsin,67 A. K. Yarritu,67 K. Yi,67 C. C. Young,67 V. Ziegler,67 P. R. Burchat,68 A. J. Edwards,68 S. A. Majewski,68 T. S. Miyashita,68 B. A. Petersen,68 L. Wilden,68 S. Ahmed,69 M. S. Alam,69 R. Bula,69 J. A. Ernst,69 B. Pan,69 M. A. Saeed,69 S. B. Zain,69 S. M. Spanier,70 B. J. Wogsland,70 R. Eckmann,71 J. L. Ritchie,71 A. M. Ruland,71 C. J. Schilling,71 R. F. Schwitters,71 J. M. Izen,72 X. C. Lou,72 S. Ye,72 F. Bianchi,73 D. Gamba,73 M. Pelliccioni,73 M. Bomben,74 L. Bosisio,74 C. Cartaro,74 F. Cossutti,74 G. Della Ricca,74 L. Lanceri,74 L. Vitale,74 V. Azzolini,75 N. Lopez-March,75 F. Martinez-Vidal,75 D. A. Milanes,75 A. Oyanguren,75 J. Albert,76 Sw. Banerjee,76 B. Bhuyan,76 K. Hamano,76 R. Kowalewski,76 I. M. Nugent,76 J. M. Roney,76 R. J. Sobie,76 P. F. Harrison,77 J. Ilic,77 T. E. Latham,77 G. B. Mohanty,77 H. R. Band,78 X. Chen,78 S. Dasu,78 K. T. Flood,78 J. J. Hollar,78 P. E. Kutter,78 Y. Pan,78 M. Pierini,78 R. Prepost,78 S. L. Wu,78 and H. 091104-1 091104-1 091104-1 PHYSICAL REVIEW D 77, 091104(R) (2008) Neal79 J. C. Dingfelder, J. Dorfan, G. P. Dubois-Felsmann, W. Dunwoodie, R. C. Field, T. Glanzman, S. J. Gowdy, M. T. Graham,67 P. Grenier,67 C. Hast,67 W. R. Innes,67 J. Kaminski,67 M. H. Kelsey,67 H. Kim,67 P. Kim,67 M. L. Kocian,67 D. W. G. S. Leith,67 S. Li,67 S. Luitz,67 V. Luth,67 H. L. Lynch,67 D. B. MacFarlane,67 H. Marsiske,67 R. Messner,67 D. R. Muller,67 S. Nelson,67 C. P. O’Grady,67 I. Ofte,67 A. Perazzo,67 M. Perl,67 B. N. Ratcliff,67 A. Roodman,67 A. A. Salnikov,67 R. H. Schindler,67 J. Schwiening,67 A. Snyder,67 D. Su,67 M. K. Sullivan,67 K. Suzuki,67 S. K. Swain,67 J. M. Thompson,67 J. Va’vra,67 A. P. Wagner,67 M. Weaver,67 W. J. Wisniewski,67 M. Wittgen,67 D. H. Wright,67 H. W. Wulsin,67 A. K. Yarritu,67 K. Yi,67 C. C. Young,67 V. Ziegler,67 P. R. Burchat,68 A. J. Edwards,68 S. A. Majewski,68 T. S. Miyashita,68 B. A. Petersen,68 L. Wilden,68 S. Ahmed,69 M. S. Alam,69 R. Bula,69 J. A. Ernst,69 B. Pan,69 M. A. Saeed,69 S. B. Zain,69 S. M. Spanier,70 B. J. Wogsland,70 R. Eckmann,71 J. L. Ritchie,71 A. M. Ruland,71 C. J. Schilling,71 R. F. Schwitters,71 J. M. Izen,72 X. C. Lou,72 S. Ye,72 F. Bianchi,73 D. Gamba,73 M. Pelliccioni,73 M. Bomben,74 L. Bosisio,74 C. Cartaro,74 F. Cossutti,74 G. Della Ricca,74 L. Lanceri,74 L. Vitale,74 V. Azzolini,75 N. Lopez-March,75 F. Martinez-Vidal,75 D. A. Milanes,75 A. Oyanguren,75 J. Albert,76 Sw. Banerjee,76 B. Bhuyan,76 K. Hamano,76 R. Kowalewski,76 I. M. Nugent,76 J. M. Roney,76 R. J. Sobie,76 P. F. Harrison,77 J. Ilic,77 T. E. Latham,77 G. B. Mohanty,77 H. R. Band,78 X. Chen,78 S. Dasu,78 K. T. Flood,78 J. J. Hollar,78 P. E. Kutter,78 Y. Pan,78 M. Pierini,78 R. Prepost,78 S. L. Wu,78 and H. Neal79 J. M. Thompson, J. Va vra, A. P. Wagner, M. Weaver, W. J. Wisniewski, M. Wittgen, D. H. Wright, H. W. Wulsin,67 A. K. Yarritu,67 K. Yi,67 C. C. Young,67 V. Ziegler,67 P. R. Burchat,68 A. J. Edwards,68 S. A. Majewski,68 T. S. Miyashita,68 B. A. Petersen,68 L. Wilden,68 S. Ahmed,69 M. S. Alam,69 R. Bula,69 J. A. Ernst,69 B. Pan,69 M. A. Saeed,69 S. B. Zain,69 S. M. Spanier,70 B. J. Wogsland,70 R. Eckmann,71 J. L. Ritchie,71 A. M. Ruland,71 C. J. Schilling,71 R. F. Schwitters,71 J. M. Izen,72 X. C. Lou,72 S. Ye,72 F. Bianchi,73 D. Gamba,73 M. Pelliccioni,73 M. Bomben,74 L. Bosisio,74 C. Cartaro,74 F. Cossutti,74 G. Della Ricca,74 L. Lanceri,74 L. PHYSICAL REVIEW D 77, 091104(R) (2008) 34, F-91898 ORSAY Cedex, Franc 37Lawrence Livermore National Laboratory, Livermore, California 94550, USA 38 38University of Liverpool, Liverpool L69 7ZE, United Kingdom ry, University of London, E1 4NS, United Kingdom Q y, y f , , g n, Royal Holloway and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 41University of Louisville, Louisville, Kentucky 40292, USA 40University of London, Royal Holloway and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 41University of Louisville, Louisville, Kentucky 40292, USA 42 y f y 42University of Manchester, Manchester M13 9PL, United Kingdom 43University of Maryland, College Park, Maryland 20742, USA y f 45Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 46McGill University, Montre´al, Que´bec, Canada H3A 2T8 47Universita` di Milano, Dipartimento di Fisica and INFN, I-20133 Milano, Italy 48University of Mississippi, University, Mississippi 38677, USA Universite´ de Montre´al, Physique des Particules, Montre´al, Que´bec, Canada H3C 3J7 50Mount Holyoke College, South Hadley, Massachusetts 01075, USA Dipartimento di Scienze Fisiche and INFN, I-8012 nal Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Nether 53 53University of Notre Dame, Notre Dame, Indiana 46556, USA 54 54Ohio State University, Columbus, Ohio 43210, USA 55 55University of Oregon, Eugene, Oregon 97403, USA e de Physique Nucle´aire et de Hautes Energies, IN2P3/CNRS, Universite´ Pierre et Marie Curie-Paris6, 61Princeton University, Princeton, New Jersey 08544, USA 62 y d Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 65 DSM/Dapnia, CEA/Saclay, F-91191 Gif-sur-Yvette, France 66University of South Carolina, Columbia, South Carolina 29208, USA 67Stanford Linear Accelerator Center, Stanford, California 94309, USA 68Stanford University, Stanford, California 94305-4060, USA 69State University of New York, Albany, New York 12222, USA 70University of Tennessee, Knoxville, Tennessee 37996, USA 71University of Texas at Austin, Austin, Texas 78712, USA 72University of Texas at Dallas, Richardson, Texas 75083, USA 73Universita` di Torino, Dipartimento di Fisica Sperimentale and INFN, I-10125 Torino, Italy 74Universita` di Trieste, Dipartimento di Fisica and INFN, I-34127 Trieste, Italy 75IFIC, Universitat de Valencia-CSIC, E-46071 Valencia, Spain 76University of Victoria, Victoria, British Columbia, Canada V8W 3P6 77Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom 78University of Wisconsin, Madison, Wisconsin 53706, USA 79Yale University, New Haven, Connecticut 06511, USA (Received 7 January 2008; published 21 May 2008) 66University of South Carolina, Columbia, South Carolina 29208, USA We present searches for the leptonic decays Bþ ! ‘þ and the lepton ﬂavor violating decays B0 ! PHYSICAL REVIEW D 77, 091104(R) (2008) ‘, where ‘ ¼ e, , with data collected by the BABAR experiment at SLAC. This search demonstrates a novel technique in which we fully reconstruct the accompanying B in ð4SÞ ! B B events, and look for a monoenergetic lepton from the signal B decay. The signal yield is extracted from a ﬁt to the signal lepton candidate momentum distribution in the signal B rest frame. Using a data sample of approximately 378 106 B B pairs (342 fb1), we ﬁnd no evidence of signal in any of the decay modes. Branching fraction upper limits of BðBþ ! eþÞ < 5:2 106, BðBþ ! þÞ < 5:6 106, BðB0 ! eþÞ < 2:8 105 and BðB0 ! þÞ < 2:2 105, are obtained at 90% conﬁdence level. DOI: 10.1103/PhysRevD.77.091104 PACS numbers: 13.25.Hw, 12.15.Hh, 11.30.Er 091104-2 091104-2 PHYSICAL REVIEW D 77, 091104(R) (2008) 33Iowa State University, Ames, Iowa 50011-3160, USA 34Johns Hopkins University, Baltimore, Maryland 21218, USA 35Universita¨t Karlsruhe, Institut fu¨r Experimentelle Kernphysik, D-76021 Karlsruhe, Germany 36Laboratoire de l’Acce´le´rateur Line´aire, IN2P3/CNRS et Universite´ Paris-Sud 11, Centre Scientiﬁque d’Orsay, B. P. PHYSICAL REVIEW D 77, 091104(R) (2008) 34, F-91898 ORSAY Cedex, France 37Lawrence Livermore National Laboratory, Livermore, California 94550, USA 38University of Liverpool, Liverpool L69 7ZE, United Kingdom 39Queen Mary, University of London, E1 4NS, United Kingdom 40University of London, Royal Holloway and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 41University of Louisville, Louisville, Kentucky 40292, USA 42University of Manchester, Manchester M13 9PL, United Kingdom 43University of Maryland, College Park, Maryland 20742, USA 44University of Massachusetts, Amherst, Massachusetts 01003, USA 45Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 46McGill University, Montre´al, Que´bec, Canada H3A 2T8 47Universita` di Milano, Dipartimento di Fisica and INFN, I-20133 Milano, Italy 48University of Mississippi, University, Mississippi 38677, USA 49Universite´ de Montre´al, Physique des Particules, Montre´al, Que´bec, Canada H3C 3J7 50Mount Holyoke College, South Hadley, Massachusetts 01075, USA 51Universita` di Napoli Federico II, Dipartimento di Scienze Fisiche and INFN, I-80126, Napoli, Italy 52NIKHEF, National Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Netherlands 53University of Notre Dame, Notre Dame, Indiana 46556, USA 54Ohio State University, Columbus, Ohio 43210, USA 55University of Oregon, Eugene, Oregon 97403, USA 56Universita` di Padova, Dipartimento di Fisica and INFN, I-35131 Padova, Italy 57Laboratoire de Physique Nucle´aire et de Hautes Energies, IN2P3/CNRS, Universite´ Pierre et Marie Curie-Paris6, Universite´ Denis Diderot-Paris7, F-75252 Paris, France 58University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 59Universita` di Perugia, Dipartimento di Fisica and INFN, I-06100 Perugia, Italy 60Universita` di Pisa, Dipartimento di Fisica, Scuola Normale Superiore and INFN, I-56127 Pisa, Italy 61Princeton University, Princeton, New Jersey 08544, USA 62Universita` di Roma La Sapienza, Dipartimento di Fisica and INFN, I-00185 Roma, Italy 63Universita¨t Rostock, D-18051 Rostock, Germany 64Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 65DSM/Dapnia, CEA/Saclay, F-91191 Gif-sur-Yvette, France 66University of South Carolina, Columbia, South Carolina 29208, USA 67Stanford Linear Accelerator Center, Stanford, California 94309, USA 68Stanford University, Stanford, California 94305-4060, USA 69State University of New York, Albany, New York 12222, USA 70University of Tennessee, Knoxville, Tennessee 37996, USA 71University of Texas at Austin, Austin, Texas 78712, USA 72University of Texas at Dallas, Richardson, Texas 75083, USA 73Universita` di Torino, Dipartimento di Fisica Sperimentale and INFN, I-10125 Torino, Italy 74Universita` di Trieste, Dipartimento di Fisica and INFN, I-34127 Trieste, Italy 75IFIC, Universitat de Valencia-CSIC, E-46071 Valencia, Spain 76University of Victoria, Victoria, British Columbia, Canada V8W 3P6 77Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom 78University of Wisconsin, Madison, Wisconsin 53706, USA 79Yale University, New Haven, Connecticut 06511, USA (Received 7 January 2008; published 21 May 2008) We present searches for the leptonic decays Bþ ! PHYSICAL REVIEW D 77, 091104(R) (2008) ‘þ and the lepton ﬂavor violating decays B0 ! ‘ where ‘ ¼ e with data collected by the BABAR experiment at SLAC This search demonstrates EARCHES FOR THE DECAYS B0 ! l . . . PHYSICAL REVIEW D 77, 091104(R) (2008) 33Iowa State University, Ames, Iowa 50011-3160, USA 4Johns Hopkins University, Baltimore, Maryland 21218, USA 35Universita¨t Karlsruhe, Institut fu¨r Experimentelle Kernphysik, D-76021 Karlsruhe, Germany ratoire de l’Acce´le´rateur Line´aire, IN2P3/CNRS et Universite´ Paris-Sud 11, Centre Scientiﬁque d , B. P. 34, F-91898 ORSAY Cedex, France 37 B. P. PHYSICAL REVIEW D 77, 091104(R) (2008) Combined with clean theoretical predictions due to the lack of QCD contributions in the ﬁnal state, such leptonic B decays present an ideal place to test the SM against NP models. In the SM, Bþ ! ‘þ decays proceed via an annihila- tion of b and u quarks into a virtual Wþ boson. In the SM the branching fraction for this type of decay is given by [5]: The searches described in this work are based on a data sample of approximately 378 106 B B pairs, correspond- ing to an integrated luminosity of 342 fb1 collected at the ð4SÞ resonance by the BABAR detector at the PEP-II asymmetric eþe storage ring. Reconstructing the accom- panying B meson in speciﬁc hadronic modes prior to the signal selection allows the missing momentum vector of the neutrino(s) to be fully determined. The resulting in- crease in the energy resolution and the ability to infer the signal B meson rest frame provide the extra kinematic handles that permit signal events to be cleanly distin- guished from the background. Previous B factory searches for Bþ ! ‘þ and B0 ! ‘þ have used an inclusive method in which the accompanying B is not explicitly reconstructed. This results in an order of magnitude gain in the selection efﬁciency, which with the current level of luminosity allows more stringent limits. However, due to the very small background achievable with the exclusive method, the two methods have a comparable sensitivity for the observation of a statistically signiﬁcant signal. The method described in this paper will be the preferred ap- proach for the high-precision studies of leptonic B decays. In particular, the SM predicted decay rate for Bþ ! þ is well within the reach of a high luminosity B factory. B ðBþ ! ‘þ‘Þ ¼ G2 FmBm2 l 8 1 m2 l m2 B f2 BjVubj2B; (1) where GF is the Fermi coupling constant, ml is the lepton mass and mB, B, and fB are the mass, lifetime and decay constant for the B meson. The Cabibbo-Kobayashi- Maskawa matrix element jVubj describes the transition from b to u quarks [6]. Within the SM, a determination of any one of the leptonic branching fractions represents a determination of the product jVubj fB, which can be directly compared with determinations from lattice calcu- lations, B-mixing, and semileptonic decay measurements [7,8]. As seen in Eq. PHYSICAL REVIEW D 77, 091104(R) (2008) BðBþ ! eþÞ < 9:8 107 and BðBþ ! þÞ < 1:7 106 [14]. Earlier studies by CLEO and BABAR collabo- rations are also available [15,16]. BðBþ ! eþÞ < 9:8 107 and BðBþ ! þÞ < 1:7 106 [14]. Earlier studies by CLEO and BABAR collabo- rations are also available [15,16]. In this paper, we present searches for the decays Bþ ! ‘þ and the lepton ﬂavor violating decays B0 ! ‘, where ‘ ¼ e, [1], using a technique in which the accom- panying B in the event is exclusively reconstructed. This method has not previously been used for searches for these modes and, although statistically limited with the present BABAR data sample, shows promise for future studies at, for example, a high luminosity Super B factory [2]. While the former decay modes are allowed in the standard model (SM) and the latter are not, both are potentially sensitive to new physics (NP) effects, such as contributions by neutral and charged non-SM Higgs [3,4]. The lepton-ﬂavor-violating (LFV) leptonic B decays, such as B0 ! ‘þ, are forbidden in the SM in the ab- sence of nonzero neutrino masses, but can occur via one- loop diagrams if neutrino oscillations are included. The rates of such processes, however, would be substantially below current or anticipated future experimental sensitiv- ities. On the other hand, many models of physics beyond the SM, in particular, supersymmetric seesaw models [4], predict dramatically higher rates for these decays. In the case of Higgs-mediated LFV processes, couplings to heav- ier leptons are favored, making B0 ! ‘þ particularly interesting. In the general ﬂavor-universal MSSM, the branching fractions allowed for B0 ! ‘þ are 2 1010 [4]. Such decays could be within the reach of a Super B factory with a data sample of 50 to 75 ab1. The current best experimental limits on the branching fractions for these two decays are BðB0 ! eþÞ < 1:1 104 and BðB0 ! þÞ < 3:8 105, set by the CLEO collaboration with 10 fb1 of data [17]. Searches for rare B decays with neutrinos in the ﬁnal state are challenging due to the limited availability of kinematic constraints. However, purely leptonic B decays involving an electron or a muon have a clear experimental signature in the form of a high momentum lepton. 091104-3 RAPID COMMUNICATIONS B. AUBERT et al. PHYSICAL REVIEW D 77, 091104(R) (2008) PHYSICAL REVIEW D 77, 091104(R) (2008) SEARCHES FOR THE DECAYS B0 ! l . . Muon identiﬁcation is provided by resistive plate chambers (partially replaced by limited streamer tubes for a subset of the data that is used in this analysis) interleaved with the passive material comprising the solenoid magnetic ﬂux return. Signal efﬁciencies and background rates are esti- mated using a Monte Carlo (MC) simulation of the BABAR detector based on GEANT4 [18]. The BABAR detector is described in detail in Ref. [19]. grounds (eþe ! ff, where f represents u; d; s; c or any charged lepton) are suppressed by requiring R2 < 0:5, where R2 is the ratio of the second to the zeroth Fox- Wolfram moment [22] computed using all charged and neutral particles in the event. Further suppression is achieved by requiring j cosTj < 0:90, where T is the angle between two thrust axes in the CM frame, the ﬁrst computed using the particles from the Btag, and the second using all other particles in the event. Reconstructed charged tracks are assigned a particle hypothesis based on information from detector subsys- tems. K0s candidates are selected by combining oppositely charged candidates and requiring that the þ invari- ant mass satisﬁes 0:47 GeV=c2 < mþ < 0:52 GeV=c2. 0 candidates are obtained from the combination of EMC clusters with no associated tracks, each with a ð4SÞ center-of-mass (CM) rest frame energy greater than 20 MeV, for which the invariant mass satisﬁes 115 MeV=c2 < m < 150 MeV=c2. All particles that are not used in the Btag reconstruction are considered candidates to be included in the reconstruc- tion of the signal B meson. Since the CM energy is pre- cisely known, reconstruction of the Btag fully determines the Bsignal 4-vector. This permits the 2-body kinematics of the signal decays to be exploited. In particular, these decays are expected to contain an electron or a muon with a momentum p, in the Bsignal rest frame, of about 2:64 GeV=c (2:34 GeV=c) for the Bþ ! ‘þ (B0 ! ‘þ) channels, very close to the kinematic endpoint for B decays. Over 96% of the time, the ð4SÞ resonance decays into a pair of B mesons [20]. Since the CM energy is precisely known at PEP-II, exclusive reconstruction of one of the two B mesons, which we denote Btag, fully determines the momentum four-vector of the other B meson in the event. PHYSICAL REVIEW D 77, 091104(R) (2008) Charged and neutral B meson candidates are reconstructed in hadronic ﬁnal states of the form B ! DðÞXhad [21]. The reconstruction procedure begins with a DðÞ0 or DðÞ seed, to which charged and neutral pions and kaons (which form the Xhad system) are then added. The combination of the DðÞ and Xhad with the lowest value of E ¼ jEB Ebeamj that satisﬁes the condition E < 0:2 GeV is chosen as the Btag candidate, where EB is the energy of the reconstructed B meson and Ebeam is the beam energy, both evaluated in the CM frame. We reconstruct Dþ in the Dþ0 and D0þ channels, and D0 in the D00 and D0 channels. The Dþ is reconstructed in the modes Kþþ, K0sþ, K0sþ0, Kþþ0, and K0sþþ. For D0 we consider the modes Kþ, Kþ0, Kþþ, and K0sþ. Signal candidate events are initially selected by requir- ing the highest momentum track in the event (excluding tracks from the Btag reconstruction) to have a momentum of 1:7 GeV=c < p < 3:0 GeV=c and to satisfy particle identiﬁcation (PID) criteria for either an electron or a muon. In events with a charged Btag, the charge of the track is required to be opposite that of the Btag, while for a neutral Btag the high-p lepton is permitted to have either positive or negative charge. Once the Btag and the signal lepton candidate are iden- tiﬁed, Bþ ! ‘þ events should ideally have no other particles in the detector, while B0 ! ‘þ events should additionally contain only the decay daughters. For the latter, the -rest frame is calculated from the observed signal lepton, assuming the nominal energy and momen- tum of the for a 2-body B0 decay. The six decay modes considered are listed in Table I. The second highest momentum track in the event (again, excluding Btag recon- struction) is assumed to be a daughter, and is required to have a charge opposite to the primary signal lepton. If this track satisﬁes electron or muon PID, the event is consid- ered to be a leptonic decay. Otherwise, the track is assumed to be a pion and the quantity E is calculated Although multiple DðÞXhad combinations may be present in a single event, this procedure permits, at most, a single Btag candidate to be retained in any given event. PHYSICAL REVIEW D 77, 091104(R) (2008) (1), the decay rates are proportional to m2 l , resulting in SM predictions for the and e modes which are suppressed by factors on the order of 250 and 107, respectively, compared with the mode. Taking the branching fraction BðBþ ! þÞ ¼ ð1:31 0:48Þ 104 from the combination of recent BABAR and BELLE results [9,10] implies BSMðBþ ! þÞ 5:2 107 and BSMðBþ ! eþeÞ 1:2 1011. New physics con- tributions to these processes can enhance or suppress the decay rates compared to the SM, and may either preserve or violate the relative rates of the three leptonic modes depending on the particular NP model [3,11]. Thus, the e and modes become particularly interesting in light of recent evidence for the Bþ ! þ decay mode. In addi- tion, the observed discrepancy of the Dþ s ! þ decay constant from its lattice QCD prediction [12,13] gives hints of new physics that may also contribute to the leptonic B decay modes. Currently, the most stringent published lim- its on Bþ ! ‘þ are from the BELLE collaboration with Charged-particle tracking and dE=dx measurements for particle identiﬁcation are provided by a ﬁve-layer double- sided silicon vertex tracker and a 40-layer drift chamber contained within the magnetic ﬁeld of a 1.5 T supercon- ducting solenoid. A ring-imaging Cherenkov detector pro- vides efﬁcient particle identiﬁcation. The energies of neutral particles are measured with an electromagnetic calorimeter (EMC) consisting of 6580 CsI(Tl) crystals arrayed in a cylindrical barrel and in a forward endcap. 091104-4 RAPID COMMUNICATIONS PHYSICAL REVIEW D 77, 091104(R) (2008) PHYSICAL REVIEW D 77, 091104(R) (2008) þ modes, but with an a 1 instead of a . The requirements of cosa1 > 0:45 and cosa1 > 0:35 are used for the two cases, respectively. There are no additional requirements on the or a1. The quantity Eextra ¼ P Etrack þ P Ecluster E‘þ P E‘;;0 describes the amount of energy recorded by the detector that is not accounted for by the high momen- tum lepton and daughters (in the case of B0 ! ‘þ). The clusters and tracks associated with the reconstruction of Btag are excluded from the sums, and only clusters with energy more than 50 MeV in the CM frame are considered. We require Eextra to be less than 1.0 GeV in the CM frame. The signal and background distributions for Eextra are shown in Fig. 1. q 1 Additional background, for both Bþ ! ‘þ and B0 ! ‘þ decays, can arise from a variety of sources, including beam backgrounds, unassociated hadronic shower frag- ments, reconstruction artifacts, bremsstrahlung, and pho- ton conversions. We demand that events have no more than two extra charged tracks and six extra neutral clusters, allowing the presence of low energy particles not neces- sarily associated with the decay of the ð4SÞ. Requirements on the missing momentum and extra energy in the event are utilized to ensure that such particles are unimportant for the analysis. Since these requirements are optimized for each signal mode individually, we quote only the values for Bþ ! ‘þ‘ modes in the text. The signal selection requirements for all signal modes are listed in Table II. The signal yields are extracted from unbinned maximum likelihood ﬁts to the signal lepton momentum distributions, as measured in the Bsignal frame. The signal and back- ground MC distributions are ﬁtted by phenomenological probability density functions (PDF). The signal distribu- tions are modeled with Crystal Ball functions [24] to account for the energy loss due to unreconstructed brems- strahlung photons. The Bþ ! ‘þ background is modeled with an exponential decay and a Gaussian distribution, while the B0 ! ‘þ background is modeled with a double Gaussian distribution. The PDF parameters are determined from simulated events. RAPID COMMUNICATIONS B. AUBERT et al. PHYSICAL REVIEW D 77, 091104(R) (2008) for the hadronic decay modes listed in Table I. E ¼ P E;0 þ p m, where m ¼ 1:777 GeV=c2, the sum is over the daughter candidates, the momentum of the neutrino is p ¼ j P ~p;0j, and all quantities are measured in the rest frame. We assign the decay mode for which jEj is smallest, requiring additional conditions for the decay modes that proceed through the intermediate resonances ! 0, a 1 ! 00, and a 1 ! þ. We calculate the quantity cos ¼ ð2EE m2 m2Þ=ð2j ~pjj ~pjÞ, where (E; ~p) and (E; ~p) are the four-momenta in the Bsignal frame, and m and m are the masses of the and . For a correctly reconstructed , this quantity peaks near unity. If the candidate does not satisfy cos > 0:70 the mode with the next smallest jEj (if one is present) is selected instead. Analogous quantities are calculated for the ! 00 and ! þ modes, but with an a 1 instead of a . The requirements of cosa1 > 0:45 and cosa1 > 0:35 are used for the two cases, respectively. There are no additional requirements on the or a1. for the hadronic decay modes listed in Table I. E ¼ P E;0 þ p m, where m ¼ 1:777 GeV=c2, the sum is over the daughter candidates, the momentum of the neutrino is p ¼ j P ~p;0j, and all quantities are measured in the rest frame. We assign the decay mode for which jEj is smallest, requiring additional conditions for the decay modes that proceed through the intermediate resonances ! 0, a 1 ! 00, and a 1 ! þ. We calculate the quantity cos ¼ ð2EE m2 m2Þ=ð2j ~pjj ~pjÞ, where (E; ~p) and (E; ~p) are the four-momenta in the Bsignal frame, and m and m are the masses of the and . For a correctly reconstructed , this quantity peaks near unity. If the candidate does not satisfy cos > 0:70 the mode with the next smallest jEj (if one is present) is selected instead. Analogous quantities are calculated for the ! 00 and ! þ modes, but with an a 1 instead of a . PHYSICAL REVIEW D 77, 091104(R) (2008) For the Btag candidate, we deﬁne the energy substituted mass, mES ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ E2 beam ~p2 B q , where ~pB is the momentum of the Btag candidate in the CM frame. Btag candidates that are correctly reconstructed peak in mES near the nominal B meson mass, while incorrectly reconstructed Btag candi- dates produce a combinatorial distribution. The signal events are required to lie within the range 5:270 GeV=c2 < mES < 5:288 GeV=c2. This reconstruction procedure re- sults in a yield of approximately 2500 (2000) correctly reconstructed B (B0) candidates per fb1 of data. TABLE I. The decays considered are listed with their branching fractions, in percent [23]. decay mode Branching Fraction e e 17:84 0:05 17:36 0:05 10:90 0:07 0 25:50 0:10 00 9:25 0:12 þ 9:33 0:08 decay mode Branching Fraction e e 17:84 0:05 17:36 0:05 10:90 0:07 0 25:50 0:10 00 9:25 0:12 þ 9:33 0:08 Because the two B mesons are produced with very little momentum in the CM frame, B B events typically produce a more isotropic distribution of particles in the detector than nonresonant (‘‘continuum’’) backgrounds. Such back- 091104-5 PHYSICAL REVIEW D 77, 091104(R) (2008) The requirements of cosa1 > 0:45 and cosa1 > 0:35 are used for the two cases, respectively. There are no additional requirements on the or a1. for the hadronic decay modes listed in Table I. E ¼ P E;0 þ p m, where m ¼ 1:777 GeV=c2, the sum is over the daughter candidates, the momentum of the neutrino is p ¼ j P ~p;0j, and all quantities are measured in the rest frame. We assign the decay mode for which jEj is smallest, requiring additional conditions for the decay modes that proceed through the intermediate resonances ! 0, a 1 ! 00, and a 1 ! þ. We calculate the quantity cos ¼ ð2EE m2 m2Þ=ð2j ~pjj ~pjÞ, where reconstruction. Pmiss is calculated in the rest frame of the parent of the neutrino(s), so that the missing momen- tum balances the sum of other signal particles’ momenta. The signal events are selected by requiring Pmiss to be less than 0:5 GeV=c. reconstruction. Pmiss is calculated in the rest frame of the parent of the neutrino(s), so that the missing momen- tum balances the sum of other signal particles’ momenta. The signal events are selected by requiring Pmiss to be less than 0:5 GeV=c. reconstruction. Pmiss is calculated in the rest frame of the parent of the neutrino(s), so that the missing momen- tum balances the sum of other signal particles’ momenta. The signal events are selected by requiring Pmiss to be less than 0:5 GeV=c. For Bþ ! ‘þ modes we also consider the direction of the missing momentum cospmiss ¼ pzmiss=pmiss, where the subscript z indicates the component of the momentum in the direction parallel to the beam pipe, as measured in the ð4SÞ CM frame. The requirement 0:76 < cospmiss < 0:92 is determined by the geometry of the detector; events where pmiss points outside of the detector acceptance in the forward or backward direction are excluded. (E; ~p) and (E; ~p) are the four-momenta in the Bsignal frame, and m and m are the masses of the and . For a correctly reconstructed , this quantity peaks near unity. If the candidate does not satisfy cos > 0:70 the mode with the next smallest jEj (if one is present) is selected instead. Analogous quantities are calculated for the ! 00 and ! SEARCHES FOR THE DECAYS B0 ! l . . . SEARCHES FOR THE DECAYS B0 ! l . . FIG. 1 (color online). Eextra distributions for the background simulation and data (left) and the signal (right) after all other selection criteria have been applied. The upper plots are for Bþ ! ‘þ modes and the lower plots are for B0 ! ‘þ modes. The background distributions show electron and muon modes together, as they are nearly identical. The background is almost completely dominated by B B events. The signal modes are shown with a branching fraction of 105. SEARCHES FOR THE DECAYS B0 ! l . . . PHYSICAL REVIEW D 77, 091104(R) (2008) PHYSICAL REVIEW D 77, 091104(R) (2008) FIG. 1 (color online). Eextra distributions for the background simulation and data (left) and the signal (right) after all other selection criteria have been applied. The upper plots are for Bþ ! ‘þ modes and the lower plots are for B0 ! ‘þ modes. The background distributions show electron and muon modes together, as they are nearly identical. The background is almost completely dominated by B B events. The signal modes are shown with a branching fraction of 105. The systematic uncertainties arising from the ﬁtting procedure are studied by repeating the procedure on addi- tional simulated samples, generated according to the PDFs, with varying number of signal events. Systematic effects are studied by repeating the procedure with PDF parame- ters varied by their uncertainties. For the case of zero signal events, we ﬁnd negligible effects on the branching fraction values, and take the standard deviation of ns and nb from their expected values in the ﬁts as systematic uncertainties. total signal selection efﬁciency and NB B is the number of BþB or B0 B0 pairs in the data sample. The signal selec- tion efﬁciencies, expected number of background events and ﬁt results are given in Table III. The number of signal events given by the ﬁts is consistent with zero for all decay modes. The uncertainties in Table III are statistical except for those shown for B which are the statistical and system- atic uncertainties added in quadrature. total signal selection efﬁciency and NB B is the number of BþB or B0 B0 pairs in the data sample. The signal selec- tion efﬁciencies, expected number of background events and ﬁt results are given in Table III. PHYSICAL REVIEW D 77, 091104(R) (2008) The ﬁt is performed using the following likelihood function: The extra momentum in the event is represented by Pmiss ¼ j ~pmiss þ P ~p‘;j, where p‘; are the momenta of the lepton or pion candidate(s) assumed to be recoiling against the neutrinos. The missing momentum is calculated according to ~pmiss ¼ ~pð4SÞ ~pBtag ~pall, where ~pall is the momentum of all tracks and clusters left after the Btag L ðns; nbÞ ¼ eðnsþnbÞ N! Y N i¼1 ½nsfsðiÞ þ nbfbðiÞ; (2) (2) where N is the total number of events in the ﬁt region, fsðiÞ and fbðiÞ are the PDFs for the signal and background, and nb and ns are the number of background and signal events. All parameters of the signal and background PDFs remain ﬁxed, while ns and nb are allowed to ﬂoat. The ﬁts are restricted to the ranges in the lepton momenta shown in Fig. 2. TABLE II. The signal selection requirements for Pmiss and Eextra, expressed in GeV=c and GeV, respectively, for each decay mode. TABLE II. The signal selection requirements for Pmiss and Eextra, expressed in GeV=c and GeV, respectively, for each decay mode. Decay mode Pmiss Eextra B0 ! ‘þ, ! 8>>>>>>>< >>>>>>>: e e 0 00 þ <1:2 <0:6 <1:2 <0:6 <0:8 <0:6 <0:9 <0:6 <1:0 <0:7 <1:0 <0:8 Bþ ! ‘þ <0:5 <1:0 The 90% conﬁdence level (C.L.) upper limit on the branching fraction B is determined by solving for B90% in 0:90 ¼ RB90% 0 LðBÞdB= R1 0 LðBÞdB for events lying in the signal regions of 2:40 GeV=c < p < 2:75 GeV=c for Bþ ! ‘þ and 2:20 GeV=c < p < 2:42 GeV=c for B0 ! ‘þ. B is related to the signal yield ns through a substitution ns ¼ tot 2 NB B B, where tot is the 091104-6 RAPID COMMUNICATIONS PHYSICAL REVIEW D 77, 091104(R) (2008) TABLE III. Signal selection efﬁciency tot determined from MC, the ﬁtted numbers of signal and background events in the signal regions ns and n b, and the branching fractions B. The uncertainties for B include statistical and systematic terms. The uncertainties for the other quantities are statistical only. TABLE III. Signal selection efﬁciency tot determined from MC, the ﬁtted numbers of signal and background events in the signal regions ns and n b, and the branching fractions B. The uncertainties for B include statistical and systematic terms. The uncertainties for the other quantities are statistical only. Signal Mode eþ þ eþ þ tot 105 135 4 120 4 32 2 27 2 n b MC 2:66 0:13 5:74 0:25 8:69 0:27 12:14 0:45 n b 2:67 0:19 5:67 0:34 9:35 0:35 13:03 0:31 ns 0:07 0:03 0:11 0:05 0:02 0:01 0:01 0:01 B 106 0:1þ2:6 1:7 0:2þ2:7 1:8 0þ15 10 0þ11 7 B90% C:L: 5:2 106 5:6 106 2:8 105 2:2 105 We ﬁnd the ﬁts to be well behaved and having no signiﬁ- cant sources of bias, introducing no additional uncertain- ties. Total uncertainties associated with the ﬁtting procedure are listed in Table III for each decay mode. Table IV lists the sources and the magnitudes of the uncertainties with their effect on B. The uncertainties are incorporated into the ﬁnal results by varying the branching fraction assumption by its uncertainty when integrating L for the 90% C.L. upper limit. p y The discrepancies between simulation and data are treated as detailed in the following paragraphs. The num- ber of correctly reconstructed Btag events in the mES signal region is compared between simulation and data. The mES distributions for simulation and data are ﬁtted with a combination of ARGUS [25] and Crystal Ball functions, allowing the number of mES peaking events to be estimated by integrating the peaking component between 5:270 GeV=c2 and 5:288 GeV=c2. We ﬁnd the simulation to underestimate the number of events with a good Btag and scale the signal selection efﬁciency by a factor of 1:11 0:06 (1:05 0:06) for events with a neutral (charged) Btag. In addition, the PID efﬁciencies in simulation are cor- rected for the 2%–5% lower efﬁciencies in data. SEARCHES FOR THE DECAYS B0 ! l . . . The number of signal events given by the ﬁts is consistent with zero for all decay modes. The uncertainties in Table III are statistical except for those shown for B which are the statistical and system- atic uncertainties added in quadrature. FIG. 2 (color online). The unbinned maximum likelihood ﬁts on the lepton momentum. The dashed line, representing the signal PDF with an arbitrary scaling, indicates where the signal is expected. FIG. 2 (color online). The unbinned maximum likelihood ﬁts on the lepton momentum. The dashed line, representing the signal PDF with an arbitrary scaling, indicates where the signal is expected. 091104-7 B. AUBERT et al. B. AUBERT et al. PHYSICAL REVIEW D 77, 091104(R) (2008) PHYSICAL REVIEW D 77, 091104(R) (2008) We assign associated uncertainties of about 2% for high momentum particles (signal lepton), and about 5% for tau daughters. The misidentiﬁcation rate of leptons and pions is found to be negligible in the simulated samples, after all selection criteria are applied. An uncertainty in the tracking algo- rithm introduces an additional 0.8% systematic uncertainty for each charged track present in any given signal mode (e.g. 1.6% for B0 ! ‘þ, ! ). The uncertain- ties for B0 ! ‘þ modes are calculated as weighted averages of all decay modes. p y The discrepancies between simulation and data are treated as detailed in the following paragraphs. The num- ber of correctly reconstructed Btag events in the mES signal region is compared between simulation and data. The mES distributions for simulation and data are ﬁtted with a combination of ARGUS [25] and Crystal Ball functions, allowing the number of mES peaking events to be estimated by integrating the peaking component between 5:270 GeV=c2 and 5:288 GeV=c2. We ﬁnd the simulation to underestimate the number of events with a good Btag and scale the signal selection efﬁciency by a factor of 1:11 0:06 (1:05 0:06) for events with a neutral (charged) Btag. We have presented searches for the rare leptonic decays Bþ ! ‘þ and B0 ! ‘, where ‘ ¼ e, , using a novel hadronic tag reconstruction technique. We ﬁnd no evidence of signal in any of the decay modes in a data sample of approximately 378 106 B B pairs (342 fb1), and set the branching fraction upper limits at BðBþ ! eþÞ < 5:2 106, BðBþ ! þÞ < 5:6 106, BðB0 ! eþÞ < 2:8 105, and BðB0 ! þÞ < 2:2 105, at 90% conﬁdence level. While these upper limits on BðBþ ! eþÞ and BðBþ ! þÞ complement the more stringent limits available from inclusive studies [14,16], the B0 ! eþ and B0 ! þ results are the most stringent published upper limits available. ( ) ( g ) tag In addition, the PID efﬁciencies in simulation are cor- rected for the 2%–5% lower efﬁciencies in data. We assign associated uncertainties of about 2% for high momentum particles (signal lepton), and about 5% for tau daughters. RAPID COMMUNICATIONS SEARCHES FOR THE DECAYS B0 ! l . . . PHYSICAL REVIEW D 77, 091104(R) (2008) PHYSICAL REVIEW D 77, 091104(R) (2008) The misidentiﬁcation rate of leptons and pions is found to be negligible in the simulated samples, after all selection criteria are applied. An uncertainty in the tracking algo- rithm introduces an additional 0.8% systematic uncertainty for each charged track present in any given signal mode (e.g. 1.6% for B0 ! ‘þ, ! ). The uncertain- ties for B0 ! ‘þ modes are calculated as weighted averages of all decay modes. We are grateful for the extraordinary contributions of our PEP-II colleagues in achieving the excellent luminos- ity and machine conditions that have made this work possible. The success of this project also relies critically on the expertise and dedication of the computing organ- izations that support BABAR. The collaborating institutions wish to thank SLAC for its support and the kind hospitality extended to them. This work is supported by the US Department of Energy and National Science Foundation, the Natural Sciences and Engineering Research Council (Canada), the Commissariat a` l’Energie Atomique and Institut National de Physique Nucle´aire et de Physique des Particules (France), the Bundesministerium fu¨r Bildung und Forschung and Deutsche Forschungs- gemeinschaft (Germany), the Istituto Nazionale di Fisica Nucleare (Italy), the Foundation for Fundamental Research on Matter (The Netherlands), the Research Council of Norway, the Ministry of Science and Technology of the Russian Federation, Ministerio de Educacio´n y Ciencia (Spain), and the Science and Technology Facilities Council (United Kingdom). Individuals have received sup- port from the Marie-Curie IEF program (European Union) and the A. P. Sloan Foundation. TABLE IV. The sources and magnitudes of systematic uncer- tainties, in percent. TABLE IV. The sources and magnitudes of systematic uncer- tainties, in percent. tainties, in percent. Signal Mode Uncertainty source e þ þ eþ þ Signal Fit 5.6 10.6 4.3 8.2 Background Fit 3.9 3.1 5.1 7.8 Btag efﬁciency 6.4 6.4 5.8 5.8 PID efﬁciency 5.3 5.8 1.0 2.0 MC Statistics 8.6 7.4 3.0 2.8 Tracking efﬁciency 1.7 1.7 0.8 0.8 NB B 1.1 1.1 1.1 1.1 091104-8 PHYSICAL REVIEW D 77, 091104(R) (2008) [1] Throughout this paper, decay modes imply also their charge conjugates. [14] N. Satoyama et al. (BELLE Collaboration), Phys. Lett. B 647, 67 (2007). [15] M. Artuso et al. (CLEO Collaboration), Phys. Rev. Lett. 75, 785 (1995). [2] Super B Collaboration, arXiv:0709.0451v2. [3] W.-S. Hou, Phys. Rev. D 48, 2342 (1993). [16] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 92, 221803 (2004). [4] A. Dedes, J. Ellis, and M. Raidal, Phys. Lett. B 549, 159 (2002). [5] D. Silverman and H. Yao, Phys. Rev. D 38, 214 (1988). [17] A. Bornheim et al. (CLEO Collaboration), Phys. Rev. Lett. 93, 241802 (2004). [6] N. Cabbibo, Phys. Rev. Lett. 10, 531 (1963); M. Kobayashi and T. Maskawa, Prog. Theor. Phys. 49, 652 (1973). [18] S. Agostinelli et al., Nucl. Instrum. Methods Phys. Res., Sect. A 506, 250 (2003). [7] E. Barberio et al. (Heavy Flavor Averaging Group), arXiv: hep-ex/0603003. [19] B. Aubert et al. (BABAR Collaboration), Nucl. Instrum. Methods Phys. Res., Sect. A 479, 1 (2002). p [8] A. Gray et al. (HPQCD Collaboration), Phys. Rev. Lett. 95, 212001 (2005). y [20] B. Barish et al. (CLEO Collaboration), Phys. Rev. Lett. 76, 1570 (1996). [9] B. Aubert et al. (BABAR Collaboration), Phys. Rev. D 76, 052002 (2007). [21] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 92, 071802 (2004). [10] K. Ikado et al. (BELLE Collaboration), Phys. Rev. Lett. 97, 251802 (2006). [22] G. C. Fox and S. Wolfram, Phys. Rev. Lett. 41, 1581 (1978). [11] A. Masiero and P. Paradisi, J. Phys. Conf. Ser. 53, 248 (2006). 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https://openalex.org/W4255314048	https://hal.archives-ouvertes.fr/hal-03451507/file/2021JA029425.pdf	English	null	Empirical selection of Auroral Kilometric Radiation during a multipoint remote observation with Wind and Cassini	null	2,021	cc-by	24,104	Key Points: Key Points: • Novel, empirically based method to extract Auroral Kilometric Radiation (AKR) from Wind/ WAVES is presented and applied to observations made during the Cassini flyby J. E. Waters1 , C. M. Jackman2 , L. Lamy3,4 , B. Cecconi3 , D. K. Whiter1 , X. Bonnin3 , K. Issautier3 , and A. R. Fogg2 1Space Environment Physics Group, School of Physics and Astronomy, University of Southampton, Southampton, UK, 2School of Cosmic Physics, DIAS Dunsink Observatory, Dublin Institute for Advanced Studies, Dublin, Ireland, 3LESIA, Observatoire de Paris, PSL Research University, CNRS, Sorbonne Université, Université de Paris, Meudon, France, 4LAM, Pythéas, Aix Marseille Université, CNRS, CNES, Marseille, France • Selected data show a distribution of AKR power with expected longitudinal and latitudinal visibility constraints • Diurnal temporal modulation observed, suggesting the dominance of a geometric viewing effect and agreeing with previous AKR observations • Diurnal temporal modulation observed, suggesting the dominance of a geometric viewing effect and agreeing with previous AKR observations Abstract Auroral Kilometric Radiation (AKR) is terrestrial radio emission that originates in particle acceleration regions along magnetic field lines, coinciding with discrete auroral arcs. AKR viewing geometry is complex due to the confinement of the source regions to nightside local times (LTs) and the anisotropy of the beaming pattern, so observations are highly dependent on spacecraft viewing position. We present a novel, empirical technique that selects AKR emission from observations made with the spin-axis aligned antenna of the Wind/WAVES instrument, based on the rapidly varying amplitude of AKR across spacecraft spin timescales. We apply the technique to Wind/WAVES data during 1999 day of year 227–257, when the Cassini spacecraft flew past Earth and provided an opportunity to observe AKR from two remote locations. We examine the AKR flux and power, with observations made from LTs of 1700–0300 hr having an average power up to 104 Wsr-1 larger than those on the dayside and an increasing AKR power observed at higher magnetic latitudes. We perform a linear cross-correlation between the Wind AKR power and the spacecraft magnetic latitude, showing positive then negative correlation as Wind travels from the Northern to Southern magnetic hemisphere. Statistically significant diurnal modulations are found in the whole 30-day period and in subsets of the data covering different local time sectors, indicative of a predominantly geometrical effect for remote AKR viewing. ©2021. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Citation: Waters, J. E., Jackman, C. M., Lamy, L., Cecconi, B., Whiter, D. K., Bonnin, X., et al. (2021). Empirical selection of Auroral Kilometric Radiation during a multipoint remote observation with wind and Cassini. Journal of Geophysical Research: Space Physics, 126, e2021JA029425. https://doi. org/10.1029/2021JA029425 Received 13 APR 2021 Accepted 28 SEP 2021 Received 13 APR 2021 Accepted 28 SEP 2021 Plain Language Summary Auroral Kilometric Radiation (AKR) is naturally occurring radio emission from the Earth's Northern and Southern polar regions, which becomes more intense as the aurora brightens. In this work, we examine data from the Wind spacecraft WAVES instrument from a 30- day interval in 1999 when a second spacecraft, Cassini, was also flying near Earth and measuring the AKR from a different viewpoint. In this work, we select the AKR using an empirical measure of the variability observed by the WAVES instrument, and compare the distribution and time profile of AKR intensity. Comparing measurements of this radio emission from different spacecraft positions help us to understand how the AKR is best viewed and illustrate the constrained beaming of the emission. This information is important for anyone wanting to attempt to interpret measurements of the AKR. J. E. Waters, J.Waters@soton.ac.uk J. E. Waters, J.Waters@soton.ac.uk Waters, J. E., Jackman, C. M., Lamy, L., Cecconi, B., Whiter, D. K., Bonnin, X., et al. (2021). Empirical selection of Auroral Kilometric Radiation during a multipoint remote observation with wind and Cassini. Journal of Geophysical Research: Space Physics, 126, e2021JA029425. https://doi. org/10.1029/2021JA029425 Key Points: The reproduction of well-known features of the AKR verifies the empirical selection and shows the promise of its application to Wind/WAVES observations. Correspondence to: Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 suggested that AKR can be observed within a cone at angles that are increasingly oblique to the magnetic field with decreasing frequency (Green et al., 1977; Kasaba et al., 1997). However, more recent observations using Cluster suggest that AKR is emitted in a more restricted geometry, with similar longitudinal extent up to a few thousand km but emitted over a narrower latitudinal region of a few tens of degrees over the auroral zone (Mutel et al., 2008). Both models of the emission geometry have been supported by in-situ observations with the Polar spacecraft, confirming the frequency-dependent, anisotropic beaming of AKR from the source region (Menietti et al., 2011). suggested that AKR can be observed within a cone at angles that are increasingly oblique to the magnetic field with decreasing frequency (Green et al., 1977; Kasaba et al., 1997). However, more recent observations using Cluster suggest that AKR is emitted in a more restricted geometry, with similar longitudinal extent up to a few thousand km but emitted over a narrower latitudinal region of a few tens of degrees over the auroral zone (Mutel et al., 2008). Both models of the emission geometry have been supported by in-situ observations with the Polar spacecraft, confirming the frequency-dependent, anisotropic beaming of AKR from the source region (Menietti et al., 2011). Furthermore, AKR is known to be fully circularly polarized, with the handedness depending on the di- rection of electron gyration in either hemisphere (Kaiser et al., 1978). Where polarization information is available, we expect to see left-handed circularly polarized (LH) emission from the Southern magnetic (Northern geographic) hemisphere and right-handed circularly polarized (RH) emission from the Northern magnetic (Southern geographic) hemisphere (assuming emission in the extraordinary mode). This has been observed at both Earth and Saturn and is a consequence of the emission mechanism (Lamy et al., 2010; Lamy, Zarka, Cecconi, Prangé, et al., 2008). The visibility of AKR is a strong function of the position of the observer. AKR and its source regions are mostly concentrated at nightside local times (LTs); AKR has been observed consistently from LTs between  E 1600–0300 hr, whereas the most intense source regions are located at 2100–2200 LT (e.g., Alexander & Kaiser, 1976; Gurnett, 1974; Panchenko, 2003). The visibility of AKR to a spacecraft at various latitudes is constrained by the beaming of the emission, as mentioned above. Journal of Geophysical Research: Space Physics Ray tracing has been used previously to examine the general propagation of AKR from the source region as well as for instances where the emission may refract from the dense plasmasphere, for example (Green et al., 1977; Mutel et al., 2004). AKR visibility with latitude has also been examined statistically for studies of hemispheric conjugacy using multiple spacecraft (Morioka et al., 2011). For a spacecraft near the equa- tor, it is possible to observe AKR emission from both hemispheres as the emission cones from sources on a given meridian overlap. In this case, the emission from each pole cannot be separated (without polarization information) and the observations must be interpreted as a global average. For closer radial distances, a spacecraft near the equator can be beneath the superposed emission cones of each hemisphere and observe no AKR. At Saturn, this equatorial shadow zone has been modeled and found at radial distances of 3.6 s E R , where 60268 S E R km is the radius of Saturn (Lamy, Zarka, Cecconi, Hess, & Prangé, 2008). At Earth, Mori- oka et al. (2011) attributed an approximate limit of 7 E E R to the equatorial shadow zone, where 6371 E E R km is the radius of Earth. Given that AKR generation is intrinsic to the magnetic field, the tilt of the planetary field with respect to the rotation axis combines with the highly directive AKR beaming to produce an illumination region that is time-dependent. Temporally, significant periodicities have been found at semi-diurnal and diurnal times- cales, the latter of which has been attributed to geometrical viewing effects as the emission region precesses, like the magnetic dipole, with respect to the rotation of the Earth (Lamy et al., 2010). Other suggestions for the source of this modulation include an intrinsic modulation due to the effect on the ionosphere of the tilt of the magnetic dipole with respect to the incoming solar wind (Panchenko et al., 2009) or a physical origin within the magnetosphere itself (Morioka et al., 2013). Discerning the origin of this variability is useful to further the understanding of the magnetosphere-ionosphere coupling. In this study, we are concerned with the extraction of AKR from the raw data of an Earth-orbiting space- craft, as well as the interpretation and quantification of any visibility effects due to the location of the space- craft relative to the radio sources. 1. Introduction Auroral kilometric radiation (AKR) describes amplified radio emission from the Earth that is generated from relativistic, precipitating electrons along magnetic field lines in the auroral zone and resonates at the electron cyclotron frequency (Wu & Lee, 1979). The emission frequency of an AKR source is close to the local electron gyrofrequency, so that lower frequency AKR emanates from a higher altitude along a field line. AKR is emitted between   30 800 E kHz and has been observed by many Earth-orbiting spacecraft such as Polar, Geotail, and Cluster (e.g., Anderson et al., 2005; Liou et al., 2000; Morioka et al., 2007; Mutel et al., 2003). The emission mechanism, the electron cyclotron maser instability, is such that AKR is emitted at angles near-perpendicular to the field lines. This leads to largely anisotropic beaming of AKR from indi- vidual field lines that has been constrained both through modeling and observations. Earliest observations WATERS ET AL. WATERS ET AL. 1 of 26 Journal of Geophysical Research: Space Physics Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 emission intensify but the frequency spectrum undergoes characteristic changes in response to substorm behavior. Observations with the Polar spacecraft have shown that the AKR source region morphology may have a dual structure, suggesting that a given field line has a more persistent AKR source at lower altitudes that suddenly extends to higher altitudes at the time of substorm onset (Morioka et al., 2007). emission intensify but the frequency spectrum undergoes characteristic changes in response to substorm behavior. Observations with the Polar spacecraft have shown that the AKR source region morphology may have a dual structure, suggesting that a given field line has a more persistent AKR source at lower altitudes that suddenly extends to higher altitudes at the time of substorm onset (Morioka et al., 2007). Before the properties of AKR can be studied in detail, the AKR-related radio signals must be disentangled from other radio emissions detected by a spacecraft's radio instrument. This non-trivial process is described in more detail in Section 2 below. Broadly speaking, an orbiting spacecraft may detect multiple possible sources of radio emission at multiple wavelengths when surveying the radio environment. At kilomet- ric wavelengths, corresponding to frequencies of 1 E MHz and below, the long, drifting tails of solar radio type III bursts can be observed, which are ubiquitous when the spacecraft is in the solar wind (Krupar et al., 2018). As well as this, characteristic frequencies of the local plasma can be observed at lower frequen- cies. This can occur both in the solar wind, at the plasma frequency and harmonics following Langmuir waves, or within the magnetosphere, where dense, turbulent plasma in the magnetosheath leads to a rise in quasi-thermal noise (QTN) (Meyer-Vernet et al., 1998, 2017). Thus AKR is often observed in superposition with other waves and must be explicitly selected, where possible, for a complete study. Goniopolarimetric (GP) inversion techniques are useful for selecting AKR, as the Stokes parameters of an incident wave can be derived using a model that accounts for the geometry of both the radio antennae and the source (Cecco- ni, 2019; Cecconi & Zarka, 2005). Then, the circular polarization can be used to discriminate against other sources; the few observations of solar radio Type III bursts at frequencies 1 E MHz show weak polarization (Pulupa et al., 2019; Reiner et al., 2007). 2. Instrumentation and Empirical Data Selection Technique The Wind spacecraft, launched in 1994 as part of the International Solar Terrestrial Physics (ISTP) mission, is equipped with various instruments designed to study the solar wind and radio emissions from both the Sun and Earth. The primary function of the spacecraft is that of a solar wind monitor, and Wind has most often observed from the Lagrangian point L1 (sunward of Earth); Wind first reached L1 in 1996 before spending time between 1998 and 2004 executing complex orbital maneuvers to explore Earth's magneto- sphere. The spacecraft returned to L1 in 2004 and has been there since. The relevant instrumentation will be described first in Section 2.1 before the appropriate calibration steps are described in Section 2.2. The method of selecting radio data pertaining to AKR is then illustrated in Section 2.3. Journal of Geophysical Research: Space Physics This has been done at Saturn, using the radio instrument on board the three-axis stabilized Cassini spacecraft to observe SKR, the Kronian analog of AKR. As the Cassini spacecraft flew by Earth, its radio instrument was turned on for a month-long period. During this time, the instrument was used to retrieve the circular polarization state of the AKR, allowing the general emission characteristics, such as the emission power and the temporal modulation to be studied (Lamy et al., 2010). For this month-long period, the Wind spacecraft was traveling on orbits that carried it through the nightside magnetosphere at perigee, allowing it to make remote observations of the AKR source region, as well as oth- er opportunities to observe AKR from other LTs. Although it is not possible to apply previously developed GP techniques for spinning spacecraft to AKR observations with Wind, a selection technique based on the observed variability on timescales of seconds has been developed and applied. This has provided an effec- tive selection of AKR emission, allowing a quantitative analysis and comparison to be performed. Here, we focus on the unique dual vantage point of this Cassini-Wind conjunction during 1999. In Section 2, we describe the instrumentation, the calibration of the radio data, and the selection technique, which we have applied to extract AKR. In Section 3, we compare and contrast the viewing geometry and observations of Wind and Cassini as they traverse the terrestrial magnetosphere on different paths. In Section 4, we sum- marize our findings and interpretation of the complementary data. Journal of Geophysical Research: Space Physics Moreover, the AKR has the potential to serve as an excellent diagnostic tool both for solar wind driving and for magnetospheric dynamics. Specifically, previous work has shown that AKR can intensify during periods of magnetospheric disturbance (Liou et al., 2000; Voots et al., 1977; Zhao et al., 2019). The generation of AKR requires the presence of strong, parallel electric fields that accel- erate electrons to the necessary relativistic speeds within the magnetosphere-ionosphere coupling region. The well-studied physical phenomenon of the magnetospheric substorm manifests in various observable signatures in both the magnetosphere and ionosphere. In the magnetosphere, the magnetic field dipolar- izes following reconnection in the magnetotail and energetic plasma flows Earthward (Juusola et al., 2011; Liou, 2002). Energetic particles are injected into the ionosphere as the substorm current wedge strength- ens the current systems at high latitudes, brightening the aurora and causing well known morphological changes in the oval (Akasofu, 1964; Kepko et al., 2015; McPherron et al., 1973). For AKR, not only does the WATERS ET AL. 2 of 26 Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 covering the whole AKR frequency spectrum and utilizes antennae that are each in the short-antenna re- gime, allowing for a beaming pattern that is independent of the observation frequency. covering the whole AKR frequency spectrum and utilizes antennae that are each in the short-antenna re- gime, allowing for a beaming pattern that is independent of the observation frequency. The RAD1 receiver can operate in one of two modes: the SEP mode allows the receiver to measure with one of the equatorial antennae (usually X) and the Z antenna independently. The SUM mode performs the elec- tronic summation of the X and Z antennae, outputting this synthetic signal as well as one with a  2 E phase shift applied to the equatorial antenna. The SUM mode thus returns two signals from the synthetic inclined dipole. In this work, the original SUM signal will be referred to as the S antenna and that with a phase shift applied as the S’ antenna. RAD1 has 256 available frequency channels between 20 and 1,040 kHz and chan- nels can be chosen to sample the radio environment using three different methods. The most often used allows the instrument to be provided with a list of frequencies to be measured over the next fixed-duration sweep cycle of samples. Each frequency channel is measured at each antenna over the respective integration time (154 ms for the S and S’ antennae and 308 ms for the Z antenna) to comprise a single observation. Measurements are then repeated at that frequency across a spacecraft spin period of 3 s in order to receive a signal that corresponds to a single period of modulation. Sampling all 256 frequency channels, while offering greater spectral reso- lution, would increase the duration of the sweep cycle and so decrease the temporal resolution. The typical total time attributed to the measurement of a single frequency for S, S’, and Z antennae, accounting for the offset incurred at the beginning of the frequency sample, is 358 ms. A sweep cycle, lasting  E 3 min, is typi- cally comprised of 64 frequency measurements, each made during one spacecraft spin period. Thus a total of  8 64 voltage spectral density measurements received by each of the S, S’, and Z antennae, as well as the corresponding times of measurement, are supplied for a single sweep cycle. 2.2. Source Flux Density Determination and Calibration 2.2. Source Flux Density Determination and Calibration Journal of Geophysical Research: Space Physics Each measurement is provided in units of   2 1 E V Hz and the preamplifier and receiver gain values have been taken into account. As well as this, general data including spacecraft attitude parameters and indicators for the mode of operation of the RAD1 receiver are supplied with each sweep cycle. L2-level data for the RAD1 instrument are provided as   480 3 min sweep cycles that comprise 24 hr of RAD1 observations. 2.1. Wind/WAVES Radio Instrumentation The WAVES investigation (Bougeret et al., 1995) is comprised of two antennae in the spin plane of the spacecraft (X and Y — with original tip-to-tip lengths of 100 and 15 m, respectively) as well as one aligned with the spin axis (Z, of length 12 m tip-to-tip). The antennae used here are of the electric dipole type, each formed of two monopolar wire lengths along the same axis on either side of the spacecraft, with the Z anten- na having a rigid structure. Of the three, WAVES radio receivers RAD1 operates between 20 and 1,040 kHz 3 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 2.2.2. Calibration Following from Equation 21 in Manning and Fainberg (1980), this gives   2 1 sin 2 Z Z P GS (1) 2 1 (1) where Z E P is the power measured by the Z antenna in  2 1 V Hz E , E G is a calibration factor, Z E S is the flux of a point source derived from the single Z antenna in   2 1 Wm Hz E and  E is the co-latitude of the radio source. For AKR, we can assume that the source region is approximated by the Earth's center and transform the source co-latitude  E to  E , the latitude of the spacecraft in geocentric-solar-ecliptic (GSE) coordinates. Given that the calibration is applied to all Wind observations during the interval, which are made from various latitudes and LT, and that we cannot determine the emission hemisphere exactly, this assumption is most broadly valid; errors in the spatial location of the source when assuming emission from either pole could be up to 1 E R given the spectral range of the instrument, the azimuthal extent of AKR sources relative to Wind and their confinement along high-latitude magnetic field lines. We have characterized the error due to assum- ing the Earth's center as the source location instead of the geographic poles and found that the maximum error implicit for this period is 1.2%. Details of this statistical error analysis are given in Appendix A. This modification gives   2 1 cos 2 Z Z P GS (2) (2) as the received signal of the Z antenna, oriented normal to the ecliptic plane, is minimized when the space- craft is above the poles. This then gives the flux density Z E S for each of the eight spin measurements, which are then averaged using the mean. The synthetically inclined S (and phase-shifted S’) antenna is longer and more sensitive than the Z antenna, making its measurements prone to spurious signals at all frequencies as receiver electronics are saturated. This is often the case when Wind observes the highly intense AKR, which for this study occurs mostly when the spacecraft is near perigee. This contamination does not occur with the shorter, less sensitive Z antenna. While the Z antenna is less prone to saturation, its lower sensitivity means that previous methods of calibra- tion with Wind/WAVES cannot be used. 2.2.2. Calibration To relate the received power of the WAVES instrument to the AKR flux density, we consider the GP tech- nique developed by Manning and Fainberg (1980). GP techniques allow for radio source parameters to be determined by inverse modeling the observations to the radio source parameters using the antenna ref- erence frame and a model of the emission geometry. In their work, spin measurements from a synthetic inclined dipole (here fulfilled by the WAVES S antenna) and a phase-shifted inclined dipole (S’) antenna are demodulated and combined with a spin-axis-aligned (Z) antenna to derive the Stokes parameters (flux density and the degrees of linear and circular polarization), angular coordinates, and the angular radius of the source, so describing the state of a partially polarized extended source. To derive these parameters, it is assumed that a single radio source is observed by the instrument and that the source parameters are constant as the spacecraft completes a spin such that the modulation pattern can be inverted. For AKR this assumption is often broken; either the intensity of the source is variable on timescales lower than that of the spin or the spacecraft is observing emission from multiple sources in each measurement as it changes position during a spin or a combination of the two (see Section 2.3). It is not possible to use the combination of the WAVES antennae in this case as the exact variability over the modulation pattern cannot be deter- mined analytically without a priori knowledge of the source parameters. However, the spin-axis-aligned Z antenna can be used to determine the source flux density after modifications are made to the original GP inversion. We assume that, for an AKR observation, the radio source is a point source and there is no linear polarization. We also assume that the AKR will be circularly polarized, but the received Z antenna power is independent of the source circular polarization using the inversion of Manning and Fainberg (1980). Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 has been determined, the relevant value is subtracted from each of the eight measurements made during a spin period, implicitly assuming isotropy of the background source. If the data are negative, so unphysical, following background subtraction, the background value at the given frequency is stored instead. has been determined, the relevant value is subtracted from each of the eight measurements made during a spin period, implicitly assuming isotropy of the background source. If the data are negative, so unphysical, following background subtraction, the background value at the given frequency is stored instead. 2.2.1. Radio Background The frequency range of the RAD1 receiver is such that background radio emission is present due to both the local plasma environment and non-thermal emission from the galactic center or disk. At lower frequencies (below 300 E kHz), the thermal motion of charged particles in the plasma surrounding the spacecraft cre- ates QTN (Meyer-Vernet & Perche, 1989) while emission from the galaxy dominates at higher frequencies (Cane, 1979; Novaco & Brown, 1978). Previous examination of measurements across the entire WAVES frequency range has consolidated the measured galactic spectrum with previously derived functional forms (Dulk et al., 2001) and more recent measurements across the RAD1 receiver show agreement with a spec- trum that falls off between 100 and 200 kHz (Hillan et al., 2010). Hillan et al. (2010) produced a complete background spectrum, combining models of both the galactic background and a two-component model of the QTN that defines the signal above and below the plasma frequency. As well as these sources of emis- sion, instrumental noise is also present due to the electronics of the antenna system and the digitization of the signal and can be derived from antenna-predeployment measurements; the galactic background (taken from a quiet period above 500 kHz) is no more than double the median RAD1 receiver noise. We assume that QTN at frequencies below 100 kHz will typically dominate the background spectrum. A similar method to that used by Zarka et al. (2004) to determine the galactic background present in the Radio and Plasma Wave Science (RPWS) instrument of the Cassini spacecraft is implemented here for all frequencies. A back- ground spectrum is formed for every 24-hr period of RAD1 data by taking the 5% quantile at each frequency channel. Although no explicitly quiet period is selected over which to take the quantile (as opposed to the method of Zarka et al., 2004), the definition of the quantile imposes that the remaining 95% of received signal is above this level. Although some examples of L2 data contain consistently high emission for the corresponding day at many frequencies, background spectra produced in this way agree well with other measured background levels observed by Wind, as well as producing the expected form due to the QTN and galactic background source described previously (Hillan et al., 2010). Once the background spectrum WATERS ET AL. 4 of 26 Journal of Geophysical Research: Space Physics 2.3. Empirical Selection of AKR As mentioned in Section 2.1, the Wind/WAVES Z antenna is spin-axis aligned. As the spacecraft rotates, antennae in the spin plane observe a modulating signal that is dictated by the rotation period and the position of the antennae with respect to the source location and other parameters. While this modulation can be used to derive source parameters using GP inversion as previously mentioned, these require that the source parameters are constant during a spacecraft rotation; a constraint which is often broken for AKR. The spin-axis aligned Z antenna sees no such modulation due to the spacecraft rotation, and so any varia- bility in the received power can be attributed to changes in the intensity of a radio source, assuming that a single source is observed. It has been mentioned that the spacecraft measures a superposition of multiple spatially separated sources, limiting the observations by the temporal and spectral resolution of the instru- ment. Although this is less true for the spin period than it is for the sweep duration, it is possible that the variability measured by the Z antenna for a spin is due to the more slowly varying intensity of separated sources. Given the generation mechanism of AKR, however, it is likely that a single source will have a high- ly varying amplitude on spin timescales of seconds. While it is nontrivial to discern between the two effects, it is important to note that this may also lead to an overestimation of the AKR selection, although this effect is negligible when data are averaged over the sweep duration. A statistical proxy for AKR can be derived from the Z antenna by modeling the 8 measurements made dur- ing a single spin as a normal distribution centered on a mean intensity, with any variability then being de- scribed by its standard deviation  E . To be able to compare the standard deviation between sources of differ- ent mean intensities, the measurements are normalized by the mean intensity of the spin. This gives Z E , the standard deviation of the spin-normalized Z antenna measurements. Z E is calculated prior to background subtraction, to retain the observed variability (it is assumed that any variability represented by Z E will be predominantly influenced by the sources of the strongest intensity). Figure 1a shows the spin-normalized measurements made by the S and Z antennae during an exemplary AKR burst. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 as given by the received power prior to antenna deployment, so the contribution from the galactic signal cannot be determined. For this reason, the initial measurements of the instrumental characteristics are used to determine the gain using as given by the received power prior to antenna deployment, so the contribution from the galactic signal cannot be determined. For this reason, the initial measurements of the instrumental characteristics are used to determine the gain using          2 2 0 a eff a s C G L Z C C (3) (3) where   0 120 E Z is the impedance of free space, 30 a E C pF and 45 s E C pF are the antenna and stray capacitances, respectively, and eff E L is the effective length of the antenna. Here, we use the physical length ( 4.65 p L m) of each monopole of the Z antenna, given that both electrical monopoles that comprise the Z antenna are in the short-dipole regime, so that  eff p E L L . To check the validity of using calibrated measurements from a linear antenna as proxy for the true flux of a radio source (here AKR), we compared observations of solar radio Type III bursts from Wind and Cassini. A modified calibration routine was prepared and applied to the linear Z antenna for these observations. We compare the flux with independent values produced by a direction-finding inversion, derived from the same initial method presented by Manning and Fainberg (1980) but bespoke to solar Type III burst observations with Wind, giving the flux density, angular coordinates and angular extent of the source with only the S and Z antennae. Initially, these independent measurements served as a scaling factor to be applied to Z E S from Equation 2, using an average spectrum of peak fluxes across a set of Type III bursts. After discussion during the review process, it became clear that this scaling factor is only relevant for Type III bursts, but not for AKR, since it essentially corrects for the size of an extended radio source. AKR can be considered as coming from a point-source when observed at large radial distances, which is why we present Z E S as an accurate (within instrumental uncertainties in the terms of Equation 3) measure of the AKR flux. Journal of Geophysical Research: Space Physics Appendix B gives explicit details of this former processing step and is retained for the interest of the reader. 2.2.2. Calibration These have employed measurements of the galactic background to determine a value of the instrument gain, using a model of the galactic emission to infer the observed flux during a quiet period and then equate this to measurements (Zarka et al., 2004; Zaslavsky et al., 2011). The shorter Z antenna typically measures the galactic background to be within 10 dB of the instrumental noise, WATERS ET AL. 5 of 26 5 of 26 of Geophysical Research: Space Physics 10.1029/2021JA029425 Journal of Geophysical Research: Space Physics 2.3. Empirical Selection of AKR Measurements ( S E P , and Z E P ) made by the synthetic-inclined (S) and spin-axis-aligned (Z) antennae of Wind/WAVES during a single spin, normalized by the average received power during this time ( S E P , Z E P ). (a) Spin observations at 124 kHz, corresponding to a rotation during the burst of Auroral Kilometric Radiation (AKR) emission between roughly 1800 and 2000 UT in panel (c). (b) Spin observations of an isolated Type III burst at 124 kHz, on 1999 day of year (DOY) 232, illustrating the modulation pattern produced in the S antenna and the lack thereof in the Z antenna, observed for measurements of a near-constant intensity source. The value of the selection metric, Z E , is given in the legend of the bottom panel for both (a) and (b). (c) Dynamic spectrograms showing 24 hr (1999 DOY 228) of the power received by the S antenna, in dB, in the top panel. Only observations with a signal-to-noise ratio (SNR) of 25 E dB are shown, for clarity. The bottom panel shows the apparent azimuth of the radio source, determined by the application of a Goniopolarimetric (GP) inversion for use with Wind AKR observations. Only observations with a signal-to-noise ratio (SNR) above 25 dB are shown. The azimuthal angle of the source is given in a non- rotating coordinate system (see text) with angles of  0 E indicating a source in the direction of the Sun, negative angles indicating a source to the right of the Wind-Sun line and positive angles indicating a source to the left of the Wind-Sun line. For the period shown in panel (c), Earth is at approximately −  130 E in this coordinate system. shows similar data for an observation of a solar radio Type III burst, with the double-peaked modulation pattern in the received power clearly visible in the top panel and the Z antenna measuring nearly constant intensity emission; illustrating measurements of a radio source more appropriate for use with a GP in- version. While a comprehensive study of the Z E distribution is not included, examination of the dynamic spectrograms show that the Z antenna consistently measures higher variability (using Z E ) during periods of AKR bursts. 2.3. Empirical Selection of AKR It is not clear from the figure that the S antenna is displaying an insufficient modulation pattern to derive the GP source parameters, and an analytic relationship between the S and Z measurements is not known. However, the variability across the Z antenna measurements shows that the observations do not meet the criteria of constant intensity for GP inversion, and the Z E value for this spin is given in the legend of the bottom panel of Figure 1a. Figure 1b 6 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 1. Measurements ( S E P , and Z E P ) made by the synthetic-inclined (S) and spin-axis-aligned (Z) antennae of Wind/WAVES during a single spin, normalized by the average received power during this time ( S E P , Z E P ). (a) Spin observations at 124 kHz, corresponding to a rotation during the burst of Auroral Kilometric Radiation (AKR) emission between roughly 1800 and 2000 UT in panel (c). (b) Spin observations of an isolated Type III burst at 124 kHz, on 1999 day of year (DOY) 232, illustrating the modulation pattern produced in the S antenna and the lack thereof in the Z antenna, observed for measurements of a near-constant intensity source. The value of the selection metric, Z E , is given in the legend of the bottom panel for both (a) and (b). (c) Dynamic spectrograms showing 24 hr (1999 DOY 228) of the power received by the S antenna, in dB, in the top panel. Only observations with a signal-to-noise ratio (SNR) of 25 E dB are shown, for clarity. The bottom panel shows the apparent azimuth of the radio source, determined by the application of a Goniopolarimetric (GP) inversion for use with Wind AKR observations. Only observations with a signal-to-noise ratio (SNR) above 25 dB are shown. The azimuthal angle of the source is given in a non- rotating coordinate system (see text) with angles of  0 E indicating a source in the direction of the Sun, negative angles indicating a source to the right of the Wind-Sun line and positive angles indicating a source to the left of the Wind-Sun line. For the period shown in panel (c), Earth is at approximately −  130 E in this coordinate system. Figure 1. 2.3. Empirical Selection of AKR Two examples of application of the empirical selection detailed in Section 2.3 to 24 hr of Wind data (3- min resolution). For each example of panel (a) (left) and panel (b) (right), the top panel shows flux density observed by Wind and derived by the method outlined in Section 2.2, the middle panel shows Z E , the statistical proxy of the source amplitude variability, and the bottom panel shows the flux density with the selection mask applied (see Section 2.3). The lower limit of the color bar of the middle panel is set at 2 10 E for visual clarity. The radial distance, ecliptic latitude, and local time for each example are shown in the ephemeris at the bottom of the plot. Figure 2. Two examples of application of the empirical selection detailed in Section 2.3 to 24 hr of Wind data (3- min resolution). For each example of panel (a) (left) and panel (b) (right), the top panel shows flux density observed by Wind and derived by the method outlined in Section 2.2, the middle panel shows Z E , the statistical proxy of the source amplitude variability, and the bottom panel shows the flux density with the selection mask applied (see Section 2.3). The lower limit of the color bar of the middle panel is set at 2 10 E for visual clarity. The radial distance, ecliptic latitude, and local time for each example are shown in the ephemeris at the bottom of the plot. of solar radio Type III bursts (multiple examples between 100 E –1,000 MHz, 0000–0400 UT, or at all frequen- cies after 2000 UT) as well as AKR. Azimuthal angles of  0 E indicate a source in the direction of the Sun, negative angles indicate a source to the right of the Wind-Sun line, and positive angles indicate a source to the left of the Wind-Sun line. The GP inversion is subject to a degeneracy in the wave vector direction, so angles that suggest an unphysical source direction for AKR have been transformed appropriately; Earth is at approximately  130 E for the data shown. For the AKR between 0800 and 2000 UT, the azimuthal angle is poorly constrained and has high variability during AKR observations due to the aforementioned variability during the spacecraft rotation. 2.3. Empirical Selection of AKR To select regions that correspond to AKR emission, a numerical threshold is chosen based on the visual identification of the dynamic spectrograms from Wind during the Cassini flyby; here  0.1 Z E . Given that the opposite limit of Z E selects sources that are less variable, Z E could be useful to select data that are more appropriate for a GP inversion with Wind. See Appendix C for further justification of the choice of the threshold value. Figure 1c shows 24 hr dynamic spectrograms of Wind AKR observations and their general unsuitability for GP applications. The top panel shows a spectrogram of the measurements made by the S antenna; relatively faint Type III bursts are seen throughout the day, whilst bursts of intense AKR are also observed. The bot- tom panel shows the result of an attempt to apply the GP inversion of Manning and Fainberg (1980) to the Wind observations. The azimuthal angle of the source is given in a fixed coordinate system, independent of the spacecraft rotation that is defined by an x axis in the direction of the Sun with the x- and y axes in the ecliptic plane, and a z axis that completes the orthonormal frame and is directed Southward of the ecliptic plane. Given that the retrieval of direction-finding parameters for radio sources is dependent on a strong signal, only observations with a signal-to-noise ratio (SNR) of 25 E dB are shown, highlighting the presence WATERS ET AL. WATERS ET AL. 7 of 26 Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 2. Two examples of application of the empirical selection detailed in Section 2.3 to 24 hr of Wind data (3- min resolution). For each example of panel (a) (left) and panel (b) (right), the top panel shows flux density observed by Wind and derived by the method outlined in Section 2.2, the middle panel shows Z E , the statistical proxy of the source amplitude variability, and the bottom panel shows the flux density with the selection mask applied (see Section 2.3). The lower limit of the color bar of the middle panel is set at 2 10 E for visual clarity. The radial distance, ecliptic latitude, and local time for each example are shown in the ephemeris at the bottom of the plot. Figure 2. 2.3. Empirical Selection of AKR While Z E acts as a proxy for the radio source here, we note that without ac- cess to the GP inversion and polarization information, we cannot make an exact physical inference and so the selection is empirical. With average Z E spectra for each sweep, a mask can then be created and applied to the calibrated flux densities to select data that meet this criteria; here the flux densities are also averaged across the 3- min sweep cycle. In this case, the consideration of multiple spatially separated sources cannot be ignored, and the flux spectra represent the spatial average of AKR emission across a relatively wide lon- gitude as well as a temporal average. Figure 2 shows two examples, (a) and (b), of 24 hr of calibrated Wind/WAVES data (top panel) with the variability proxy Z E (middle panel) and the application of the resulting mask (bottom panel). Wind obser- vations in both (a) and (b) contain many Solar Type III bursts with various drift rates that at times cover the entire RAD1 frequency range, as well as increases in QTN and emission at the plasma frequency at fre- quencies  E 100 kHz. The middle panel shows the effectiveness of Z E as an identifier of AKR emission, with observations of Type III bursts exhibiting a standard deviation less than that of the AKR by about an order of magnitude. AKR is generally more intense than Type III bursts, and this couples with the aforemen- tioned considerations of the AKR generation mechanism and viewing geometry to produce the observed discrepancy in the Z E distributions of each source, as shown here by the middle panel of Figure 2. While not shown here, the 52 kHz channel has persistently higher Z E due to radio frequency interference (RFI). In Figure 2 (and Figure 5), the 52 kHz channel has been removed and the values of Z E in neighboring channels used to interpolate an updated “background” value to increase visual clarity. For the remaining analysis, after selecting the AKR data with the Z E criterion, flux densities from the 52 kHz channel are removed to avoid contamination. AKR can be seen in both examples; in (a), Wind is approaching perigee at around WATERS ET AL. 2.3. Empirical Selection of AKR 8 of 26 Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 1600–1700 LT and  4 E latitude, and emission is observed for 11 hr between   80 800 E kHz; it exits perigee in (b), crossing 0800–0900 LT around  6 E latitude and observing more sporadic emission patches throughout the day across a narrower frequency range,   100 500 E kHz. Given that the metric for selection, Z E is determined using the variance of measurements made during a spin, there are other sources of emission that could be responsible for the retention of a particular sample. Instru- mental RFI is an example of this, but particularly rapid changes in in- tensity due to energetic fluctuations in the local plasma can also produce signatures similar to those produced by AKR. Particularly when Wind travels through the turbulent, dense plasma in the magnetosheath (see  E DOY 229–231 and 251–254 in Figure 5), the majority of the emission at lower frequencies is retained. At times when Wind is on the dayside, within the solar wind, emission is occasionally selected that has no ob- served, corresponding AKR signal at higher frequencies and is assumed to be caused by similar local and temporal variations in the plasma. Inten- sification of the signal at other characteristic frequencies of the plasma are also occasionally seen and retained by the selection, such as emission at the plasma frequency. The retention of non-AKR signals is lessened at higher frequencies, where Wind typically sees contributions from AKR, Jovian hectometric or kilometric radiation, which is often faint (Zarka et al., 2004), or solar radio bursts that have little variability across the spacecraft spin period. As well as other sources of emission that could be responsible for the variability, there is also the possibility that Wind is subject to incident waves from multiple radio sources. Due to this, it is not guaranteed that only AKR will be present in any of the selected data, although the most intense emission determines the modulation pattern in these cases. AKR is typically more intense than other radio sources for Wind observations, and this is the case particularly when Wind is at a favorable viewing position for AKR. Observations where solar radio type III bursts are the most intense emission arriving at the Z antenna, for example, are thus removed using the selection with Z E . 2.3. Empirical Selection of AKR The former is given by the model of Shue et al. (1998), using solar wind data from OMNI with average parameters. The latter surface is derived using similar data and the model of Wu et al. (2000). 2.3. Empirical Selection of AKR AKR is typically more intense than other radio sources for Wind observations, and this is the case particularly when Wind is at a favorable viewing position for AKR. Observations where solar radio type III bursts are the most intense emission arriving at the Z antenna, for g the selection with Z E . Although fine structure and variability has been II bursts and there is no complete way to discriminate between this vari- AKR observations, applications of the selection such as those in Figure 2 ns where the Type III dominates are removed. Given the ubiquity of Type ess variable emission and the simplicity of the Z E selection criteria used dying AKR emission, as shown in the following section and conversely for R in studies of solar radio bursts. Applying the flux-density calibration and S observations since 1994 can form a unique data set to investigate AKR takes advantage of the selection to make an initial comparison to AKR Cassini in 1999 Figure 3. Orbital trajectory of Wind for 1999 day of year (DOY) 227–257, projected onto the ecliptic plane. The start of each day for the Wind trajectory is marked by crosses. Also shown in gray is the Cassini trajectory for the closest approach, with the start of DOY 230 marked by the triangle. The Cassini trajectory can be found to a greater extent in figure 2a of Lamy et al. (2010). The arrows indicate the direction of travel for both spacecraft and the colored markers indicate the start of DOY 230. Average modeled magnetopause and bowshock surfaces are shown as thin solid and dashed lines, respectively; the Sun is to the right. The former is given by the model of Shue et al. (1998), using solar wind data from OMNI with average parameters. The latter surface is derived using similar data and the model of Wu et al. (2000). trajectory is marked by crosses. Also shown in gray is the Cassini trajectory for the closest approach, with the start of DOY 230 marked by the triangle. The Cassini trajectory can be found to a greater extent in figure 2a of Lamy et al. (2010). The arrows indicate the direction of travel for both spacecraft and the colored markers indicate the start of DOY 230. Average modeled magnetopause and bowshock surfaces are shown as thin solid and dashed lines, respectively; the Sun is to the right. 2.3. Empirical Selection of AKR Although fine structure and variability has been observed in the spectra of Type III bursts and there is no complete way to discriminate between this vari- ability and that exhibited during AKR observations, applications of the selection such as those in Figure 2 show that, on average, observations where the Type III dominates are removed. Given the ubiquity of Type III bursts and sources of other less variable emission and the simplicity of the Z E selection criteria used here, the technique is utile for studying AKR emission, as shown in the following section and conversely for removing the contribution of AKR in studies of solar radio bursts. Applying the flux-density calibration and selection of AKR to Wind/WAVES observations since 1994 can form a unique data set to investigate AKR characteristics. The next section takes advantage of the selection to make an initial comparison to AKR radio measurements obtained by Cassini in 1999. or 1999 day of year (DOY) 227–257, start of each day for the Wind hown in gray is the Cassini trajectory of DOY 230 marked by the triangle. a greater extent in figure 2a of Lamy irection of travel for both spacecraft tart of DOY 230. Average modeled are shown as thin solid and dashed ht. The former is given by the model data from OMNI with average ed using similar data and the model E E Figure 3. Orbital trajectory of Wind for 1999 day of year (DOY) 227–257, projected onto the ecliptic plane. The start of each day for the Wind trajectory is marked by crosses. Also shown in gray is the Cassini trajectory for the closest approach, with the start of DOY 230 marked by the triangle. The Cassini trajectory can be found to a greater extent in figure 2a of Lamy et al. (2010). The arrows indicate the direction of travel for both spacecraft and the colored markers indicate the start of DOY 230. Average modeled magnetopause and bowshock surfaces are shown as thin solid and dashed lines, respectively; the Sun is to the right. The former is given by the model of Shue et al. (1998), using solar wind data from OMNI with average parameters. The latter surface is derived using similar data and the model of Wu et al. (2000). 2.3. Empirical Selection of AKR E E 1600–1700 LT and  4 E latitude, and emission is observed for 11 hr between   80 800 kHz; it exits perigee in (b), crossing 0800–0900 LT around  6 E latitude and observing more sporadic emission patches throughout the day across a narrower frequency range,   100 500 E kHz. 1600–1700 LT and  4 E latitude, and emission is observed for 11 hr between   80 800 kHz; it exits perigee in (b), crossing 0800–0900 LT around  6 E latitude and observing more sporadic emission patches throughout the day across a narrower frequency range,   100 500 E kHz. Given that the metric for selection, Z E is determined using the variance of measurements made during a spin, there are other sources of emission that could be responsible for the retention of a particular sample. Instru- mental RFI is an example of this, but particularly rapid changes in in- tensity due to energetic fluctuations in the local plasma can also produce signatures similar to those produced by AKR. Particularly when Wind travels through the turbulent, dense plasma in the magnetosheath (see  DOY 229–231 and 251–254 in Figure 5), the majority of the emission at lower frequencies is retained. At times when Wind is on the dayside, within the solar wind, emission is occasionally selected that has no ob- served, corresponding AKR signal at higher frequencies and is assumed to be caused by similar local and temporal variations in the plasma. Inten- sification of the signal at other characteristic frequencies of the plasma are also occasionally seen and retained by the selection, such as emission at the plasma frequency. The retention of non-AKR signals is lessened at higher frequencies, where Wind typically sees contributions from AKR, Jovian hectometric or kilometric radiation, which is often faint (Zarka et al., 2004), or solar radio bursts that have little variability across the spacecraft spin period. As well as other sources of emission that could be responsible for the variability, there is also the possibility that Wind is subject to incident waves from multiple radio sources. Due to this, it is not guaranteed that only AKR will be present in any of the selected data, although the most intense emission determines the modulation pattern in these cases. 3.1. Spacecraft Ephemerides Entering the nightside magnetosphere for the second time during the period, Wind reached a second perigee on DOY 252 at 0000 LT and close to the ecliptic plane. Wind then exited the magnetosphere once more, and the final observations that are conjunct with Cassini are made at around 1000 LT with GSE latitude of   3.0 E and a radial distance of 67 E E R . As mentioned in Section 1, GP inversions have been successfully applied to observations from Cassini and allowed the circular polarization of the radio emission to be determined and thus the hemisphere of origin. For the empirical selection of AKR emission used here with Wind, there is no unambiguous way to deter- mine circular polarization from flux measurements. Due to the anisotropic, widely beamed emission from the AKR source regions, visibility of the emission from either pole is highly dependent on the magnetic lat- itude of Wind and inferences of the origin of the emission can be made based on this. While Wind is on the dayside, approximately between the two perigees at DOY 231 and DOY 252, it covers a range of low magnet- ic latitudes in first the Southern then the Northern magnetic hemispheres, crossing the magnetic equator near the apogee (see Figure 4). In both cases, where Wind crosses the nightside and where AKR is expected to be most visible, the spacecraft approaches from the dusk flank in the Northern magnetic hemisphere and crosses into the Southern magnetic hemisphere at perigee. Magnetic coordinates are as defined in Lamy et al. (2010), with positive magnetic latitudes in the Southern geographic (Northern magnetic) hemisphere. The magnetic coordinate system used here has the z-direction oppositely defined to that in solar magnetic (SM) coordinates. Given that the magnetic latitude does not exceed  30 E , it is uncertain whether or not the spacecraft will be in either or both of the regions illuminated by emission from either hemisphere. Previous examination of the average AKR source region has suggested that emission from both hemispheres can be observed at distances 12 E E R in the equatorial plane (Gallagher & Gurnett, 1979), with the approximate perigee distance of Wind ( 13 E E R ) implying that this will be the case for all nightside observations for this period. In their study, Lamy et al. 3.1. Spacecraft Ephemerides Figure 3 shows the Wind trajectory projected onto the ecliptic plane for the period studied here, as well as part of the Cassini trajectory. The start of day of year (DOY) 230 is indicated in color for each spacecraft, during which Cassini reached its closest approach to Earth at a radial distance of 1.2 E E R . After traversing the dawn flank of the magnetosphere and passing Earth, Cassini continued downtail between 0100 and 0200 LT at 9 R h E / . Figure 4 shows the latitudes of both spacecraft in both geocentric solar ecliptic (GSE) and magnetic coordinates. The latter refers to the coordinate system used in Lamy et al. (2010), which defines positive latitudes as those in the Northern magnetic (Southern rotational) hemisphere. 9 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 4. Latitudes of the Wind (a) and Cassini (b) spacecraft for the period shown in geocentric (GSE) coordinates with the gray, dashed line and magnetic coordinates with the black, solid line. Magnetic coordinates are as defined in Lamy et al. (2010), with positive magnetic latitudes in the Southern geographic (Northern magnetic) hemisphere. The magnetic coordinate system used here has the z-direction oppositely defined to that in solar magnetic (SM) coordinates, indicated by   SM E in the legend. Markers show the beginning of each day. Figure 4. Latitudes of the Wind (a) and Cassini (b) spacecraft for the period shown in geocentric (GSE) coordinates with the gray, dashed line and magnetic coordinates with the black, solid line. Magnetic coordinates are as defined in Lamy et al. (2010), with positive magnetic latitudes in the Southern geographic (Northern magnetic) hemisphere. The magnetic coordinate system used here has the z-direction oppositely defined to that in solar magnetic (SM) coordinates, indicated by   SM E in the legend. Markers show the beginning of each day. During Cassini's flyby of Earth and the 30- day period studied by Lamy et al. (2010), from August 15 to September 14, 1999 (DOY 227–257), Wind completed close to two petal orbits with a perigee radii of 13 E E R and apogee radii of 88 E E R . 3.1. Spacecraft Ephemerides At the start of this period, Wind approached its first perigee from a position duskward of Earth at roughly 1500 LT on DOY 227, with a GSE latitude of  3.0 E and a radial distance of 67 E E R . From there, Wind approached the magnetosphere and crossed the bow shock and magnetopause before the first perigee was reached. Wind reached perigee around 0100 LT, at a GSE latitude of  1 E , while traversing the magnetotail. Wind then exited the magnetosphere, covering the dawn flank and reaching apogee on DOY 241 at a GSE latitude of   0.4 E around 1200 LT. After 23 days, Wind reached 1500 LT once more at a closer ra- dial distance of 44 E E R and a GSE latitude of  4.6 E . Entering the nightside magnetosphere for the second time during the period, Wind reached a second perigee on DOY 252 at 0000 LT and close to the ecliptic plane. Wind then exited the magnetosphere once more, and the final observations that are conjunct with Cassini are made at around 1000 LT with GSE latitude of   3.0 E and a radial distance of 67 E E R . During Cassini's flyby of Earth and the 30- day period studied by Lamy et al. (2010), from August 15 to September 14, 1999 (DOY 227–257), Wind completed close to two petal orbits with a perigee radii of 13 E E R and apogee radii of 88 E E R . At the start of this period, Wind approached its first perigee from a position duskward of Earth at roughly 1500 LT on DOY 227, with a GSE latitude of  3.0 E and a radial distance of 67 E E R . From there, Wind approached the magnetosphere and crossed the bow shock and magnetopause before the first perigee was reached. Wind reached perigee around 0100 LT, at a GSE latitude of  1 E , while traversing the magnetotail. Wind then exited the magnetosphere, covering the dawn flank and reaching apogee on DOY 241 at a GSE latitude of   0.4 E around 1200 LT. After 23 days, Wind reached 1500 LT once more at a closer ra- dial distance of 44 E E R and a GSE latitude of  4.6 E . 3.1. Spacecraft Ephemerides (2010) use the polarisation information to assess the average AKR power 10 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Journal of Geophysical Research: Space Physics Geophysical Research: Space Physics 10.1029/2021JA029425 10.1029/2021JA029425 Figure 5. Dynamic spectrogram showing the flux density measured by Wind/WAVES (top) and Cassini (bottom) for 1999 day of year (DOY) 227–257 and normalized to a distance of 1 AU. The top panel shows the average flux density at a 3- min resolution as selected with the Z E threshold described in Section 2.3. The flux density is computed by calibrating the power received by the spin-axis aligned Z antenna, as outlined in Section 2.2.2. The bottom panel shows the flux density observed by the Cassini spacecraft, namely the maximum of the left-handed circularly polarized or right-handed circularly polarized Auroral Kilometric Radiation (AKR) emission of a given frequency at a 90- s resolution (for complete details of the calibration and selection of AKR with Cassini, see Lamy et al., 2010). The radial distance, ecliptic latitude, and local time of each spacecraft are shown in the ephemeris at the bottom of each panel. Figure 5. Dynamic spectrogram showing the flux density measured by Wind/WAVES (top) and Cassini (bottom) for 1999 day of year (DOY) 227–257 and normalized to a distance of 1 AU. The top panel shows the average flux density at a 3- min resolution as selected with the Z E threshold described in Section 2.3. The flux density is computed by calibrating the power received by the spin-axis aligned Z antenna, as outlined in Section 2.2.2. The bottom panel shows the flux density observed by the Cassini spacecraft, namely the maximum of the left-handed circularly polarized or right-handed circularly polarized Auroral Kilometric Radiation (AKR) emission of a given frequency at a 90- s resolution (for complete details of the calibration and selection of AKR with Cassini, see Lamy et al., 2010). The radial distance, ecliptic latitude, and local time of each spacecraft are shown in the ephemeris at the bottom of each panel. from each hemisphere when Cassini is above a given magnetic latitude finding that LH AKR was close to 4 orders of magnitude weaker than the RH AKR when the spacecraft had a magnetic latitude    10 m E . 3.1. Spacecraft Ephemerides This suggests that Wind is likely illuminated by emission from AKR sources in both hemispheres whilst near the equatorial plane, with Southern, LH emission likely to be dominant prior to perigee and Northern, RH emission afterward. 3.2. AKR Flux Density and Power Figure 5 shows the AKR flux density of both Wind and Cassini for the entire 30- day period studied here. The AKR flux density with Wind is obtained using the calibration and selection outlined in Section 2. The AKR flux density with Cassini contains that of both LH- and RH-circularly polarized AKR, obtained using a GP inversion technique and selecting data with \| \| . V 0 2 , where E V is the normalized Stokes parameter describing circular polarization (Lamy et al., 2010). The maximum of either the LH or RH AKR is shown at 90 s resolution in the Cassini spectrogram. Both flux densities are scaled for the distance from the ap- proximate source (Earth's center) and normalized to 1 AU to enable a comparison between the two data sets. The general effect of viewing position on the AKR observations from each spacecraft can be seen here; emission is stronger and more consistent for Cassini as it travels away from Earth downtail, remaining on the nightside, while a periodic variability is seen in the Wind spectrogram as it passes the nightside during its perigees. While the day-to-day variability in the observed emission is not studied here, it is interesting to note differences between the two perigee observations. Wind sees more persistent, stronger emission for the first perigee (  E DOY 229) than the second (  E DOY 251). While this variability between orbits is not surpris- ing, given the changing solar wind conditions that affect AKR, it demonstrates the differences between ob- servations made at different times, which will clearly bias any average result with a limited selection of data. Figure 6 shows AKR flux density spectra from both spacecraft given by statistical thresholds, namely the median spectra and those for the 90% and 99% quantiles or increasing intensity thresholds. The top pan- el shows Cassini flux densities for both LH and RH circularly polarized AKR, reproducing figure 6a of 11 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 6. Reduced flux density spectra comparing Auroral Kilometric Radiation (AKR) observations from Cassini during the whole period (top panel) with those made by Wind when in a comparable viewing position (middle and bottom panels). For this, the Wind data were further selected such that the spacecraft was located from      23 16 m and within 0000–0200 hr LT. 3.2. AKR Flux Density and Power Flux density data in each case are reduced to give the measured intensity that reached 50% (median — black), 10% (red), and 1% (orange) of the time. The top panel shows spectra for both the LH and RH circularly polarized AKR as given by the GP inversion applied to Cassini. The middle panel shows AKR- and position-selected data from Wind during the 30-day period studied here. The bottom panel shows AKR- and position-selected data from Wind for all of 1999, to increase the statistical rigor of the selection verification (see main text). Figure 6. Reduced flux density spectra comparing Auroral Kilometric Radiation (AKR) observations from Cassini during the whole period (top panel) with those made by Wind when in a comparable viewing position (middle and bottom panels). For this, the Wind data were further selected such that the spacecraft was located from      23 16 m and within 0000–0200 hr LT. Flux density data in each case are reduced to give the measured intensity that reached 50% (median — black), 10% (red), and 1% (orange) of the time. The top panel shows spectra for both the LH and RH circularly polarized AKR as given by the GP inversion applied to Cassini. The middle panel shows AKR- and position-selected data from Wind during the 30-day period studied here. The bottom panel shows AKR- and position-selected data from Wind for all of 1999, to increase the statistical rigor of the selection verification (see main text). Lamy et al. (2010). The middle panel shows Wind flux densities returned by the selection (described in Section 2.3) applied to data from 1999 DOY 227–257. Given the anisotropy of the AKR beaming and the longitudinal distribution of AKR source regions, it cannot be assumed that each spacecraft will observe the same emission region at a given time from different viewing positions in space. To elicit a valid comparison of the reduced spectra between the spacecraft, we select Wind data such that only observations made from a similar viewing position are included; Cassini was in the region with a magnetic latitude      23 16 m E and within 0000–0200 hr LT during its flyby. A complete discussion of the flux densities observed by Cassini can be found in Lamy et al. (2010), but here we compare the main features with those of Wind. Lamy et al. (2010). Journal of Geophysical Research: Space Physics Again comparing the Wind spectra in the middle panel with Cassini in the top panel of Figure 6, the peak of the Wind median spectrum agrees, existing at a frequency close to 200 kHz and between the peak flux of the Cassini LH and RH AKR median spectra; also in agreement with initial AKR observations (Kaiser & Alexander, 1977). While the differences at lower frequencies are likely due to contamination, the Wind median spectrum falls off more rapidly than the RH median spectrum of Cassini and is more comparable to the LH median spectrum. At frequencies 700 E kHz, the Wind median spectrum is more closely comparable to the RH AKR median spectrum, although the aforementioned limitations prevent close physical interpre- tation of the differences or similarities between the spectra. Generally, however, the Wind median spectrum has a minimum at the highest observed frequencies, which again agrees with the Cassini measurements. Each of the Wind spectra that show the higher intensity thresholds in the middle panel show generally good agreement in magnitude with those of Cassini; a similar increase of 2 orders of magnitude between the median and highest intensity spectra is seen in the selected Wind data. This is interesting considering the small amount of time Wind spent in the region relative to Cassini and suggests that the limited Wind measurements here are characteristic of the AKR that Cassini observes for the whole period. There is also evidence of broadening of the spectral peaks to higher frequencies with increasing intensity in the middle panel as observed by Cassini. Given that Wind spends the least amount of time on the nightside during perigee for this period, it is impor- tant to consider the limiting effect of the position selection. To highlight this, while Wind spends 1.2% E of the time in the specified region during this 30- day period, 5.1% of the AKR data selected here is observed in this region. However, this increase shows the efficacy of the empirical selection in reflecting the preferential location of the nightside for observing AKR emission and the AKR sources themselves. Although we cannot compare the Cassini spectra with Wind observations made outside of the temporal range covered by the Cassini flyby, increasing the scope of the data included can allow us to characterize the selected data more rigorously by comparing the general features of the spectra. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 the viewing of high altitude, low frequency AKR sources. The Z E selection is limited at low frequencies due to contamination by QTN and local plasma waves; although we assume that the aforementioned effect dominates, given that Wind is likely inside the magnetosphere for the selected observations (as shown by the average magnetopause boundary in Figure 3), where these emissions have less of a presence than in other regions. Figure 6 also shows better agreement between the two spacecraft at 100 E kHz, the resulting range including the typical spectral peak of AKR. The AKR emission at higher frequencies is known to be more temporally consistent and can be said to be better representative of the average AKR signal compared between remotely observing spacecraft. For these reasons, in the following, only the selected signal above 100 kHz is considered. At frequencies higher than this, discrepancies between the spectra may exist simply due to the two spacecraft primarily observing different AKR source regions as we do not expect the AKR spectrum to be constant at all LT. the viewing of high altitude, low frequency AKR sources. The Z E selection is limited at low frequencies due to contamination by QTN and local plasma waves; although we assume that the aforementioned effect dominates, given that Wind is likely inside the magnetosphere for the selected observations (as shown by the average magnetopause boundary in Figure 3), where these emissions have less of a presence than in other regions. Figure 6 also shows better agreement between the two spacecraft at 100 E kHz, the resulting range including the typical spectral peak of AKR. The AKR emission at higher frequencies is known to be more temporally consistent and can be said to be better representative of the average AKR signal compared between remotely observing spacecraft. For these reasons, in the following, only the selected signal above 100 kHz is considered. At frequencies higher than this, discrepancies between the spectra may exist simply due to the two spacecraft primarily observing different AKR source regions as we do not expect the AKR spectrum to be constant at all LT. 3.2. AKR Flux Density and Power The middle panel shows Wind flux densities returned by the selection (described in Section 2.3) applied to data from 1999 DOY 227–257. Given the anisotropy of the AKR beaming and the longitudinal distribution of AKR source regions, it cannot be assumed that each spacecraft will observe the same emission region at a given time from different viewing positions in space. To elicit a valid comparison of the reduced spectra between the spacecraft, we select Wind data such that only observations made from a similar viewing position are included; Cassini was in the region with a magnetic latitude      23 16 m E and within 0000–0200 hr LT during its flyby. A complete discussion of the flux densities observed by Cassini can be found in Lamy et al. (2010), but here we compare the main features with those of Wind. While the Cassini spectra for both LH and RH AKR fluxes see a mostly shallow increase up to the peak at 200 kHz, this is not the case for Wind. A consistent plateau is seen in each of the spectra in the middle panel below 50 kHz, after which the flux density increases more sharply for each quantile. Discrepancies in the spectra can be seen at lower frequencies, which is likely due to a combination of the nature of low frequency AKR emission and the fragmented viewing by Wind when in the appropriate position. While Cassini makes 30 days of remote observations in this magnetic latitude and LT range, Wind spends  E 9 hr in the same position, with each observation  E 20 hr apart. Without considering the viewing effects, the transience of AKR below 100 kHz alone could produce the discrepancies in the average spectra. As well as this, Wind is closer Earth and passes the nightside while crossing the magnetic equator, which could affect 12 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 3.3. AKR Viewing Geometry Figure 7 shows the integrated power data described in the previous Sec- tion 3.2 after taking the mean power in 1 hr spacecraft LT (GSE) bins and plotting the 10 log E power as a rose plot; the noon and midnight sectors are on the right and left of the figure, respectively, and the dawn and dusk sectors are at the bottom and on the top of the figure, respectively. A large asymmetry can be seen in the selected power with a 3–4 order of mag- nitude increase of the LT bins between 1700 and 0300 over those on the dayside that have the lowest powers, namely the LT sector at 1100 hr. The broad picture of Figure 7 is thus consistent with previous findings, which suggest that the source regions are located on the nightside and beam ani- sotropically (Alexander & Kaiser, 1976; Gallagher & Gurnett, 1979; Mutel et al., 2004, 2008). Also of interest here are the bins at 2100 and 2200 hr LT, in the center of the range of intense emission. This corresponds to previous observations of the LT of the source regions most favored by AKR sources (Mutel et al., 2004; Panchenko, 2003), as well as the average LT of the most intense AKR source region at 2200 MLT (Green, 2004). Given the illumination region of an AKR source, and that the observations here are made from comparatively large radial distances and with a swept frequency receiver, it is expected that the emission from an AKR source could be measured by Wind from a neighboring LT sector. At Saturn, this longitudinal difference has been observed to be up to 2 hr LT (Kimura et al., 2013; Lamy, Zarka, Cecconi, Prangé, et al., 2008). The location of the centroid of the most intense average power in Figure 7 is an indication of the effectiveness of the AKR selection used here. ted across the range 100–650 kHz AKR) flux (as selected by 2.3) and wide. The sun and noon sector is to to the left. Colors show the number etained by the empirical selection in Figure 7. Log power in 1 E Wsr integrated across the range 100–650 kHz for the Auroral Kilometric Radiation (AKR) flux (as selected by 2.3) and averaged in local time (LT) bins 1 hr wide. The sun and noon sector is to the right, while the midnight sector is to the left. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 E Figure 7. Log power in 1 E Wsr integrated across the range 100–650 kHz for the Auroral Kilometric Radiation (AKR) flux (as selected by 2.3) and averaged in local time (LT) bins 1 hr wide. The sun and noon sector is to the right, while the midnight sector is to the left. Colors show the number of 3-minute-resolution observations, retained by the empirical selection in each LT bin. the integrated powers of Wind and Cassini, it enables general characteris- tics of the AKR, such as the viewing geometry and temporal modulations, to be studied. With a more refined selection of AKR signal at frequencies below 100 kHz, the power can be integrated over a frequency range such as 30–100 kHz to investigate the lower frequency AKR component. Given that the integration time of each flux density measurement is the spin period of the spacecraft, the power can be integrated by simply taking the mean of the flux densities and integrating over the frequency channels. The flux densities have been normalized to 1 AU, so this distance is used as the effective area for the integration. Although we derive the AKR flux as that from a point source, the AKR is emitted in a wide geometry and so we present the AKR power as a fraction of the true beaming in units of 1 Wsr . Journal of Geophysical Research: Space Physics For this reason, the bottom panel of Figure 6 shows spectra defined by the same thresholds but applied to Wind data from the entirety of 1999 after select- ing the AKR as described. Although the magnitude of the spectra is lower (which is expected as more AKR emission is included, assuming that the more extreme events will happen less regularly), the broadening of the spectral peak to higher frequencies with increasing intensity is present. There is a larger increase of 3 orders of magnitude between the median and highest quantile spectra in the bottom panel, suggesting that the observations made in the 30- day period here are more intense than other times in the same year. There is also a separate, much shorter peak around 30–40 kHz that exists in the highest intensity spectra of the bottom panel, which could be indicative of the average state of the magnetosphere or solar wind throughout 1999 as consistent Langmuir wave excitations may be seen close to the local plasma frequency in this region. The AKR source region, as discussed in Section 1, is typically found at altitudes of roughly 2,000–10,000 km along a given field line, corresponding to emission at frequencies 100–800 kHz. This higher frequency emis- sion is much less transient than that of higher altitude sources that emit between   30 100 E kHz and are well correlated with substorm onset (Morioka et al., 2007). To characterize the AKR observed by Wind and facilitate the comparison between the two spacecraft, the selected flux data are integrated over the frequen- cy range 100–650 kHz. While the frequency range used by Lamy et al. (2010) (30–650 kHz) encompasses both the lower frequency and main band of AKR, we increase the lower frequency limit here to mitigate the inclusion of spurious data as mentioned above. While this does not allow for a direct comparison between WATERS ET AL. 13 of 26 Journal of Geophysical Research: Space Physics Journal of Geophysical Research: Space Physics Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 E Figure 8. Distribution of Auroral Kilometric Radiation (AKR) power (100–650 kHz) with magnetic latitude. The color bar shows the local time (LT) of Wind in geocentric-solar-ecliptic (GSE) coordinates, with observations in the Northern magnetic hemisphere made from the dusk flank, prior to perigee, and those in the Southern hemisphere from the dawn sector. The top panel shows all selected observations from day of year (DOY) 227–257, whilst the bottom panel shows a subset of observations made between 1900 and 0100 LT to account for poor viewing. The dashed black line shows the mean of the log-power after binning in 5° wide latitude bins. The solid black lines show linear-log fits to the average data for negative and positive magnetic latitudes, where we expect dominant emission from left-handed circularly polarized (LH) to right- handed circularly polarized (RH) AKR, respectively. The legend indicates the coefficients (in ascending rank order) of the linear-log fit of the form shown in Equation 4. The dotted black line in each panel shows the analytic form found for Cassini RH AKR observations (see Equation 4; A  4.2, B = 0.29) (Lamy et al., 2010). E LT 0600 ) are retained by the selection. The variation in the number of selected data in the noon sector in Figure 7 shows that the distribution here may not be indicative of the true average AKR power, as temporal variability of the state of the magnetosphere system will bias this count as the spacecraft crosses the dayside once during the 30- day period studied here. As discussed in Section 3.1, Wind crosses the nightside twice during this period, so the data contributing to the average on the nightside are comprised of two separate samples of this region. Further discussion of the power distributions that produced the averages for each LT bin can be found in Appendix D. In addition to the viewing constraints introduced by the interaction of the terrestrial magnetosphere with the solar wind in Figure 7, the latitu- dinal distribution of AKR sources is such that we expect better viewing of AKR from individual magnetic hemispheres at higher latitudes. Journal of Geophysical Research: Space Physics Figure 8 shows the distribution of AKR power with magnetic latitude with the LT of Wind indicated; while Wind was at relatively low magnetic latitudes for the period, it approached the nightside from the dusk flank at increas- ingly higher magnetic latitudes in the Northern magnetic hemisphere before crossing the magnetic equator and traveling to mid-latitudes as it leaves via the dusk flank. This can be seen in the top panel of Figure 8, which shows all selected data for the period. While powers were often observed as low as  2 1 10 Wsr E for all LT, the azimuthal viewing constraints of AKR are also seen as the average AKR power in the Southern magnetic hemisphere is relatively constant, indicating that Wind is not best situat- ed to observe the most intense LH emission as it travels to the dayside, as shown also in Figure 7. The bottom panel of Figure 8 shows a smaller subset of the selected AKR data and attempts to mitigate the effect of the LT viewing, with only observations made between 1900 and 0100 LT included. While this LT range is slightly more restrictive than suggested by the distribution in Figure 7, it is chosen for a better characterization of the AKR for these observations. In both plots, the mean of the log-power observations made in 5° wide magnetic latitude bins is shown, with a linear-logarithmic fit of the form used in equation 1 of Lamy et al. (2010), repeated here Figure 8. Distribution of Auroral Kilometric Radiation (AKR) power (100–650 kHz) with magnetic latitude. The color bar shows the local time (LT) of Wind in geocentric-solar-ecliptic (GSE) coordinates, with observations in the Northern magnetic hemisphere made from the dusk flank, prior to perigee, and those in the Southern hemisphere from the dawn sector. The top panel shows all selected observations from day of year (DOY) 227–257, whilst the bottom panel shows a subset of observations made between 1900 and 0100 LT to account for poor viewing. The dashed black line shows the mean of the log-power after binning in 5° wide latitude bins. The solid black lines show linear-log fits to the average data for negative and positive magnetic latitudes, where we expect dominant emission from left-handed circularly polarized (LH) to right- handed circularly polarized (RH) AKR, respectively. Journal of Geophysical Research: Space Physics The legend indicates the coefficients (in ascending rank order) of the linear-log fit of the form shown in Equation 4. The dotted black line in each panel shows the analytic form found for Cassini RH AKR observations (see Equation 4; A  4.2, B = 0.29) (Lamy et al., 2010).    , log W m P A B (4) (4) where E P is the AKR power for a given hemisphere (e.g., LH or RH emis- sion) in 1 Wsr E , and  , W m E is the magnetic latitude of Wind in degrees. Lamy et al. (2010) found, using the polarization to characterize each hemisphere, that the average emission from the Northern magnetic hem- isphere, when integrated over 15 minutes, gave values A  E 4.2, B = 0.29. Close to the magnetic equator, it is expected that AKR from both hem- ispheres will be observed, but their observations also showed that RH emission can be observed at   2 1 10 Wsr E from magnetic latitudes of up to  5 E in the opposite hemisphere of origin (Southern). Without the polar- ization state we cannot discern the exact origin of emission, so the fit in Equation 4 is performed on data subset by negative or positive magnetic latitude, corresponding to assumed LH and RH AKR emission, respectively. The observations in the Southern hemisphere, made mostly on the dawn flank, show little dependence with latitude for reasons previously discussed. The differences between the positions of the spacecraft and the LT of Wind also clearly affect the observations made at positive magnetic latitudes, seen by the difference between the fit to positive latitudes in the two panels of Figure 8. The bottom panel shows better agreement with the RH power dependence observed by Cassini, with a shallower increase of the av- erage power with latitude. In addition to Cassini being at a much greater distance from Earth and therefore, observing less low power AKR bursts, poorer temporal and spatial sampling from ideal viewing positions by g p g ck lines show linear-log fits to the magnetic latitudes, where we expect circularly polarized (LH) to right- R, respectively. 3.3. AKR Viewing Geometry Colors show the number of 3-minute-resolution observations, retained by the empirical selection in each LT bin. While AKR has been previously observed from the dayside, it can be difficult, without polarization infor- mation, to discern whether this emission is attributable to the illumination of the spacecraft by a source on the nightside via either emission viewing geometry or scattering or that of a dayside source (Hanasz et al., 2003; Mutel et al., 2004; Schreiber et al., 2017). Deducing the exact origin of the source of emission is complex and is not possible within the scope of this study. However, the dynamic spectrogram in Figure 5 as well as the distribution of average power in Figure 7 show that we can observe AKR at all LT, with some of the most intense observations on the dayside made when Wind was within the LT sector 1300–1400 hrs and near the ecliptic plane. The color of each LT bin reflects the number of data used to compute the average, where each data point represents the power of each 3- min frequency sweep. The orbital dynamics of the spacecraft produce the overall color distribution, with perigee on the nightside limiting the number of observations that can be made and vice versa on the dayside. For this reason, more total observations will be made on the dayside, but fewer of those observations will be selected given the preferred nightside location of the AKR source regions. This is seen when taking the ratio of the number of observations made in a local time sector with the number of selected data from the same sector. For the observations made on the dayside (  0600 E LT 1800 ), an average of 38% of data is retained by the selection, while 83% of nightside observations (  1800 E WATERS ET AL. 14 of 26 Journal of Geophysical Research: Space Physics The legend indicates der) of the linear-log fit of the form k line in each panel shows the KR observations (see Equation 4; A where E P is the AKR power for a given hemisphere (e.g., LH or RH emis- sion) in 1 Wsr E , and  , W m E is the magnetic latitude of Wind in degrees. Lamy et al. (2010) found, using the polarization to characterize each hemisphere, that the average emission from the Northern magnetic hem- isphere, when integrated over 15 minutes, gave values A  E 4.2, B = 0.29. Close to the magnetic equator, it is expected that AKR from both hem- ispheres will be observed, but their observations also showed that RH emission can be observed at   2 1 10 Wsr E from magnetic latitudes of up to  5 E in the opposite hemisphere of origin (Southern). Without the polar- ization state we cannot discern the exact origin of emission, so the fit in Equation 4 is performed on data subset by negative or positive magnetic latitude, corresponding to assumed LH and RH AKR emission, respectively. The observations in the Southern hemisphere, made mostly on the dawn flank, show little dependence with latitude for reasons previously discussed. The differences between the positions of the spacecraft and the LT of Wind also clearly affect the observations made at positive magnetic latitudes, seen by the difference between the fit to positive latitudes in the two panels of Figure 8. The bottom panel shows better agreement with the RH power dependence observed by Cassini, with a shallower increase of the av- erage power with latitude. In addition to Cassini being at a much greater distance from Earth and therefore, observing less low power AKR bursts, poorer temporal and spatial sampling from ideal viewing positions by g p g k lines show linear-log fits to the magnetic latitudes, where we expect circularly polarized (LH) to right- R, respectively. The legend indicates der) of the linear-log fit of the form k line in each panel shows the KR observations (see Equation 4; A g p  5 E in the opposite hemisphere of origin (Southern). Without the polar- ization state we cannot discern the exact origin of emission, so the fit in Equation 4 is performed on data subset by negative or positive magnetic latitude, corresponding to assumed LH and RH AKR emission, respectively. 3.4. AKR Temporal Modulation To quantify the temporal variability of the AKR whilst accounting for the viewing, we perform a Fourier analysis on the entire time series as well as three subsets that comprise the two nightside passes/perigees of Wind during DOY 228–233 and 250–255, defined by a five-day peri- od approximately centered on the midnight meridian and the dayside traversal during DOY 232–251. Figure 9 shows the result of a fast fourier transform (FFT) when applied to the Wind AKR power, integrated over 100–650 kHz, for the four aforementioned timeframes. The AKR power is initially averaged over a 3- hr window to smooth the data and remove local temporal variability. Given that the integrated power can vary over several orders of magnitude, the FFT is performed after log-transform- ing the data to reduce the weight of this variability on the analysis. The relative spectral power is shown, having normalized the FFT output by the maximum spectral power found at the maximum period relevant to each timeframe. For example, the FFT output of the entire period in Fig- ure 9a has the maximum peak at a period of 24 days (576 hr), denoted by the vertical gray dotted line and corresponding to the approximate orbital period of Wind during this time. The presence of a peak at this period can be explained with reference to Figures 3b and 5; the perigee of Wind precesses downward across the nightside magnetosphere, measuring in- tensifications in the AKR as it passes the nightside across two to three days, separated by the dayside apogee. We do not comment on the origin of the maximum peaks of the FFT in Figures 9b–9d. The vertical dashed gray line in each panel of Figure 9 indicates a period of 24 hr. For Figure 9a, covering the entire 30- day period, the FFT output is noisy, particularly at lower frequencies but also at periods between 1 and 24 days. A peak of comparable power to the maximum at a 24- day period is seen at a period of 24 hr, showing the observation of the diurnal modulation of AKR. Figures 9b and 9d, including data corresponding to the two nightside perigees, also both show peaks at 24- hr periods, with the spectral resolution being limited by the length of these time series. Journal of Geophysical Research: Space Physics The observations in the Southern hemisphere, made mostly on the dawn flank, show little dependence with latitude for reasons previously discussed. The differences between the positions of the spacecraft and the LT of Wind also clearly affect the observations made at positive magnetic latitudes, seen by the difference between the fit to positive latitudes in the two panels of Figure 8. The bottom panel shows better agreement with the RH power dependence observed by Cassini, with a shallower increase of the av- erage power with latitude. In addition to Cassini being at a much greater distance from Earth and therefore, observing less low power AKR bursts, poorer temporal and spatial sampling from ideal viewing positions by WATERS ET AL. 15 of 26 Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 9. Fast Fourier Transforms (FFT) of the Auroral Kilometric Radiation (AKR) power, integrated over 100–650 kHz, for the whole 30- day period (a), a 5- day period that spans Wind's first nightside perigee (b), a 19- day period spanning Wind's apogee on the dayside (c) and a 5- day period spanning Wind's second nightside perigee (d). The legends in each panel show the 1999 day of year (DOY) for each subset, exclusive of the last date in each case. The integrated power is input at a 3- min resolution; data where no AKR is present are set to    8 1 10 Wsr E P to include them in the analysis. Analysis is performed on the integrated powers after applying a 3- hr rolling window and log-transforming the data. The relative spectral power is shown; data of each panel are normalized by the value at the respective peak. The vertical gray dotted line is at a period of 24 days, the approximate orbital period of Wind, highlighting the peak period of Panel (a). The vertical gray dashed line shows a period of 24 hr. Wind are assumed to produce the observed discrepancy. A full statistical characterization of the latitudinal dependence is out of the scope of this study and is not included. 3.4. AKR Temporal Modulation Figure 9c, corresponding to apogee on the dayside, also shows a diurnal modulation; each period was tested for statistical significance with boot- strapping, where the time series is shuffled randomly before undergoing the same analysis and comparing the spectral peak, allowing the null hy- pothesis to be tested. We found that the peak at 24 hr for each panel in Figure 9 has a p-value of 5 10 E and so is significant. While the presence of diurnal modulation has been previously observed and is a validation of the empirical selection used here in itself, a significant peak in Wind observations on the dayside further corroborates the hypothesis that the diurnal modulation is predominantly due to a geometric viewing effect. Given that the diurnal modulation is seen during observations near noon, and near the magnetic equator, we expect that intrinsic variability of the AKR sources is unobservable and so such a modulation is likely due to the extremes of the emission cones illuminating Wind as the magnetic dipole tilts. Figure 9. Fast Fourier Transforms (FFT) of the Auroral Kilometric Radiation (AKR) power, integrated over 100–650 kHz, for the whole 30- day period (a), a 5- day period that spans Wind's first nightside perigee (b), a 19- day period spanning Wind's apogee on the dayside (c) and a 5- day period spanning Wind's second nightside perigee (d). The legends in each panel show the 1999 day of year (DOY) for each subset, exclusive of the last date in each case. The integrated power is input at a 3- min resolution; data where no AKR is present are set to    8 1 10 Wsr E P to include them in the analysis. Analysis is performed on the integrated powers after applying a 3- hr rolling window and log-transforming the data. The relative spectral power is shown; data of each panel are normalized by the value at the respective peak. The vertical gray dotted line is at a period of 24 days, the approximate orbital period of Wind, highlighting the peak period of Panel (a). The vertical gray dashed line shows a period of 24 hr. 16 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure 10. Comparative time series of Auroral Kilometric Radiation (AKR) power for the eight days (day of year [DOY] 227–235) that comprise Wind's first perigee, taking it across the nightside. 3.4. AKR Temporal Modulation The top panel shows the Cassini integrated power (30–650 kHz) averaged over a moving 3- hr window and including both left-handed circularly polarized (LH) and left-handed circularly polarized (RH) emission. The middle panel shows the 3-hour-averaged Wind integrated power (100–650 kHz). Shown in orange on the right ordinate of both the top and middle panels is the magnetic latitude of the respective spacecraft. Also shown in the top and middle panels are lines indicating the magnetic equator and  5 E . The bottom panel shows the linear cross-correlation between the AKR power as observed by Wind and the magnetic latitude of the spacecraft, with a 24- hr moving window. Figure 10. Comparative time series of Auroral Kilometric Radiation (AKR) power for the eight days (day of year [DOY] 227–235) that comprise Wind's first perigee, taking it across the nightside. The top panel shows the Cassini integrated power (30–650 kHz) averaged over a moving 3- hr window and including both left-handed circularly polarized (LH) and left-handed circularly polarized (RH) emission. The middle panel shows the 3-hour-averaged Wind integrated power (100–650 kHz). Shown in orange on the right ordinate of both the top and middle panels is the magnetic latitude of the respective spacecraft. Also shown in the top and middle panels are lines indicating the magnetic equator and  5 E . The bottom panel shows the linear cross-correlation between the AKR power as observed by Wind and the magnetic latitude of the spacecraft, with a 24- hr moving window. For a highly directional emission such as AKR, with spatially complex source regions and triggering via coupling to the dynamic magnetosphere, we can examine the temporal variability observed from each spacecraft's position to attempt to further explain these diurnal modulations. Figure 10 shows time series of the AKR power observed by Wind and Cassini for DOY 227–235, during which Wind traveled across the nightside from the dusk to dawn sectors, crossing the magnetic equator around DOY 231. The power shown for Cassini in the top panel is for both LH and RH AKR, is integrated over the range 30–650 kHz, and is averaged similarly over a 3- h window. 3 h averages of the Wind AKR power are shown in the middle panel, but again integrated over the range 100–650 kHz for reasons given in Section 3.2. 3.4. AKR Temporal Modulation A particularly strong example of this is seen in the peak   3 1 10 Wsr E in the AKR power observed by Wind when between −  20 E and −  30 E magnetic latitude, centered on DOY 233.5, whilst Cassini sees a peak of   7 1 10 Wsr E whilst close to the magnetic equator at positive latitudes. the contamination from low frequencies. Given the dependence of viewing of the AKR from a particular hemisphere on the magnetic latitude as observed by Cassini and shown in figure 8 of Lamy et al. (2010), we can assume that the dominant AKR observed by a spacecraft at a magnetic latitude \| \| m   5 will be of the corresponding magnetic hemisphere, with little contribution from the other hemisphere if observed. Visual inspection of the AKR power from both spacecraft shows that the bursts are not only seen to be antiphased when in opposite hemispheres, but that they can also be phased on various timescales regardless of the spacecraft position. A particularly strong example of this is seen in the peak   3 1 10 Wsr E in the AKR power observed by Wind when between −  20 E and −  30 E magnetic latitude, centered on DOY 233.5, whilst Cassini sees a peak of   7 1 10 Wsr E whilst close to the magnetic equator at positive latitudes. Given the significant diurnal modulation, we can attempt to discern its origin by examining the correlation of the AKR bursting with the magnetic latitude of the observing spacecraft, here Wind. The bottom panel of Figure 10 shows the linear cross-correlation of the AKR power with the magnetic latitude over a 24- hr moving window. While a similar analysis is performed over a 5- day window in Lamy et al. (2010), the data coverage of Wind while in an appropriate viewing position is insufficient for this. While the choice of the window size is arbitrary, this was chosen to examine correlation features on a similar timescale to the mod- ulation in question. 3.4. AKR Temporal Modulation While the correlation fluctuates, the AKR power is mostly correlated positively with the magnetic latitude of Wind, with the correlation increasing as Wind travels to higher latitudes in the Northern magnetic hemisphere, away from the region in which the emission from either hemisphere is ob- servable and of a comparable intensity (cf., figure 8 of Lamy et al. (2010)). Once wind crosses the magnetic equator at the start of DOY 231, the AKR power becomes anti-correlated with Wind's magnetic latitude after perigee; whilst the AKR power decreases as Wind travels away from the nightside and into the dawn sector, the time series clearly shows the dominant emission from the Southern magnetic hemisphere after Wind crosses the magnetic equator. These observations are consistent with those of Cassini, which showed an anti-correlated diurnal modulation of the RH and LH AKR power, with each correlating with the magnetic latitude of the corresponding magnetic hemisphere. This was interpreted as a result of the diurnal rocking of the AKR source region and the beaming pattern, and this is assumed to be the dominant source of the modulation observed here. On the other hand, the presence of phased bursts in observations of both space- craft regardless of magnetic latitude is contrary to this. While AKR observations with Wind alone appear to be consistent with those of Cassini, with peak AKR power observed at extremes of the spacecraft magnetic latitude and suggesting dominance of a viewing effect, the phasing between AKR bursts observed by both spacecraft brings another new and important constraint to the origin of the diurnal modulation. While the combination of the Wind and Cassini observations here has again illustrated the value of multipoint meas- urements of AKR, the detailed investigation of the burst phasing is beyond the scope of this study. 3.4. AKR Temporal Modulation Although we expect some discrepancy in the magnitude of the average power for this reason, the effects of this will be negligible compared to those introduced by the viewing positions and it is preferable to compare this range to reduce For a highly directional emission such as AKR, with spatially complex source regions and triggering via coupling to the dynamic magnetosphere, we can examine the temporal variability observed from each spacecraft's position to attempt to further explain these diurnal modulations. Figure 10 shows time series of the AKR power observed by Wind and Cassini for DOY 227–235, during which Wind traveled across the nightside from the dusk to dawn sectors, crossing the magnetic equator around DOY 231. The power shown for Cassini in the top panel is for both LH and RH AKR, is integrated over the range 30–650 kHz, and is averaged similarly over a 3- h window. 3 h averages of the Wind AKR power are shown in the middle panel, but again integrated over the range 100–650 kHz for reasons given in Section 3.2. Although we expect some discrepancy in the magnitude of the average power for this reason, the effects of this will be negligible compared to those introduced by the viewing positions and it is preferable to compare this range to reduce 17 of 26 WATERS ET AL. WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 the contamination from low frequencies. Given the dependence of viewing of the AKR from a particular hemisphere on the magnetic latitude as observed by Cassini and shown in figure 8 of Lamy et al. (2010), we can assume that the dominant AKR observed by a spacecraft at a magnetic latitude \| \| m   5 will be of the corresponding magnetic hemisphere, with little contribution from the other hemisphere if observed. Visual inspection of the AKR power from both spacecraft shows that the bursts are not only seen to be antiphased when in opposite hemispheres, but that they can also be phased on various timescales regardless of the spacecraft position. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 of the spectral peak at around 200 kHz in the median spectrum to higher frequencies for the most intense emission, agreeing with the accepted AKR spectrum and the previous Cassini observations. of the spectral peak at around 200 kHz in the median spectrum to higher frequencies for the most intense emission, agreeing with the accepted AKR spectrum and the previous Cassini observations. Accounting for the aforementioned limitations, we integrate the flux densities measured by Wind between 100 and 650 kHz for each 3- min sweep to represent the confident selection of AKR, covering the main frequency range and allowing a comparison with previous results from Cassini. We have accounted for the viewing geometry of AKR in the observations by averaging the integrated power measured in each one- hour LT bin shown in Figure 7. This reproduces quantitatively the day-night asymmetry that can be seen in Figure 5 and is again expected, with a 4 order of magnitude increase from    4 8 1 10 10 Wsr E . This also provides confidence in the selection of AKR by this method, given that it also reproduces the dawn-dusk asymmetry associated with the close correlation between the AKR source region and the auroral region in the ionosphere. Emission is selected at all LTs, showing that we can observe AKR from many of Wind's various viewing positions in the magnetosphere and solar wind and shows promise for future studies where the selection can be applied to a larger Wind data set and aid statistical analyses of events. We also examine in Figure 8 the dependence of the average AKR power with the magnetic latitude of the observation. While we cannot confirm a latitudinal dependence of the AKR power for sources observed from the dayside, Wind observations made at ideal LT show general agreement with the relationship found for RH AKR observed by Cassini in the Northern magnetic hemisphere. Using the selected integrated power, we examined the temporal variability of the Wind AKR observations, confirming the presence of a diurnal modulation with an FFT analysis shown in Figure 9. Journal of Geophysical Research: Space Physics We observe a statistically significant (   5 10 E p ) peak at 24 hr for the entire period, as well as for three subsets of the data corresponding to the first perigee from DOY 228–233, the traversal of the dayside from DOY 232–251 and the second perigee from DOY 250 to 255. In Figure 10, we compared in more detail the AKR power between Wind and Cassini during the first perigee of Wind, which placed it ideally to observe intrinsic AKR modu- lation over a few days. A linear cross-correlation between the Wind AKR power and the magnetic latitude of the spacecraft showed that the AKR is generally correlated in the Northern magnetic hemisphere and then anti-correlated as it crosses into the Southern, lending weight to the visibility of the AKR emission cone in each hemisphere as the predominant source of the diurnal modulation. On the contrary, however, AKR bursts are also observed that are in phase whilst the spacecraft are in separate magnetic hemispheres, although only a short time period is shown here and further inference is out of the scope of the study. Given the ease of masking the Wind data with this technique, longer-term studies of diurnal and semi-diurnal modulations can be conducted with Wind alone, which has 2 E decades of observations from a variety of po- sitions. With the verification of the empirical selection as seen here, statistical analyses can also be conduct- ed between resulting AKR data set and lists of substorm onsets that cover decades and are complimentary to Wind's lifetime. 4. Conclusion We have described a new method of selecting AKR emission from the complex radio environment observed by Wind, using a statistical measure of the variability of radio flux across the spin-axis-aligned Z antenna during a spacecraft spin. Examination of individual spins and the flux density dynamic spectrograms during a 30- day trajectory of Wind shows that the selection is effective at removing ubiquitous solar Type III bursts from the data and isolating AKR (Figures 1 and 2). Although there are limitations at lower frequencies as RFI and sources of high temporal variability from the local plasma can contaminate the selection, the selection criterion employed here is based on a simple numeric threshold and can be readily applied to the extensive data set of Wind. Here, we applied the AKR selection to Wind data for an interval overlapping with the Earth flyby of the Cassini spacecraft. The Cassini data have previously been treated with a full GP inversion (Lamy et al., 2010), but here they provide context for the sensitivity of radio observations to the viewing position of the spacecraft. After considering the discrepancies between the viewing positions of the two spacecraft during the period (Figures 3 and 4), the flux density dynamic spectrograms of the 30- day period between Wind and Cassini are compared in Figure 5. This shows the expected reduction in observed emission as Wind traverses the dayside and is no longer illuminated by the most intense AKR sources located on the nightside. Examining the data more closely, we compare the flux density spectra of Wind observations made from a comparable region to that of Cassini for the period and find that the general char- acteristics of the AKR spectrum are reproduced well at frequencies 100E kHz. Figure 6 shows the broadening 18 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics Appendix A: Errors Due to Assumption of Source Direction As described in Section 2.2.2, we do not have an exact indicator of the AKR emission hemisphere and expect that both LH and RH AKR will illuminate Wind during certain times is dependent on its viewing position. AKR source regions are known to be confined to nightside LT along high latitude magnetic field lines, which allow the geographic or magnetic poles to be used as a reasonable assumption for the source center. While an appropriate pole could be chosen based on the sign of the spacecraft magnetic latitude, for exam- ple, it is favourable to apply a consistent assumption to all observations for this general selection algorithm. Figure A1 shows the distribution of errors introduced by using the Earth's center over the poles. P E , the apparent latitude of Wind when translating the ecliptic plane to either geographic pole, gives a proxy of the geographic pole direction from Wind. From Equation 2, we compare the ratio   2 2 cos cos P GSE E to characterize the implicit error introduced here and find that the maximum value in the distribution is 1.2%. GSE mplicit error introduced here and find that the maximum value in the distribution is 1.2%. 19 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure A1. Statistical characterization of the error implicit in the assumption of Earth's center as the direction of the Auroral Kilometric Radiation (AKR) source. Presented data relates only to the 30- day period studied here and shows the fraction of Wind observations that would be affected by a given error factor.  2 cos GSE E is the term used in Equation 2, while  2 cos P E is the term calculated using the apparent latitude of Wind with respect to geographic poles. Figure A1. Statistical characterization of the error implicit in the assumption of Earth's center as the direction of the Auroral Kilometric Radiation (AKR) source. Presented data relates only to the 30- day period studied here and shows the fraction of Wind observations that would be affected by a given error factor.  2 cos GSE E is the term used in Equation 2, while  2 cos P E is the term calculated using the apparent latitude of Wind with respect to geographic poles. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure B1. Comparing flux densities of a single Type III burst at comparable frequency channels between spacecraft (left) and the resulting ratio between the two datasets from Wind, using the peak flux spectra from the Type III burst (right). Included in the left panel are data derived from Wind using the calibration method described in Section 2.2.2 ( Z S , blue circles), modified to account for the change in radio source (see Appendix B), data from Cassini using a GP inversion that treats Type III bursts (green crosses) and data from Wind using a similarly modified GP inversion at the original 90 s resolution of the frequency channel (  T III E S ), orange triangles. The panel on the right shows the ratio  Z T III S S , where each data point is given by the ratio of the peak flux between data sets during DOY 240 18:00–18:30. The gray, horizontal dashed line is at unity. Figure B1. Comparing flux densities of a single Type III burst at comparable frequency channels between spacecraft (left) and the resulting ratio between the two datasets from Wind, using the peak flux spectra from the Type III burst (right). Included in the left panel are data derived from Wind using the calibration method described in Section 2.2.2 ( Z S , blue circles), modified to account for the change in radio source (see Appendix B), data from Cassini using a GP inversion that treats Type III bursts (green crosses) and data from Wind using a similarly modified GP inversion at the original 90 s resolution of the frequency channel (  T III E S ), orange triangles. The panel on the right shows the ratio  Z T III S S , where each data point is given by the ratio of the peak flux between data sets during DOY 240 18:00–18:30. The gray, horizontal dashed line is at unity. Figure B1 shows the comparison of calibrated flux densities for an example of a Type III burst observed by Wind and Cassini during 1999 DOY 240. The Wind observations are given at different resolutions; the data used here are averaged over the 3- min sweep, while the original resolution of each frequency channel is retained in the data from the Type III GP inversion with Wind. Appendix B: Cross-Checking Flux Densities Using Type III Bursts Appendix B: Cross-Checking Flux Densities Using Type III Bursts As mentioned in Section 2.2.2, we initially compared the flux density resulting from Equation 2, derived from the linear Z antenna, with those from a full GP inversion. Although the scaling factor that results from this work is not applied, we retain the results here as a point of interest, given the observed discrepancies. We have access to results from a GP inversion that assumes the source parameters of a solar radio Type III burst (namely that they are unpolarized) and utilizes the Wind/WAVES system such that the total flux den- sity is retrieved. The S’ antenna, used explicitly to retrieve the degree of circular polarization, is thus ignored in this inversion. To compare the fluxes from the Z antenna to GP fluxes, we must omit the  2 cos E term from Equation 2 to reflect the fact that the source direction is no longer assumed to be at Earth, and the Type III burst source region is sufficiently far enough away and close to the ecliptic plane that the Z antenna is always perpendicular to the emission. Explicitly, the Z antenna is pre-calibrated using 1 2 Z Z P GS (B1) 1 2 Z Z P GS (B1) for the observations presented in this section. The fluxes from the Type III inversion with Wind are derived using a calibration from Zarka et al. (2004), which has since been corrected by a factor 2 by Zaslavsky et al. (2011) and is accounted for here. The fluxes from the Type III inversion with Cassini are similarly derived but have already accounted for this factor (Cecconi et al., 2017). for the observations presented in this section. The fluxes from the Type III inversion with Wind are derived using a calibration from Zarka et al. (2004), which has since been corrected by a factor 2 by Zaslavsky et al. (2011) and is accounted for here. The fluxes from the Type III inversion with Cassini are similarly derived but have already accounted for this factor (Cecconi et al., 2017). 20 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics Journal of Geophysical Research: Space Physics The peak of the primary Type III burst at 272 kHz is seen close to 18:10 UT in this example, and while good agreement is seen between the Type III GP inversions with Cassini and Wind, the Z antenna-calibrated data have an approximately constant offset following the peak. By defining the bounding UT of the Type III burst as well as its frequency limits fol- lowing visual examination of the relevant dynamic spectrogram, we can track the peak flux of the Type III burst for each set of fluxes and produce a spectrum of the ratio  Z T III S E S , where Z E S is the flux from Equation B1 Figure B1 shows the comparison of calibrated flux densities for an example of a Type III burst observed by Wind and Cassini during 1999 DOY 240. The Wind observations are given at different resolutions; the data used here are averaged over the 3- min sweep, while the original resolution of each frequency channel is retained in the data from the Type III GP inversion with Wind. The peak of the primary Type III burst at 272 kHz is seen close to 18:10 UT in this example, and while good agreement is seen between the Type III GP inversions with Cassini and Wind, the Z antenna-calibrated data have an approximately constant offset following the peak. By defining the bounding UT of the Type III burst as well as its frequency limits fol- lowing visual examination of the relevant dynamic spectrogram, we can track the peak flux of the Type III burst for each set of fluxes and produce a spectrum of the ratio  Z T III S E S , where Z E S is the flux from Equation B1 and  T III E S is the flux from the Type III GP inversion with Wind. The example in Figure B1 shows the entire Wind/WAVES RAD1 spectrum, including the lower frequencies that the Type III emission does not reach and showing the discrepancy between the two data below 70 E kHz. While we do not explore it in detail, it is interesting to note that the Cassini observations, derived with an inversion adapted to AKR sources, sees good agreement with the Wind observations from the Type III inversion. 21 of 26 WATERS ET AL. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure B2. Journal of Geophysical Research: Space Physics The fact that the linear Z antenna observes calibrated fluxes larger than those from two independent sets of observations using GP inversions with Wind and Cassini is puzzling, but we do not explore the details for this here. Journal of Geophysical Research: Space Physics Spectrum of the average ratio between peak flux spectra for a total of 19 Type III bursts. As detailed in section Appendix B, each Type III example is described by a start and end time as well as its frequency limits. A spectrum is formed for each example by taking the ratio of the peak fluxes in each channel between the bounding times (see text). The average spectrum shows the mean of the ratios of each frequency channel, with error bars showing the standard error of the mean. Figure B2. Spectrum of the average ratio between peak flux spectra for a total of 19 Type III bursts. As detailed in section Appendix B, each Type III example is described by a start and end time as well as its frequency limits. A spectrum is formed for each example by taking the ratio of the peak fluxes in each channel between the bounding times (see text). The average spectrum shows the mean of the ratios of each frequency channel, with error bars showing the standard error of the mean. Figure B2. Spectrum of the average ratio between peak flux spectra for a total of 19 Type III bursts. As detailed in section Appendix B, each Type III example is described by a start and end time as well as its frequency limits. A spectrum is formed for each example by taking the ratio of the peak fluxes in each channel between the bounding times (see text). The average spectrum shows the mean of the ratios of each frequency channel, with error bars showing the standard error of the mean. Figure B2 shows the mean  Z T III S E S spectrum from a set of 19 Type III bursts. The uncertainties on the cross-calibration spectrum are given by the standard error of the mean and show the better-constrained val- ues at frequencies 50 E kHz for which the majority of the selected Type III bursts are emitting. Z T III S E S tends T III to increase at the lowest frequencies, with the most pronounced increase seen at 20 kHz. This is likely due to the inclusion of spurious emission from the visual examination, as well as the limited number of Type III bursts (7) that were emitting at this frequency. Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure C1. Distribution of Z E values for all observations made at 272 kHz during the 30- day period studied here. The black, vertical dashed line shows the value of the threshold (  0.1 Z E ) chosen to select Auroral Kilometric Radiation (AKR) data. Figure C1. Distribution of Z E values for all observations made at 272 kHz during the 30- day period studied here. The black, vertical dashed line shows the value of the threshold (  0.1 Z E ) chosen to select Auroral Kilometric Radiation (AKR) data. Appendix C: Z Threshold Justification Figure C1 shows the distribution of Z E values observed at 272 kHz and during the 30- day period stud- ied here. This frequency channel was chosen to present as it is a good representation of the typical peak frequency of the AKR spectrum. Shown in the plot is the value used to select AKR data as described in Section 2.3; at values lower than this the majority of the emission is found, with a strong Gaussian profile centered roughly on 1.5 10 E representing the majority of other emission such as Type III bursts and back- ground sources, while at values higher than 1 10 E , a second population is seen that contains AKR as well as low frequency contaminants. While determining the exact Z E distribution of AKR observations for the entire spectral range is out of the scope of this study, examinations of this metric at other frequencies exhibit similar distributions that lend weight to this choice of the threshold. 22 of 26 WATERS ET AL. Appendix D: AKR Power Distributions With Spacecraft LT Figure D1 shows the distribution of integrated power observations (100–650 kHz) for each LT bin used to compute the average values for Figure 7. From the top and bottom rows, corresponding mostly to LT sectors that Wind traveled through on two, separate occasions, evidence can be seen that suggests that the distributions are comprised of two separate events of AKR emission which differ in intensity, whether due to intrinsic differences due to the current state of the magnetosphere or other effects. Some of the bins exhibit two distributions that are separated by 6 orders of magnitude in some cases (e.g., 01–02 LT), as well as non-Gaussian distributions (e.g., 08–09 LT). Applying the selection technique over a longer period will remove some of these local effects and better characterize the statistical AKR power distribution with LT. Taking the mean of the distributions here gives an average that is skewed toward higher values and repre- sents more closely the larger extreme of the total distribution of data, as seen by its position with respect to the 75% quantile in most panels of Figure D1. The median of these distributions would be a more statistical- ly rigorous measure of the average. Here, however, where the aim is to demonstrate the empirical selection of AKR data and not to rigorously define the average AKR power of these observations with LT, the mean is sufficient. WATERS ET AL. 23 of 26 Journal of Geophysical Research: Space Physics 10.1029/2021JA029425 Figure D1. Normalized histograms showing the distribution of log integrated power in each 1 hr local time (LT) bin used to create Figure 7. Each histogram has black dashed and red dashed vertical lines to represent the median and lower and upper quartiles of the distributions, while the black dotted line shows the mean. Figure D1. Normalized histograms showing the distribution of log integrated power in each 1 hr local time (LT) bin used to create Figure 7. Each histogram has black dashed and red dashed vertical lines to represent the median and lower and upper quartiles of the distributions, while the black dotted line shows the mean. Data Availability Statement The Cassini RPWS data from the GP inversion during the period studied here are included in Lamy et al. (2010) and can be found online (https://doi.org/10.25935/5JFX-DH49 (Cecconi et al., 2017)). The data used in this study can be found online (https://doi.org/10.25935/wxv0-vr90), as can the code used to cali- brate and apply the AKR selection (https://github.com/WatersJE/WindWaves_AKR_calibration_selection). References Acknowledgments J. E. Waters's work was supported by the EPSRC Centre for Doctoral Training in Next Generation Computational Modelling Grant No. EP/L015382/1. C. M. Jackman's work is supported by the Science Foundation Ireland Grant 18/ FRL/6199. D. K. Whiter was supported by the Natural Environment Research Council of the UK under grant NE/ S015167/1. The authors acknowledge CNES (Centre National d'Etudes Spatiales), CNRS (Centre National de la Recherche Scientifique), and Observa- toire de Paris for support to the Wind/ Waves team and the CDPP (Centre de Données de la Physique des Plasmas) for the provision of the Wind/Waves RAD1 L2 data. 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https://openalex.org/W4394616898	https://ikprress.org/index.php/AJOMCOR/article/download/8635/7840	English	null	Kirchhoff Biharmonic System with Choquard Nonlinearity and Singular Weights	Deleted Journal	2,024	cc-by	14,669	Asian Journal of Mathematics and Computer Research Asian Journal of Mathematics and Computer Research Volume 31, Issue 2, Page 8-28, 2024; Article no.AJOMCOR.12012 Volume 31, Issue 2, Page 8-28, 2024; Article no.AJOMCOR.12012 ISSN: 2456-477X KirchhoﬀBiharmonic System with Choquard Nonlinearity and Singular Weights aSchool of Mathematics and Statistics, Southwest University, 400715, China. p y This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, diﬀerent versions of the manuscript, comments of the editors, etc are available here: https://prh.ikprress.org/review-history/12012 Received: 20/01/2024 Accepted: 27/03/2024 Published: 08/04/2024 Received: 20/01/2024 Accepted: 27/03/2024 Original Research Article Published: 08/04/2024 Original Research Article Corresponding author: E-mail: wuwuluying@163.com; Corresponding author: E-mail: wuwuluying@163.com; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024 Abstract The aim of this paper is to ﬁnd the existence of solutions for the following Kirchhoﬀtype biharmonic system with exponential nonlinearity and singular weights          m ∥u∥2 + ∥v∥2 ∆2u = h Iµ ∗F (x,u,v) \|x\|α i f1(x,u,v) \|x\|α in Ω; m ∥u∥2 + ∥v∥2 ∆2v = h Iµ ∗F (x,u,v) \|x\|α i f2(x,u,v) \|x\|α in Ω; u = 0, v = 0, ∇u = 0, ∇v = 0 on ∂Ω, where Ωis a bounded domain in R4 containing the origin with smooth boundary, µ ∈(0, 4), 0 < α < µ 2 , Iµ(x) = 1 \|x\|4−µ , m is a Kirchhoﬀtype function, ∥u∥2 = R Ω\|∆u\|2dx, fi behaves like eβ0s2 when \|s\| →∞for some β0 > 0, and there is C1 function F : R2 →R such that ∂F (x,u,v) ∂u , ∂F (x,u,v) ∂v = (f1(x, u, v), f2(x, u, v)). We establish suﬃcient conditions for the solutions of the above system by using variational methods with Adams inequality. *Corresponding author: E-mail: wuwuluying@163.com; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024 Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Keywords: Biharmonic equation; Kirchhoﬀtype system; choquard nonlinearity; critical exponential growth. 2010 Mathematics Subject Classiﬁcation: 35J35, 35B33. Keywords: Biharmonic equation; Kirchhoﬀtype system; choquard nonlinearity; critical exponential growth. 1 Introduction In this paper, we are concerned with the existence of solutions for the following biharmonic Kirchhoﬀsystem with exponential nonlinearity and singular weights          m ∥u∥2 + ∥v∥2 ∆2u = h Iµ ∗F (x,u,v) \|x\|α i f1(x,u,v) \|x\|α in Ω; m ∥u∥2 + ∥v∥2 ∆2v = h Iµ ∗F (x,u,v) \|x\|α i f2(x,u,v) \|x\|α in Ω; u = 0, v = 0, ∇u = 0, ∇v = 0 on ∂Ω, (1.1) (1.1) where Ωis a bounded domain in R4 containing the origin with smooth boundary, µ ∈(0, 4), 0 < α < µ 2 , and ∥u∥2 = R Ω\|∆u\|2dx. Iµ is deﬁned as Iµ(x) = 1 \|x\|4−µ . m : R+ →R+ is a Kirchhoﬀtype function. F satisﬁes suitable growth assumptions and f1 = ∂F ∂u , f2 = ∂F ∂v . where Ωis a bounded domain in R4 containing the origin with smooth boundary, µ ∈(0, 4), 0 < α < µ 2 , and ∥u∥2 = R Ω\|∆u\|2dx. Iµ is deﬁned as Iµ(x) = 1 \|x\|4−µ . m : R+ →R+ is a Kirchhoﬀtype function. F satisﬁes suitable growth assumptions and f1 = ∂F ∂u , f2 = ∂F ∂v . Problems involving biharmonic equations have been studied extensively by many researchers until now. For instance, Rani and Goyal in [1] considered the following biharmonic critical Choquard equation: Problems involving biharmonic equations have been studied extensively by many researchers until now. For instance, Rani and Goyal in [1] considered the following biharmonic critical Choquard equation:    ∆2u = λf(x)\|u\|q−2u + g(x) R Ω g(y)\|u(y)\|2∗ α \|x−y\|α dy \|u\|2∗ α−2u, in Ω; u = 0, ∇u = 0, on ∂Ω, (1.2) (1.2) where Ωis a bounded domain in RN (N ≥5) with smooth boundary, 1 < q < 2, 0 < α < N, 2∗ α = 2N−α N−4 and f, g : ¯Ω→R are continuous sign-changing weight functions. They proved the existence of two nontrivial solutions for the problem (1.2) in a suitable range of λ. Speciﬁcally the readers expressing an interest in the above part we refer to [2, 3, 4, 5, 6, 7] and the references therein for the existence and multiplicity of solutions for biharmonic equations. 1 Introduction 8-28, 2024; Article no.AJOMCOR.12012 where ζn,m is sharp and given by where ζn,m is sharp and given by where ζn,m is sharp and given by ζn,m =        n ωn−1 πn/22mΓ( m+1 2 ) Γ( n−m+1 2 ) n n−m when m is odd, n ωn−1 πn/22mΓ( m 2 ) Γ( n−m 2 ) n n−m when m is even. when m is odd, when m is even. And the symbol ∇mu denotes the mth-order gradient of u and is deﬁned as ∇mu = ( ∇∆(m−1)/2u; if m is odd, ∆(m)/2u; if m is even, if m is even, where ∆and ∇denote the usual Laplacian and gradient operators respectively. With regard to the problem (1.1) in this paper, we consider the case m = 2, n = 4, and we will use the following inequality [11]: sup u∈H2 0 (Ω),∥∆u∥L2(Ω)≤1 Z Ω exp β\|u\|2 dx < ∞, for all 0 < β ≤32π2, Ω⊂R4. Moreover, in the case n = 4, m = 2, we say that f(t) has critical exponential growth at inﬁnity if there exists β0 > 0 such that lim \|t\|→∞ \|f(t)\| eβt2 = 0, for all β > β0; and lim \|t\|→∞ \|f(t)\| eβt2 = ∞, for all β < β0. At this point, if Ωis a smooth bounded domain in R4, in [12], Robert and Struwe considered the biharmonic equations involving critical exponential growth    ∆2uϵ = λuϵe32π2u2 ϵ, in Ω; uϵ = ∂uϵ ∂n = 0, on ∂Ω,  uϵ = ∂uϵ ∂n = 0, on ∂Ω, where λ ∈R. They described the asymptotics of uϵ as ϵ →0, supposing that uϵ →0 weakly in a suitable space H2 2,0(Ω) when supΩuϵ →∞. As for the whole Euclidean space R4, we refer the reader to [13, 14] for the existence results on biharmonic equations with critical exponential nonlinearities. where λ ∈R. They described the asymptotics of uϵ as ϵ →0, supposing that uϵ →0 weakly in a suitable space H2 2,0(Ω) when supΩuϵ →∞. As for the whole Euclidean space R4, we refer the reader to [13, 14] for the existence results on biharmonic equations with critical exponential nonlinearities. 1 Introduction where Ωis a bounded domain in RN (N ≥5) with smooth boundary, 1 < q < 2, 0 < α < N, 2∗ α = 2N−α N−4 and f, g : ¯Ω→R are continuous sign-changing weight functions. They proved the existence of two nontrivial solutions for the problem (1.2) in a suitable range of λ. Speciﬁcally the readers expressing an interest in the above part we refer to [2, 3, 4, 5, 6, 7] and the references therein for the existence and multiplicity of solutions for biharmonic equations. Biharmonic equations involving critical exponential nonlinearities have been also investigated recently. In fact, let Ωbe a smooth bounded domain in Rn, we know the classical Sobolev space embedding shows that W 1,p 0 (Ω) ,→L¯p(Ω) if n > p, where ¯p = np n −p. W 1,p 0 (Ω) ,→L¯p(Ω) if n > p, where ¯p = np n −p. As for n = p, we say W 1,n 0 (Ω) ,→Ls(Ω) for all 1 ≤s < ∞, however L∞(Ω) does not hold. Later, Pohozaev[8] and Trudinger[9] found the function φ(t) = e\|t\| n n−1 −1 such that sup ∥u∥W 1,n 0 (Ω)≤1 Z Ω φ(u)dx < ∞. Then Moser in [10] further improved the above result and obtained the following inequality Then Moser in [10] further improved the above result and obtained the following inequality sup ∥u∥W 1,n 0 (Ω)≤1 Z Ω exp β\|u\| n n−1 dx < ∞, u ∈W 1,n 0 (Ω), if and only if β ≤βn, sup u∥W 1,n(Ω)≤1 Z Ω exp β\|u\| n n−1 dx < ∞, u ∈W 1,n 0 (Ω), if and only if β ≤βn, where Ω⊂Rn is a bounded domain, βn = nω 1 n−1 n and ωn is the surface area for unit ball of Rn. After that, Adams [11] extended this result to higher order Sobolev spaces. That is, let Ωbe a bounded domain in Rn and n, m ∈N satisfying m < n, then for all 0 ≤ζ ≤ζn,m and u ∈W m, n m 0 (Ω), it follows that sup ∥∇mu∥ L n m (Ω) ≤1 Z Ω exp ζ\|u\| n n−m dx < ∞, 9 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 1 Introduction Due to the nonlocal term m ∥u∥2 + ∥v∥2 , Kirchhoﬀproblems were ﬁrstly mentioned in 1883 by Kirchhoﬀ, see [15], which the typical equation is known as follows ρ∂2u ∂t2 − P0 h + E 2L Z L 0 ∂u ∂x 2 dx ! ∂2u ∂x2 = g(x, u), where ρ is the mass density, P0 is the inital tension, h is the area of cross-section, E is the Young modulus of the material and L is the length of the string. Besides this, some problems concerning the nonlocal term are also applied in various ﬁelds, mostly in biological and physical domains. The readers interested in these aspects we refer to the articles [16, 17, 18, 19]. Actually new problems involved with Kirchhoﬀtype emerged from the above researches and authors obtained the existence results of solutions for the Kirchhoﬀtype equations involving critical exponential nonlinearities via variational methods. In relation to Kirchhoﬀproblems, the existence and multiplicity of solutions for elliptic equations inolving critical exponential nonlinearity can be found in the literatures [20, 21] and a class of elliptic equations with a small nonhomogeneous term was studied in the articles [22, 23, 24] in a bounded domain of R2. For biharmonic equations we refer to [25, 26] in the whole Euclidean space R4. where ρ is the mass density, P0 is the inital tension, h is the area of cross-section, E is the Young modulus of the material and L is the length of the string. Besides this, some problems concerning the nonlocal term are also applied in various ﬁelds, mostly in biological and physical domains. The readers interested in these aspects we refer to the articles [16, 17, 18, 19]. Actually new problems involved with Kirchhoﬀtype emerged from the above researches and authors obtained the existence results of solutions for the Kirchhoﬀtype equations involving critical exponential nonlinearities via variational methods. In relation to Kirchhoﬀproblems, the existence and multiplicity of solutions for elliptic equations inolving critical exponential nonlinearity can be found in the literatures [20, 21] and a class of elliptic equations with a small nonhomogeneous term was studied in the articles [22, 23, 24] in a bounded domain of R2. For biharmonic equations we refer to [25, 26] in the whole Euclidean space R4. Another common nonlocal problem is the Choquard nonlinear term. 1 Introduction Later, L¨u [27] studied the non-degenerate Choquard equation with Kirchhoﬀfunction in R3 and obatined the ground state solution via the method of Nehari manifold. Meanwhile, Arora et al. in [28] established the existence of solutions for the Kirchhoﬀ-Choquard type 10 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 problem involving critical exponential nonlinearities by using the Mountain-pass theorem and the boundness of the corresponding Palais-Smale sequence. We recall that the existence solutions for the nonlinear Choquard N-Laplacian equation in a bounded domain of RN can be found in [28], and for the polyharmonic problem we refer to [29, 30]. problem involving critical exponential nonlinearities by using the Mountain-pass theorem and the boundness of the corresponding Palais-Smale sequence. We recall that the existence solutions for the nonlinear Choquard N-Laplacian equation in a bounded domain of RN can be found in [28], and for the polyharmonic problem we refer to [29, 30]. Recently, applications of system involving critical exponential nonlinearity had attracted many researchers to join in this ﬁeld. In [31], Arora et al. infered the relevant Adams-Moser inequality in W m, n m 0 (Ω) × W m, n m 0 (Ω). And they established the existence solutions for the following Kirchhoﬀsystem by using the method of Nehari manifold          −m R Ω\|∇u\|ndx ∆nu = R Ω F (y,u,v) \|x−y\|µ dy f1(x, u, v), u > 0 in Ω; −m R Ω\|∇v\|ndx ∆nv = R Ω F (y,u,v) \|x−y\|µ dy f2(x, u, v), v > 0 in Ω; u = 0, v = 0, on ∂Ω, where Ωis a bounded domain, n ≥2, 0 < µ < n, m : R+ →R+ is a continuous function, ∆nu := div(\|∇u\|n−2∇u), F satisﬁes suitable growth assumptions and f1 = ∂F ∂u , f1 = ∂F ∂v . Furthermore, in [23], the authors extended to k(∈N) equations for singular Trudinger-Moser growth in Ωwhich is a smooth bounded domain in R2 containing the origin with smooth boundary. And they obtained the multiplicity of solutions of the following elliptic Kirchhoﬀsystem    −m Pk i=1( R Ω\|∇uj\|2dx) ∆u = fi(x,u1,...,uk) \|x\|β + εhi(x) in Ω; i = 1..., k, u1 = u2 = ... 1 Introduction = uk = 0, on ∂Ω, where β ∈[0, 2), m is a continuous function, fi behaves like eα0s2 when \|s\| →∞for some α0 > 0, and there is C1 function F : Ω× Rk →R such that ∂F ∂u1 , ..., ∂F ∂uk = (f1, ..., fk), hi ∈((H1 0(Ω))∗, ∥· ∥∗), ε is a small positive parameter. We also refer to [21] for Kirchhoﬀtype elliptic system involving critical exponential growth. where β ∈[0, 2), m is a continuous function, fi behaves like eα0s2 when \|s\| →∞for some α0 > 0, and there is C1 function F : Ω× Rk →R such that ∂F ∂u1 , ..., ∂F ∂uk = (f1, ..., fk), hi ∈((H1 0(Ω))∗, ∥· ∥∗), ε is a small positive parameter. We also refer to [21] for Kirchhoﬀtype elliptic system involving critical exponential growth. Motivated by the above results, in this paper, we consider the existence of solutions for the Kirchhoﬀtype biharmonic system (1.1). Then the Kirchhoﬀterm is a diﬃculty which implies that the equation in problem (1.1) is no longer a pointwise identity. It means that we need to overcome the lack of compactness due to Choquard nonlinearity involving critical exponential growth as well as the Kirchhoﬀterm. Especially important, we ﬁrstly need to obtain the improved Adams-Trudinger inequality involving two variables. In order to treat the system problem (1.1), now we give some deﬁnitions as follows. We introduce H2 0(Ω) with the scalar product Z ⟨u, v⟩= Z Ω ∆u∆vdx for each u, v ∈H2 0(Ω). Then we denote for each u, v ∈H2 0(Ω). Then we denote H2 0(Ω, R2) := H2 0(Ω) × H2 0(Ω), endowed with the scalar product ⟨U, V ⟩= Z Ω ∆u1∆v1dx + Z Ω ∆u2∆v2dx, where U = (u1, u2) and V = (v1, v2), to which corresponds the norm ∥U∥= ⟨U, U⟩1/2 = (∥u1∥2 + ∥u2∥2)1/2, then H2 0(Ω, R2) is well deﬁned and also a Hilbert space. That is, for any (u, v) ∈H2 0(Ω, R2), where U = (u1, u2) and V = (v1, v2), to which corresponds the norm ∥U∥= ⟨U, U⟩1/2 = (∥u1∥2 + ∥u2∥2)1/2, then H2 0(Ω, R2) is well deﬁned and also a Hilbert space. That is, for any (u, v) ∈H2 0(Ω, R2), ∥(u, v)∥= (∥u1∥2 H2 0 (Ω) + ∥u2∥2 H2 0 (Ω)) 1 2 , where ∥u∥H2 0 (Ω) = R Ω\|∆u\|2dx 1/2. 1 Introduction Moreover, for all 1 ≤p < ∞we deﬁne Lp(Ω, R2) as Lp(Ω, R2) := Lp(Ω) × Lp(Ω), where ∥u∥H2 0 (Ω) = R Ω\|∆u\|2dx 1/2. Moreover, for all 1 ≤p < ∞we deﬁne Lp(Ω, R2) as Lp(Ω, R2) := Lp(Ω) × Lp(Ω), where ∥u∥H2 0 (Ω) = R Ω\|∆u\|2dx 1/2. Moreover, for all 1 ≤p < ∞we deﬁne Lp(Ω, R2) as Lp(Ω, R2) := Lp(Ω) × Lp(Ω), 11 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 where Lp(Ω) is the standard Lp-space, we can know Lp(Ω, R2) is well deﬁned and for any (u, v) ∈Lp(Ω, R2), we deﬁne ∥(u, v)∥p = Z Ω \|u\|pdx + Z Ω \|v\|pdx 1 p , and \|(u, v)\| = \|u\|2 + \|v\|2 1 2 . For any 1 ≤p < ∞, by Sobolev embedding theorem, we can know that the embedding H2 0(Ω, R2) ,→Lp(Ω, R2) is compact. Now, let us introduce the precise assumptions under what our problem is studied. For this, we deﬁne M(t) = R t 0 m(s)ds, the primitive of m so that M(0) = 0. The hypotheses on Kirchhoﬀfunction m : R+ →R+ are the following: (m1) There exists m0 > 0 such that m(t) ⩾m0 for t ⩾0 and m(t) is non-decreasing on [0, +∞); (m2) There exists θ > 1 such that m(t) tθ−1 is non-increasing for all t ∈(0, +∞). Remark. (1) By m(t) is nondecreasing for t ≥0, we have R t1+t2 t1 m(s)ds ≥ R t2 0 m(s)ds for all t1, t2 ≥0, then it holds that R t1 0 m(s)ds + R t1+t2 t1 m(s)ds ≥ R t1 0 m(s)ds + R t2 0 m(s)ds, i.e. M(t1 + t2) ≥M(t1) + M(t2). (2) From (m2), we can see that Remark. (1) By m(t) is nondecreasing for t ≥0, we have R t1+t2 t1 m(s)ds ≥ R t2 0 m(s)ds for all t1, t2 ≥0, then it holds that R t1 0 m(s)ds + R t1+t2 t1 m(s)ds ≥ R t1 0 m(s)ds + R t2 0 m(s)ds, i.e. M(t1 + t2) ≥M(t1) + M(t2). (2) From (m ) we can see that θM(t) −m(t)t ≥0, for all t ≥0. θM(t) −m(t)t ≥0, for all t ≥0. 1 Introduction (f1) fi(x, t, s) = 0 when either t ≤0 or s ≤0 and fi(x, t, s) > 0 when t, s > 0 for all x ∈Ωand i = 1, 2. (f2) For i = 1, 2, fi has critical exponential growth at inﬁnity, that is, there exists β0 > 0 such that (f2) For i = 1, 2, fi has critical exponential growth at inﬁnity, that is, there exists β0 > 0 s f2) For i = 1, 2, fi has critical exponential growth at inﬁnity, that is, there exists β0 > 0 such that lim \|(t,s)\|→+∞ \|fi(x, t, s)\| eβ\|(t,s)\|2 = ( 0, ∀β > β0; +∞, ∀β < β0. lim \|(t,s)\|→+∞ \|fi(x, t, s)\| eβ\|(t,s)\|2 = ( 0, ∀β > β0; +∞, ∀β < β0. lim \|(t,s)\|→+∞ eβ\|(t,s)\|2 = ( +∞, ∀β < β0. (f3) There exists l > θ such that the maps u 7→ f1(x,t,s) \|t\|l , v 7→ f2(x,t,s) \|s\|l are increasing functions of t and s respectively. (f3) There exists l > θ such that the maps u 7→ f1(x,t,s) \|t\|l , v 7→ f2(x,t,s) \|s\|l are increasing functions of t respectively. (f3) There exists l > θ such that the maps u 7→ f1(x,t,s) \|t\|l , v 7→ f2(x,t,s) \|s\|l are increasing functions of t and s respectively. (f4) There exist q ∈(0, 1], a0, b0, M0 > 0 such that 0 < \|t\|qF(x, t, s) ≤M0f1(x, t, s) for all \|t\| ⩾a0 and 0 < \|s\|qF(x, t, s) ≤M0f2(x, t, s) for all \|s\| ⩾b0 uniformly in x ∈Ω. (f4) There exist q ∈(0, 1], a0, b0, M0 > 0 such that 0 < \|t\|qF(x, t, s) ≤M0f1(x, t, s) for all \|t\| ⩾a0 and 0 < \|s\|qF(x, t, s) ≤M0f2(x, t, s) for all \|s\| ⩾b0 uniformly in x ∈Ω. (f5) There exists r > 0 such that lim\|(t,s)\|→(0,0) fi(x,t,s) \|t\|r+\|s\|r = 0 holds for i = 1, 2. (f5) There exists r > 0 such that lim\|(t,s)\|→(0,0) fi(x,t,s) \|t\|r+\|s\|r = 0 holds for i = 1, 2. lim inf t,s→∞ (\|t\| + \|s\|)F(x, t, s) eβ0(\|t\|2+\|s\|2) > κ >   4π2(4 + µ −2α)e 21(4+µ−2α)−8 8 m 4π2(4+µ−2α) β0 β2 0Cµd4+µ−2α   1 2 Uniformly in x ∈Ω, where Cµ = 24π4 µ(µ+1)(µ+2)(µ+3)(µ+4), and 2d is the radius of the largest open ball containing the origin contained in Ω. Remark. 1 Introduction (1.3) (1.3) Indeed, for any 0 < t1 < t2, we deduce that θM(t1) −m(t1)t1 = θM(t2) −θ Z t2 t1 m(t)dt −m(t1) tθ−1 1 tθ 1 ⩽θM(t2) −m(t2) tθ−1 2 (tθ 2 −tθ 1) −m(t2) tθ−1 2 tθ 1 = θM(t2) −m(t2)t2. = θM(t2) −m(t2)t2. Therefore, θM(t) −m(t)t is nondecreasing for t ≥0. In particular, θM(t) −m(t)t ≥0 for all t ≥0. θM(t) −m(t)t ≥0 for all t ≥0. θM(t) −m(t)t ≥0 for all t ≥0. M(t) ≥M(1)tθ for 0 ≤t ≤1; M(t) ≤M(1)tθ for t ≥1. M(t) ≤C1tθ + C2, for all t ≥0. M(t) ≤C1tθ + C2, for all t ≥0. (1.4) (1.4) Moreover, by (m2), for t1, t2 > 0 one has M(t1 + t2) = Z t1+t2 0 m(s)ds = Z t1 0 m(s)ds + Z t1+t2 t1 m(s)ds = M(t1) + Z t2 0 m(t1 + s)ds ≤M(t1) + m(t1) tθ−1 1 Z t2 0 (t1 + s)θ−1ds = M(t1) + t1m(t1) θ " 1 + t2 t1 θ −1 # . (1.5) Remark. A typical example of a function m satisfying the conditions (m1)−(m2) is given by m(t) = m0+atθ−1 with θ > 1 and a ≥0. Remark. A typical example of a function m satisfying the conditions (m1)−(m2) is given by m(t) = m0+atθ−1 with θ > 1 and a ≥0. Throughout this paper, we assume fi: Ω× R2 →R for i = 1, 2 are continuous functions satisfying the following conditions: Throughout this paper, we assume fi: Ω× R2 →R for i = 1, 2 are continuous functions satisfying the fo conditions: Throughout this paper, we assume fi: Ω× R2 →R for i = 1, 2 are continuous functions satisfying the following conditions: 12 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 (f1) fi(x, t, s) = 0 when either t ≤0 or s ≤0 and fi(x, t, s) > 0 when t, s > 0 for all x ∈Ωand i = 1, 2. (f2) For i = 1, 2, fi has critical exponential growth at inﬁnity, that is, there exists β0 > 0 such that (f1) fi(x, t, s) = 0 when either t ≤0 or s ≤0 and fi(x, t, s) > 0 when t, s > 0 for all x ∈Ωand i = 1, 2. 1 Introduction In view of the above results, we found that It means that we can derive tF(x, t) ≥K−ε 2β0 eβ0t2 when f(x, t) ≥(K −ϵ)eβ0t2 for t large enough and ε > 0 small enough. In view of the above results, we found that lim inf t→∞ tf(x, t)F(x, t) e2β0t2 > K2 2β0 . lim inf t→∞ tf(x, t)F(x, t) e2β0t2 > K2 2β0 . Then according to the above analysis, for the system (1.1) we know (f6) is weaker than (1.6). Theorem 1.1. Suppose that m satisﬁes (m1) −(m2), f satisﬁes (f1) −(f6). Then (1.1) has a ground state solution (u, v) ∈N such that Φ(u, v) = b := infN Φ, where N = {(u, v) ∈H2 0(Ω, R2)\{(0, 0)} : ⟨Φ′(u, v), (u, v)⟩= 0}. (1.7) (1.7) The remainder of this paper is organized as follows. We prove the Adams inequality and the version of Lion’s Lemma in the Sobolev spaces, namely H2 0(Ω, R2) in Section 2. In Section 3, we give the energy functional and prove that the system (1.1) satisﬁes the geometric conditions of the Mountain-pass theorem and the corresponding Palais-Smale sequence is bounded. In this article, we denote that C, Ci, ci are some positive constants. 1 Introduction By (f3), for any 1 < p ≤l, it holds that tf1(x, t, s) −pF(x, t, s) > 0, sf2(x, t, s) −pF(x, t, s) > 0, for all (x, t, s) ∈Ω× R2. In fact, for any 1 < p ≤l, from (f3) we have f1(x,t,s) tp−1 is increasing functions of t > 0 uniformly of s > 0. Let s > 0 and 0 < t1 < t2 be ﬁxed, then it holds that t1f1(x, t1, s) −pF(x, t1, s) < f1(x, t2, s) tp−1 2 tp 1 −pF(x, t2, s) + p Z t2 t1 f1(x, t, s)dt. On the other hand, p Z t2 t1 f1(x, t, s)dt < pf1(x, t2, s) tp−1 2 Z t2 t1 tp−1dt = f1(x, t2, s) tp−1 2 (tp 2 −tp 1). From the above inequalities, we derive that t1f1(x, t1, s) −pF(x, t1, s) < t2f1(x, t2, s) −pF(x, t2, s). Then we obtain tf1(x, t, s) −pF(x, t, s) > 0 for all (x, t, s) ∈Ω× R2. Similarly, we also obtain sf2(x, t, s) − pF(x, t, s) > 0 for all (x, t, s) ∈Ω× R2. Remark. Here in [31], the condition for the estimate on energy level is as follows lim t,s→∞ (f1(x, t, s)t + f2(x, t, s)s) F(x, t, s) exp(q(\|t\| n n−1 + \|s\| n n−1 )) = ∞ uniformly in x ∈Ω, (1.6) lim t,s→∞ (f1(x, t, s)t + f2(x, t, s)s) F(x, t, s) exp(q(\|t\| n n−1 + \|s\| n n−1 )) = ∞ uniformly in x ∈Ω, (1.6) (1.6) for some q > 2. For a single equation, we know if the condition for the estimation method is lim inf t→∞ f(x, t) eβ0t2 > K > 0, lim inf t→∞ f(x, t) eβ0t2 > K > 0, then we obtain then we obtain lim inf t→∞ tF(x, t) eβ0t2 ≥lim inf t→∞ R t 0 sf(x, s)ds eβ0t2 = lim inf t→∞ f(x, t) 2β0eβ0t2 . 13 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 It means that we can derive tF(x, t) ≥K−ε 2β0 eβ0t2 when f(x, t) ≥(K −ϵ)eβ0t2 for t large enough and ε > 0 small enough. 2 Preliminaries and Auxiliary Results In this section, we introduce some famous inequalities as follows, and inspired by these we conclude some similar forms of inequalities and give some preliminaries. Lemma 2.1. ([11]) For each u ∈H2 0(Ω), Ω⊂R4 is a bounded domain, then for any β > 0, Z Ω exp β\|u\|2 dx < ∞. Moreover, we have sup ∥u∥⩽1 Z Ω exp β\|u\|2 dx < ∞, provided β ≤32π2. Lemma 2.2. For each (u, v) ∈H2 0(Ω, R2), Ω⊂R4 is a bounded domain, then for any β > 0, Z Ω exp β \|u\|2 + \|v\|2 dx < ∞. Lemma 2.2. For each (u, v) ∈H2 0(Ω, R2), Ω⊂R4 is a bounded domain, then for any β > 0, Z Ω exp β \|u\|2 + \|v\|2 dx < ∞. Lemma 2.2. For each (u, v) ∈H2 0(Ω, R2), Ω⊂R4 is a bounded domain, then for any β > 0, Moreover, we have sup ∥(u,v)∥=1 Z Ω exp β \|u\|2 + \|v\|2 dx < ∞, provided β ≤32π2. Proof. From Lemma 2.1 and Young’s inequality, for each (u, v) ∈H2 0(Ω, R2), and for any β > 0, we obtain Proof. From Lemma 2.1 and Young’s inequality, for each (u, v) ∈H2 0(Ω, R2), and for any β > 0, we obta Z Ω eβ(\|u\|2+\|v\|2)dx = Z Ω eβ\|u\|2eβ\|v\|2dx ≤ Z Ω e2β\|u\|2dx + Z Ω e2β\|v\|2dx < ∞. Now for any (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥= 1, then ∥u∥2, ∥v∥2 ⩽1. Let r1 = ∥u∥2, r2 = ∥v∥2, then we know r1 + r2 = 1. Hence by using H¨older inequality and Lemma 2.1 we obtain Z Ω eβ(\|u\|2+\|v\|2)dx ⩽ Z Ω eβ\|u\|2/r1dx r1 Z Ω eβ\|v\|2/r2dx r2 < C, thus the proof is completed. thus the proof is completed. 14 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Lemma 2.3. Let {(un, vn)} be a sequence in H2 0(Ω, R2) satisfying ∥(un, vn)∥= 1 such that (un, vn) ⇀(u, v) ̸= 0 weakly in H2 0(Ω, R2). Then for any 0 < β < 32π2 1−∥(u,v)∥2 , we have Lemma 2.3. Let {(un, vn)} be a sequence in H2 0(Ω, R2) satisfying ∥(un, vn)∥= 1 such that (un, vn) ⇀(u, v) ̸= 0 weakly in H2 0(Ω, R2). Then for any 0 < β < 32π2 1−∥(u,v)∥2 , we have sup n∈N Z Ω exp β \|un\|2 + \|vn\|2 dx < ∞. 2 Preliminaries and Auxiliary Results then we obtain R Ωexp β \|un\|2 + \|vn\|2 dx is bounded, and this lemma is proved. then we obtain R Ωexp β \|un\|2 + \|vn\|2 dx is bounded, and this lemma is proved. Lemma 2.4. For any (u, v) ∈H2 0(Ω, R2), if β > 0, s > 0 satisfying ∥(u, v)∥⩽M such that βM 2 < 32π2, then there exists C = C(β, M, s) > 0 such that ma 2.4. For any (u, v) ∈H2 0(Ω, R2), if β > 0, s > 0 satisfying ∥(u, v)∥⩽M such that βM 2 < 32π2, then xists C = C(β, M, s) > 0 such that 2.4. For any (u, v) ∈H2 0(Ω, R2), if β > 0, s > 0 satisfying ∥(u, v)∥⩽M such that βM 2 < 32π2, then s C = C(β, M, s) > 0 such that Z Ω \|(u, v)\|seβ\|(u,v)\|2dx ⩽C∥(u, v)∥s. Proof. For each (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥⩽M, we choose r > 1 close to 1 such that rβM 2 ⩽32π2 and sq > 1, where q = s s−1. By using H¨older inequality and Lemma 2.2, we obtain Proof. For each (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥⩽M, we choose r > 1 close to 1 such that rβM 2 ⩽32π2 and sq > 1, where q = s s−1. By using H¨older inequality and Lemma 2.2, we obtain Proof. For each (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥⩽M, we choose r > 1 close to 1 such that rβM 2 ⩽32π2 and sq > 1, where q = s s−1. By using H¨older inequality and Lemma 2.2, we obtain Z Ω \|(u, v)\|seα\|(u,v)\|2dx ⩽ Z Ω erβ\|(u,v)\|2dx 1/r ∥(u, v)∥s qs ⩽C∥(u, v)∥s qs. Since sq > 1, using the continuous embedding H2 0(Ω, R2) ,→Lqs(Ω, R2), we ﬁnish the proof. g the continuous embedding H2 0(Ω, R2) ,→Lqs(Ω, R2), we ﬁnish the proof. Since sq > 1, using the continuous embedding H2 0(Ω, R2) ,→Lqs(Ω, R2), we ﬁnish the proof. Proposition 2.5. ([32]) Let t, r > 1 and 0 < µ < N with m, n ⩾0, 1 t + µ+m+n N + 1 r = 2, µ + m + n ⩽N. Then there exists a constant C(m, n, t, µ, r) > 0 which is dependent of f ∈Lt(RN), h ∈Lr(RN) such that Proposition 2.5. 2 Preliminaries and Auxiliary Results sup n∈N Z Ω exp β \|un\|2 + \|vn\|2 dx < ∞. sup n∈N Z Ω exp β \|un\|2 + \|vn\|2 dx < ∞. Proof. For (un, vn) ⇀(u, v) ̸= 0 in H2 0(Ω, R2) satisfying ∥(un, vn)∥= 1, it is easy to see that lim n→∞∥(un −u, vn −v)∥2 = lim n→∞ 1 −2⟨un, u⟩−2⟨vn, v⟩+ ∥(u, v)∥2 = 1 −∥(u, v)∥2 < 32π2 β , (2.1) (2.1) and u2 n ≤(un −u)2 + ϵu2 n + Cϵu2, and u2 n ≤(un −u)2 + ϵu2 n + Cϵu2, (2.2) f ll h h C l d h b ( ) h u2 n ≤(un −u)2 + ϵu2 n + Cϵu2, (2.2) (2.2) for ϵ small enough, where Cϵ is a positive constant related to ϵ. Then by using (2.2) we have ough, where Cϵ is a positive constant related to ϵ. Then by using (2.2) we have Z Ω eβ(\|un\|2+\|vn\|2)dx ≤ Z Ω eβ((un−u)2+(vn−v)2)eβϵ(u2 n+v2 n)eβCϵ(u2+v2)dx. Now we take r1, r2, r3 > 1 such that 1 r1 + 1 r2 + 1 r3 = 1, and by using H¨older inequality we obtain Now we take r1, r2, r3 > 1 such that 1 r1 + 1 r2 + 1 r3 = 1, and by using H¨older inequality we obtain e r1, r2, r3 > 1 such that 1 r1 + 1 r2 + 1 r3 = 1, and by using H¨older inequality we obtain Z Ω eβ(\|un\|2+\|vn\|2)dx ≤ Z Ω eβr1((un−u)2+(vn−v)2)dx 1/r1 Z Ω eϵβr2(u2 n+v2 n)dx 1/r2 × Z Ω eβCϵr3(u2+v2)dx 1/r3 . Using Lemma 2.2, then we can choose ϵ small enough such that Z Ω eϵβr2(u2 n+v2 n)dx ≤C, Z Ω eβCϵr3(u2+v2)dx ≤C. Z Ω Z Ω Moreover, we choose r1 > 1 close to 1 such that βr1∥(un −u, vn −v)∥2 < 32π2, then according to (2.1) and Lemma 2.2 we obtain Moreover, we choose r1 > 1 close to 1 such that βr1∥(un −u, vn −v)∥2 < 32π2, then according to (2.1) and Lemma 2.2 we obtain Z Ω exp βr1 (un −u)2 + (vn −v)2 dx = Z Ω exp " βr1 un −u ∥(un −u, vn −v)∥ 2 + vn −v ∥(un −u, vn −v)∥ 2! ∥(un −u, vn −v)∥2 # dx < C, then we obtain R Ωexp β \|un\|2 + \|vn\|2 dx is bounded, and this lemma is proved. 3 The Variational Framework and the Minimax Estimate 3 We now consider the energy functional Φ(u, v) given by Φ(u, v) = 1 2M ∥(u, v)∥2 −1 2 Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx. (3.1) (3.1) Lemma 3.1. Assume that (f2) and (f3) hold, then we have that Φ(u, v) is well deﬁned on H2 0(Ω, R2). Moreover, ⟨Φ′(u, v), (φ, ψ)⟩= m ∥(u, v)∥2 Z Ω ∆u∆φdx + Z Ω ∆v∆ψdx − Z Ω Iµ ∗F(x, u, v) \|x\|α (f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, (3.2) (3.2) for any (φ, ψ) ∈H2 0(Ω, R2). for any (φ, ψ) ∈H2 0(Ω, R2). Proof. For (u, v) ∈H2 0(Ω, R2), we know fi has critical exponential growth, then by (f2) we choose β > β0 and there exists C1 > 0 such that \|fi(x, u, v)\| ⩽C1eβ\|(u,v)\|2, for all (x, u, v) ∈Ω× R2, i = 1, 2. (3.3) given ϵ > 0 there exist C2, C3 > 0 and δ > 0 such that (3.3) \|fi(x, u, v)\| ⩽C1e , for all (x, u, v) ∈Ω× R , i 1, 2. (3.3) Moreover, by (f3), given ϵ > 0 there exist C2, C3 > 0 and δ > 0 such that f1(x, u, v) ≤C2\|u\|, f2(x, u, , v) ≤C3\|v\| always that \|u\|, \|v\| ≤δ. (3.4) Then combing (3.3) and (3.4), we have f1(x, u, v) ≤C2\|u\|, f2(x, u, , v) ≤C3\|v\| always that \|u\|, \|v\| ≤δ. (3.4) Then combing (3.3) and (3.4), we have f1(x, u, v) ≤C2\|u\|, f2(x, u, , v) ≤C3\|v\| always that \|u\|, \|v\| ≤δ. (3.4) Then combing (3.3) and (3.4), we have (3.4) \|F(x, u, v)\| ⩽C4\|(u, v)\|eβ\|(u,v)\|2 + C5\|(u, v)\|2, (3.5) \|F(x, u, v)\| ⩽C4\|(u, v)\|eβ\|(u,v)\|2 + C5\|(u, v)\|2, (3.5) for all (u v) ∈H2 0(ΩR2) Now using (3 5) and Proposition 2 5 with N = 4 t = r = 8 and m = n = α (3.5) for all (u, v) ∈H2 0(Ω, R2). Now using (3.5) and Proposition 2.5 with N = 4, t = r = 8 4+µ−2α and m = n = α, we obtain for all (u, v) ∈H2 0(Ω, R2). 2 Preliminaries and Auxiliary Results ([32]) Let t, r > 1 and 0 < µ < N with m, n ⩾0, 1 t + µ+m+n N + 1 r = 2, µ + m + n ⩽N. Then there exists a constant C(m, n, t, µ, r) > 0 which is dependent of f ∈Lt(RN), h ∈Lr(RN) such that Z RN Z RN f(x)h(y) \|x −y\|µ\|y\|m\|x\|n dxdy ⩽C(m, n, t, µ, r)∥f∥Lt(RN )∥h∥Lr(RN ). Z RN Z RN f(x)h(y) \|x −y\|µ\|y\|m\|x\|n dxdy ⩽C(m, n, t, µ, r)∥f∥Lt(RN )∥h∥Lr(RN ). 15 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Lemma 3.2. Under the assumptions (m1), (m2) and (f1). (i) there exists R0, Υ > 0 such that Φ(u, v) ⩾Υ for any (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥= R0. (ii) there exists a (eu, ev) ∈H2 0(Ω, R2) with ∥(eu, ev)∥> R0 such that Φ(eu, ev) < 0. there exists R0, Υ > 0 such that Φ(u, v) ⩾Υ for any (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥= R0. ) there exists a (eu, ev) ∈H2 0(Ω, R2) with ∥(eu, ev)∥> R0 such that Φ(eu, ev) < 0. (i) there exists R0, Υ > 0 such that Φ(u, v) ⩾Υ for any (u, v) ∈H2 0(Ω, R2) satisfying ∥(u, v)∥= R0. (ii) th i t (e e) ∈H2(ΩR2) ith ∥(e e)∥> R h th t Φ(e e) < 0 ( ) 0, ( , ) ⩾ f y ( , ) 0( , ) fy g ∥( , )∥ 0 (ii) there exists a (eu, ev) ∈H2 0(Ω, R2) with ∥(eu, ev)∥> R0 such that Φ(eu, ev) < 0. 3 The Variational Framework and the Minimax Estimate Now using (3.5) and Proposition 2.5 with N = 4, t = r = 8 4+µ−2α and m = n = α, we obtain Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx ⩽C(µ, α)∥F(x, u, v)∥2 8 4+µ−2α ⩽C(µ, α) Z Ω \|(u, v)\|2 + \|(u, v)\| exp β\|(u, v)\|2 8 4+µ−2α dx 4+µ−2α 4 ⩽C(µ, α) Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 16 4+µ−2α exp 8β\|(u, v)\|2 4 + µ −2α dx 4+µ−2α 4 . According to Lemma 2.4 and the continuous embedding H2 0(Ω, R2) ,→Ls(Ω, R2) with s ≥1, we know Φ(u, v) is well deﬁned, and we can see Φ ∈C1(H2 0(Ω, R2), R). According to Lemma 2.4 and the continuous embedding H2 0(Ω, R2) ,→Ls(Ω, R2) with s ≥1, we know Φ(u, v) is well deﬁned, and we can see Φ ∈C1(H2 0(Ω, R2), R). From Lemma 3.1, we have that critical points of the functional Φ are precisely weak solutions of problem (1.1). Then we will verify that the functional Φ satisﬁes the conditions of Mountain-pass theorem. 16 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Proof. Let (u, v) ∈H2 0(Ω, R2) such that ∥(u, v)∥= R0. Similarly, taking Proposition 2.5 with N = 4, t = r = 8 4+µ−2α and m = n = α, by (3.5) and H¨older inequality we obtain Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx ⩽C(µ, α)∥F(x, u, v)∥2 8 4+µ−2α ⩽C(µ, α) Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 16 4+µ−2α exp 8β\|(u, v)\|2 4 + µ −2α dx 4+µ−2α 4 ⩽C(µ, α) (Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 32 4+µ−2α dx 1 2 × "Z Ω exp 16β∥(u, v)∥2 4 + µ −2α \|(u, v)\| ∥(u, v)∥ 2! dx # 1 2    4+µ−2α 4 . 3 The Variational Framework and the Minimax Estimate Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx ⩽C(µ, α)∥F(x, u, v)∥2 8 4+µ−2α ⩽C(µ, α) Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 16 4+µ−2α exp 8β\|(u, v)\|2 4 + µ −2α dx 4+µ−2α 4 ⩽C(µ, α) (Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 32 4+µ−2α dx 1 2 × "Z Ω exp 16β∥(u, v)∥2 4 + µ −2α \|(u, v)\| ∥(u, v)∥ 2! dx # 1 2    4+µ−2α 4 . ⩽C(µ, α)∥F(x, u, v)∥ 8 4+µ−2α ⩽C(µ, α) Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 16 4+µ−2α exp 8β\|(u, v)\|2 4 + µ −2α dx 4+µ−2α 4 ⩽C(µ, α) (Z Ω \|(u, v)\| 16 4+µ−2α dx + Z Ω \|(u, v)\| 32 4+µ−2α dx 1 2 × "Z Ω exp 16β∥(u, v)∥2 4 + µ −2α \|(u, v)\| ∥(u, v)∥ 2! dx # 1 2    4+µ−2α 4 . Now we choose suitable R0 > 0 such that 16βR2 0 4+µ−2α ⩽32π2, then by using Sobolev imbedding and Lemma 2.2 we have Now we choose suitable R0 > 0 such that 16βR2 0 4+µ−2α ⩽32π2, then by using Sobolev imbedding and Lemma 2.2 h Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx ⩽C(µ, α) ∥(u, v)∥ 16 4+µ−2α + ∥(u, v)∥ 16 4+µ−2α 4+µ−2α 4 ⩽C(µ, α)∥(u, v)∥4. Then for any ∥(u, v)∥= R0 < q 2π2(4+µ−2α) β , by (m1) we know Φ(u, v) ⩾m0 2 ∥(u, v)∥2 −C(µ, α)∥(u, v)∥4. Φ(u, v) ⩾m0 2 ∥(u, v)∥2 −C(µ, α)∥(u, v)∥4. Φ(u, v) ⩾m0 2 ∥(u, v)∥2 −C(µ, α)∥(u, v)∥4. So we choose ∥(u, v)∥= R0 small enough so that Φ(u, v) ⩾Υ for some Υ > 0 and hence (i) follows. So we choose ∥(u, v)∥= R0 small enough so that Φ(u, v) ⩾Υ for some Υ > 0 and hence (i) follows. Now we choose q ∈R such that θ < q < l in Remark 1, then we obtain tf1(x, t, s) −qF(x, t, s) > 0 and sf2(x, t, s) −qF(x, t, s) > 0 for all (x, t, s) ∈Ω× R2. 3 The Variational Framework and the Minimax Estimate So we obtain So we choose ∥(u, v)∥= R0 small enough so that Φ(u, v) ⩾Υ for some Υ > 0 and h Now we choose q ∈R such that θ < q < l in Remark 1, then we obtain tf1(x, t, s) −qF(x, t, s) > 0 and sf2(x, t, s) −qF(x, t, s) > 0 for all (x, t, s) ∈Ω× R2. So we obtain F(x, u, v) ≥c1\|u\|q −c2, F(x, u, v) ≥c3\|v\|q −c4, thus we conclude that F(x, u, v) ≥c1\|u\|q + c3\|v\|q −c5, for all (x, u, v) ∈Ω× R2. (3.6) (3.6) Then using (3.6) it follows that Z Ω Iµ ∗F(x, tu, tv) \|x\|α F(x, tu, tv) \|x\|α dx ⩾ Z Ω Z Ω (c1\|tu\|q + c3\|tv\|q −c4)2 \|x\|α\|y\|α\|x −y\|µ dxdy ⩾c6t2q −c7tq + c8. By using (1.4) we have M(t) ≤C1tθ + C2, then using (3.1) we obtain By using (1.4) we have M(t) ≤C1tθ + C2, then using (3.1) we obtain Φ(tu, tv) ⩽c9t2θ −c10t2q + c11tq −c12. Φ(tu, tv) ⩽c9t2θ −c10t2q + c11tq −c12. Since q > θ we obtain Φ(tu, tv) →−∞as t →∞. Then there exists (eu, ev) = (t0u, t0v) ∈H2 0(Ω, R2) with t0 large enough such that ∥(eu, ev)∥> R0 and Φ(eu, ev) < 0, hence (ii) holds. Then according to Lemma 3.2, we know Φ(u, v) satisﬁes the geometric conditions of the Mountain-pass theorem, let Then according to Lemma 3.2, we know Φ(u, v) satisﬁes the geometric conditions of the Mountain-pass theorem, let c∗= inf γ∈Γ max t∈[0,1] Φ(γ(t)) > 0 (3.7) (3.7) 17 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 be the minimax level of Φ, where be the minimax level of Φ, where Γ = {γ ∈C([0, 1], H2 0(Ω, R2)) : γ(0) = 0, Φ(γ(1)) < 0}. Then there exists a Palais-Smale sequence {(un, vn)} ⊂H2 0(Ω, R2) satisfying Φ(un, vn) →c∗, Φ′(un, vn) →0, (3.8) (3.8) as n →∞. as n →∞. as n →∞. Lemma 3.3. Assume that (f3) and (m1) hold, then every Palais-Smale sequence of Φ is bounded in H2 0(Ω, R2). Proof. Let {(un, vn)} be a Palais-Smale sequence of Φ for c∗∈R in H2 0(Ω, R2), then it follows that Φ(un, vn) →c∗, Φ′(un, vn) →0 as n →∞. 3 The Variational Framework and the Minimax Estimate Therefore we have Φ(un, vn) = 1 2M ∥(un, vn)∥2 −1 2 Z Ω Iµ ∗F(x, un, vn) \|x\|α F(x, un, vn) \|x\|α dx = c∗+ δn, (3.9) where δn →0 as n →∞, and (3.9) ⟨Φ′(un, vn), (φ, ψ)⟩= m ∥(un, vn)∥2 Z Ω (∆un∆φ + ∆vn∆ψ) dx − Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)φ + f2(x, un, vn)ψ \|x\|α dx ⩽ϵn∥(φ, ψ)∥, (3.10) for all (φ, ψ) ∈H2 0(Ω, R2). By Remark 1, we obtain that for all (φ, ψ) ∈H2 0(Ω, R2). By Remark 1, we obtain that lF(x, t, s) ⩽uf1(x, t, s), and lF(x, t, s) ⩽vf2(x, t, s) for all (x, t, s) ∈Ω× R2. (3.11) Then by (1.3), (3.9), (3.10), (3.11) and (m1), for large n we have lF(x, t, s) ⩽uf1(x, t, s), and lF(x, t, s) ⩽vf2(x, t, s) for all (x, t, s) ∈Ω× R2. (3.11) Then by (1.3), (3.9), (3.10), (3.11) and (m1), for large n we have lF(x, t, s) ⩽uf1(x, t, s), and lF(x, t, s) ⩽vf2(x, t, s) for all (x, t, s) ∈Ω× R2. (3.11) Then by (1.3), (3.9), (3.10), (3.11) and (m1), for large n we have (3.11) c∗+ ϵn∥(un, vn)∥≥Φ(un, vn) −1 4l ⟨Φ′(un, vn), (un, vn)⟩ = 1 2M ∥(un, vn)∥2 −1 4l m ∥(un, vn)∥2 ∥(un, vn)∥2 −1 2 Z Ω Iµ ∗F(x, un, vn) \|x\|α F(x, un, vn) \|x\|α dx + 1 4l Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)un + f2(x, un, vn)vn \|x\|α dx ⩾1 2M ∥(un, vn)∥2 −1 4l m ∥(un, vn)∥2 ∥(un, vn)∥2 + 1 4l Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)un + f2(x, un, vn)vn −2lF(x, un, vn) \|x\|α dx ⩾1 2θ θM ∥(un, vn)∥2 −m ∥(un, vn)∥2 ∥(un, vn)∥2 + 1 2θ −1 4l m ∥(un, vn)∥2 ∥(un, vn)∥2 ⩾ 1 2θ −1 4l m0∥(un, vn)∥2, thus we know {(un, vn)} is bounded in H2 0(Ω, R2) with l > θ. thus we know {(un, vn)} is bounded in H2 0(Ω, R2) with l > θ. 18 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Now we give a precise estimation about the Mountain pass level c∗deﬁned by (3.7). 3 The Variational Framework and the Minimax Estimate Inspired by [33] and [14], we give the deﬁnite Adams functions eφn(x) supported in B2d(0) ⊂Ωas follows. Now we give a precise estimation about the Mountain pass level c∗deﬁned by (3.7). Inspired by [33] and [14], we give the deﬁnite Adams functions eφn(x) supported in B2d(0) ⊂Ωas follows. eφn(x) =            q ln n 8π2 − n2 δ2√ 32π2 ln n\|x\|2 + 1 √ 32π2 ln n, for \|x\| ⩽d n; ln(d/\|x\|) √ 8π2 ln n, for d n < \|x\| ⩽d; ηn(x), for d < \|x\| ≤2d; 0, for \|x\| > 2d, (3.12) (3.12) where ηn(x) = − 1 2 √ 2π2 ln nd3 (\|x\| −d)3 + 1 √ 2π2 ln nd2 (\|x\| −d)2 − 1 2 √ 2π2 ln nd(\|x\| −d) and d was given in (f6). Moreover, ηn(x) is a radial function on annulus B2d \ Bd satisfying the boundary condition where ηn(x) = − 1 2 √ 2π2 ln nd3 (\|x\| −d)3 + 1 √ 2π2 ln nd2 (\|x\| −d)2 − 1 2 √ 2π2 ln nd(\|x\| −d) and d was given in (f6). Moreover, ηn(x) is a radial function on annulus B2d \ Bd satisfying the boundary condition ηn(x)\|∂Bd = 0, ∂ηn(x) ∂ν \|∂Bd = − 4 d √ 128π2 ln n , ηn(x)\|∂Bd = 0, ∂ηn(x) ∂ν \|∂Bd = − 4 d √ 128π2 ln n , and and ηn(x)\|∂B2d = 0, ∂ηn(x) ∂ν \|∂B2d = 0. and ηn(x)\|∂B2d = 0, ∂ηn(x) ∂ν \|∂B2d = 0. ηn(x)\|∂B2d = 0, ∂ηn(x) ∂ν \|∂B2d = 0. a d ηn(x)\|∂B2d = 0, ∂ηn(x) ∂ν \|∂B2d = 0. Then straightforward calculations show that Then straightforward calculations show that Then straightforward calculations show that ∥eφn∥2 = 1 + 21 8 ln n. (3.13) φn(x) = eφn(x) ∥eφn∥ , ∥eφn∥2 = 1 + 21 8 ln n. (3.13) ∥eφn∥2 = 1 + 21 8 ln n. (3.13) Now we set φn(x) = eφn(x) ∥eφn∥ , then it follows that ∥φ ∥= 1 (3 14) ∥eφn∥2 = 1 + 21 8 ln n. ∥eφn∥2 = 1 + 21 8 ln n. ( ∥eφn∥2 = 1 + 21 8 ln n. (3.13) Now we set e (3.13) Now we set then it follows that then it follows that ∥φn∥= 1. (3.14) then it follows that ∥φn∥= 1. (3.14) then it follows that ∥φn∥= 1. 3 The Variational Framework and the Minimax Estimate (3.14) M i d b [20] h h f ll i l Motivated by [20], we have the following lemma: Motivated by [20], we have the following lemma: Motivated by [20], we have the following lemma: Lemma 3.4. Assume that (m1), (m2) and (f1) −(f6) hold. Then there exists n ∈N such that Lemma 3.4. Assume that (m1), (m2) and (f1) −(f6) hold. Then there exists n ∈N such that c∗⩽max t⩾0 Φ √ 2 2 tφn(x), √ 2 2 tφn(x) < 1 2M 4π2(4 + µ −2α) β0 . (3.15) (3.15) Proof. By a straight estimation, we have Z Bd/n 1 \|y\|α dy Z Bd/n 1 \|x\|α\|x −y\|4−µ dx = d4+µ−2α Z B1/n 1 \|y\|α dy Z B1/n 1 \|x\|α\|x −y\|4−µ dx ⩾d4+µ−2α 1 n −2α Z B1/n dx Z B1/n 1 \|x −y\|4−µ dy ⩾d4+µ−2α 1 n −2α Z B1/n dx Z B1/n 1 \|z\|4−µ dz ⩾d4+µ−2α 1 n −2α Z B1/n dx Z B1/n−\|x\| 1 \|z\|4−µ dz = d4+µ−2α 1 n −2α 4π4 µ Z 1 n 0 1 n −r µ r3dr = 24π4 µ(µ + 1)(µ + 2)(µ + 3)(µ + 4) d n 4+µ−2α . 19 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Brieﬂy, we deduce Z Bd/n 1 \|y\|α dy Z Bd/n 1 \|x\|α\|x −y\|4−µ dx ⩾Cµ d n 4+µ−2α , (3.16) where Cµ = 24π4 µ(µ+1)(µ+2)(µ+3)(µ+4). According to the Remark 1 and (f6), we know lim inf t,s→∞ (\|t\| + \|s\|)F(x, t, s) eβ0(\|t\|2+\|s\|2) > κ, for any x ∈Ω, where  4π2(4 + µ −2α)e 21(4+µ−2α)−8 8 m 4π2(4+µ−2α) β 1 2 Brieﬂy, we deduce Z Bd/n 1 \|y\|α dy Z Bd/n 1 \|x\|α\|x −y\|4−µ dx ⩾Cµ d n 4+µ−2α , (3.16) where Cµ = 24π4 µ(µ+1)(µ+2)(µ+3)(µ+4). According to the Remark 1 and (f6), we know (\|t\| + \| \|)F( t ) Brieﬂy, we deduce Brieﬂy, we deduce Z Bd/n 1 \|y\|α dy Z Bd/n 1 \|x\|α\|x −y\|4−µ dx ⩾Cµ d n 4+µ−2α , (3.16) 24 4 Z Bd/n 1 \|y\|α dy Z Bd/n 1 \|x\|α\|x −y\|4−µ dx ⩾Cµ d n 4+µ−2α , (3.16) (3.16) Z Bd/n \|y\|α Z Bd/n \|x\|α\|x −y\|4 µ n where Cµ = 24π4 µ(µ+1)(µ+2)(µ+3)(µ+4). According to the Remark 1 and (f6), we know ere Cµ = 24π4 µ(µ+1)(µ+2)(µ+3)(µ+4). 3 The Variational Framework and the Minimax Estimate According to the Remark 1 and (f6), we know lim inf t,s→∞ (\|t\| + \|s\|)F(x, t, s) eβ0(\|t\|2+\|s\|2) > κ, for any x ∈Ω, lim inf t,s→∞ (\|t\| + \|s\|)F(x, t, s) eβ0(\|t\|2+\|s\|2) > κ, for any x ∈Ω, κ >   4π2(4 + µ −2α)e 21(4+µ−2α)−8 8 m 4π2(4+µ−2α) β0 β2 0Cµd4+µ−2α   1 2 in (f6), we choose ε > 0 such that in (f6), we choose ε > 0 such that in (f6), we choose ε > 0 such that (κ −ε)2 (1 + ε)2 > 4π2(4 + µ −2α)e 21(4+µ−2α) 8 β2 0Cµd4+µ−2αe m 4π2(4 + µ −2α) β0 (3.17) 21(4 + µ −2α) 8 + ln 4π2(1 + ε)2(4 + µ −2α)m 4π2(4+µ−2α) β0 (κ −ε)2β2 0Cµd4+µ−2α < 1 −ε 1 + ε. (3.18) (κ −ε)2 (1 + ε)2 > 4π2(4 + µ −2α)e 21(4+µ−2α) 8 β2 0Cµd4+µ−2αe m 4π2(4 + µ −2α) β0 (3.17) (3.17) and 21(4 + µ −2α) 8 + ln 4π2(1 + ε)2(4 + µ −2α)m 4π2(4+µ−2α) β0 (κ −ε)2β2 0Cµd4+µ−2α < 1 −ε 1 + ε. (3.18) (3.18) Using (f6), we know that there exists tε > 0 such that Using (f6), we know that there exists tε > 0 such that (\|t1\| + \|t2\|)F(x, t1, t2) ⩾(κ −ε)eβ0\|(t1,t2)\|2, ∀x ∈Ω, \|t1\|, \|t2\| ⩾tε. (3.19) (3.19) er are four possible cases as follows. From now on, in the sequel, all inequalities hold for large n ∈N. q Case i) t ∈ 0, q 2π2(4+µ−2α) β0 . Then it follows that Case i) t ∈ 0, q 2π2(4+µ−2α) β0 . Then it follows that Φ √ 2 2 tφn, √ 2 2 tφn = 1 2M t2∥φn∥2 −1 2 Z Ω " Iµ ∗F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α # F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α dx ≤1 2M 2π2(4 + µ −2α) β0 . Clearly, there exists n ∈N such that (3.15) holds. Clearly, there exists n ∈N such that (3.15) holds. Case ii) t ∈ q 2π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), it follows that Clearly, there exists n ∈N such that (3.15) holds. 3 The Variational Framework and the Minimax Estimate (3.22) sing (3.22) we obtain m(t2 n) = Cµβ2 0(κ −ε)2d4+µ−2α(ln n)2 π2(4 + µ −2α) (4(ln n)2 + 4 ln n + 1) n4+µ−2α e(4π2∥eφn∥2)−1β0t2 n ln n. (3.22) m(t2 n) = Cµβ2 0(κ −ε)2d4+µ−2α(ln n)2 π2(4 + µ −2α) (4(ln n)2 + 4 ln n + 1) n4+µ−2α e(4π2∥eφn∥2)−1β0t2 n ln n. (3.22) (3.22) Then by using (3.22) we obtain lim n→∞t2 n = 4π2(4 + µ −2α) β0 . (3.23) Then by using (3.22) we obtain Then by using (3.22) we obtain lim n→∞t2 n = 4π2(4 + µ −2α) β0 . (3.23) lim n→∞t2 n = 4π2(4 + µ −2α) β0 . (3.23) Set Set Set A := ln 4π2(1 + ε)(4 + µ −2α)m 4π2(4+µ−2α) β0 Cµβ2 0(κ −ε)2d4+µ−2α . (3.24) Set A := ln 4π2(1 + ε)(4 + µ −2α)m 4π2(4+µ−2α) β0 Cµβ2 0(κ −ε)2d4+µ−2α . (3.24) Then (3.17) and (3.18) show that A := ln 4π2(1 + ε)(4 + µ −2α)m 4π2(4+µ−2α) β0 Cµβ2 0(κ −ε)2d4+µ−2α . (3.24) Cµβ2 0(κ −ε)2d4+µ−2α ( ) Then (3.17) and (3.18) show that Then (3.17) and (3.18) show that Then (3.17) and (3.18) show that Then (3.17) and (3.18) show that (1 + ε) max 21(4 + µ −2α) 8 + A, 0 −(1 −ε) < 0. (3.25) (3.25) From (3.22), (3.23) and (3.24), we have From (3.22), (3.23) and (3.24), we have t2 n = 4π2(4 + µ −2α)∥eφn∥2 β0 " 1 + ln (4 + µ −2α)π2m(t2 n) 4(ln n)2 + 4 ln n + 1 (4 + µ −2α) ln n −ln((ln n)2) + ln Cµβ0(κ −ε)2d4+µ−2α (4 + µ −2α) ln n # ⩽4π2(4 + µ −2α) β0 + 4π2A β0 ln n + 21(4 + µ −2α)π2 2β0 ln n + O 1 (ln n)2 (3.26) # ⩽4π2(4 + µ −2α) β0 + 4π2A β0 ln n + 21(4 + µ −2α)π2 2β0 ln n + O 1 (ln n)2 (3.26) and ϕn(t) ⩽ϕn(tn) = 1 2M(t2 n) −2π2∥eφn∥2m(t2 n) β0 ln n , ∀t ≥0. (3.27) and and ϕn(t) ⩽ϕn(tn) = 1 2M(t2 n) −2π2∥eφn∥2m(t2 n) β0 ln n , ∀t ≥0. (3.27) ϕn(t) ⩽ϕn(tn) = 1 2M(t2 n) −2π2∥eφn∥2m(t2 n) β0 ln n , ∀t ≥0. 3 The Variational Framework and the Minimax Estimate Case ii) t ∈ q 2π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), it follows that Case ii) t ∈ q 2π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), it follows that Case ii) t ∈ q 2π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), it follows that Z Ω " Iµ ∗F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α # F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α dx ⩾ Z Bd/n Z Bd/n F(x, √ 2 2 tφn(x), √ 2 2 tφn(x))F(y, √ 2 2 tφn(y), √ 2 2 tφn(y)) \|x\|α\|x −y\|4−µ\|y\|α dxdy ⩾(κ −ε)2 2t2 Z Bd/n Z Bd/n eβ0t2φ2 n(x)+β0t2φ2 n(y) φn(x)φn(y)\|x\|α\|x −y\|4−µ\|y\|α dxdy ⩾ β0(κ −ε)2 8π2(4 + µ −2α) Z Bd/n Z Bd/n eβ0t2φ2 n(x)+β0t2φ2 n(y) φn(x)φn(y)\|x\|α\|x −y\|4−µ\|y\|α dxdy ⩾ 4Cµβ0∥eφn∥2(κ −ε)2d4+µ−2α ln n (4 + µ −2α) (4(ln n)2 + 4 ln n + 1) n4+µ−2α e(4π2∥eφn∥2)−1β0t2 ln n. (3.20) (3.20) 20 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Then from (3.1), (3.14) and (3.20), it holds that Then from (3.1), (3.14) and (3.20), it holds that Then from (3.1), (3.14) and (3.20), it holds that Φ √ 2 2 tφn, √ 2 2 tφn = 1 2M t2∥φn∥2 −1 2 Z Ω " Iµ ∗F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α # F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α dx ⩽1 2M(t2) − 2Cµβ0∥eφn∥2(κ −ε)2d4+µ−2α ln n (4 + µ −2α) (4(ln n)2 + 4 ln n + 1) n4+µ−2α e(4π2∥eφn∥2)−1β0t2 ln n =: ϕ(t). (3.21) (3.21) Let tn > 0 such that ϕ′ n(tn) = 0, thus Let tn > 0 such that ϕ′ n(tn) = 0, thus m(t2 n) = Cµβ2 0(κ −ε)2d4+µ−2α(ln n)2 π2(4 + µ −2α) (4(ln n)2 + 4 ln n + 1) n4+µ−2α e(4π2∥eφn∥2)−1β0t2 n ln n. M(t1 + t2) ⩽M(t1) + t1 θ " 1 + t2 t1 θ −1 # m(t1), ∀t1, t2 > 0. 3 The Variational Framework and the Minimax Estimate Clearly, in this case, the above estimate implies that there exists n large enough such that (3.15) holds. ii) t ∈ q 4π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 (1 + ε) . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, f6), (3.12), (3.16) and (3.19), the process is similar to case ii, we delete it. Case iii) t ∈ q 4π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 (1 + ε) . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), the process is similar to case ii, we delete it. Case iv) t ∈ q 4π2(4+µ−2α) β0 (1 + ε), +∞ . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (3.1), (3.12), (3.14), (3.16) and (3.19), it follows that Case iii) t ∈ q 4π2(4+µ−2α) β0 , q 4π2(4+µ−2α) β0 (1 + ε) . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (f6), (3.12), (3.16) and (3.19), the process is similar to case ii, we delete it. Case iv) t ∈ q 4π2(4+µ−2α)(1 + ε) +∞ Then √ 2tφ (x) ⩾t for x ∈B (0) and for large n ∈N from from (f6), (3.12), (3.16) and (3.19), the process is similar to case ii, we delete it. Case iv) t ∈ q 4π2(4+µ−2α) β0 (1 + ε), +∞ . Then √ 2 2 tφn(x) ⩾tε for x ∈Bd/n(0) and for large n ∈N, from (3.1), (3.12), (3.14), (3.16) and (3.19), it follows that Φ √ 2 2 tφn, √ 2 2 tφn = 1 2M(t2∥φn∥2) −1 2 Z Ω " Iµ ∗F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α # F(x, √ 2 2 tφn, √ 2 2 tφn) \|x\|α dx ⩽1 2M(t2) −8Cµπ2∥eφn∥2(κ −ε)2d4+µ−2α ln n (4(ln n)2 + 4 ln n + 1) t2n4+µ−2α e(4π2∥eφn∥2)−1β0t2 ln n ⩽1 2M 4π2(4 + µ −2α)(1 + ε) β0 −2Cµβ0 ln n∥eφn∥2(κ −ε)2d4+µ−2αe (8ε ln n−21)(4+µ−2α) ln n 8 ln n+21 (4 + µ −2α)(1 + ε) (4(ln n)2 + 4 ln n + 1) ⩽1 3M 4π2(4 + µ −2α) β0 , then there exists n ∈N such that (3.15) holds. 3 The Variational Framework and the Minimax Estimate In the above derivation process, we use the fact that the function 1 2M(t2) −8Cµπ2∥eφn∥2(κ−ε)2d4+µ−2α ln n (4(ln n)2+4 ln n+1)t2n4+µ−2α e(4π2∥eφn∥2)−1β0t2 ln n is decreasing on t ∈ q 4π2(4+µ−2α) β0 (1 + ε), +∞ , ( ) since its stagnation points tend to q 4π2(4+µ−2α) β0 as n →∞. ( ) since its stagnation points tend to q 4π2(4+µ−2α) β0 as n →∞. 3 The Variational Framework and the Minimax Estimate (3.27) (3.27) By (1.5) in Remark 1, we know By (1.5) in Remark 1, we know M(t1 + t2) ⩽M(t1) + t1 θ " 1 + t2 t1 θ −1 # m(t1), ∀t1, t2 > 0. 21 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Then using (m1), (1.5), (3.23), (3.26) and (3.27), we obtain Then using (m1), (1.5), (3.23), (3.26) and (3.27), we obtain Then using (m1), (1.5), (3.23), (3.26) and (3.27), we obtain (m1), (1.5), (3.23), (3.26) and (3.27), we obtain ϕn(t) ⩽ϕn(tn) = 1 2M(t2 n) −2π2∥eφn∥2m(t2 n) β0 ln n ⩽1 2M 4π2(4 + µ −2α) β0 + 4π2A β0 ln n + 21(4 + µ −2α)π2 2β0 ln n + O 1 (ln n)2 − 2π2(1 −ε)m 4π2(4+µ−2α) α0 β0 ln n ⩽1 2M 4π2(4 + µ −2α) β0 + (1 + ε) max n 21(4+µ−2α) 8 + A, 0 o −(1 −ϵ) β0 ln n × 2π2m 4π2(4 + µ −2α) β0 + O 1 (ln n)2 . (3.28) ϕn(t) ⩽ϕn(tn) = 1 2M(t2 n) −2π2∥eφn∥2m(t2 n) β0 ln n ⩽1 2M 4π2(4 + µ −2α) β0 + 4π2A β0 ln n + 21(4 + µ −2α)π2 2β0 ln n + O 1 (ln n)2 − 2π2(1 −ε)m 4π2(4+µ−2α) α0 β0 ln n ⩽1 2M 4π2(4 + µ −2α) β0 + (1 + ε) max n 21(4+µ−2α) 8 + A, 0 o −(1 −ϵ) β0 ln n × 2π2m 4π2(4 + µ −2α) β0 + O 1 (ln n)2 . (3 ⩽1 2M 4π2(4 + µ −2α) β0 + (1 + ε) max n 21(4+µ−2α) 8 + A, 0 o −(1 −ϵ) β0 ln n × 2π2m 4π2(4 + µ −2α) β0 + O 1 (ln n)2 . (3.28) (3.28) Hence, combining (3.21) with (3.28), one has Hence, combining (3.21) with (3.28), one has Φ √ 2 2 tφn, √ 2 2 tφn ⩽1 2M 4π2(4 + µ −2α) β0 + (1 + ε) max n 21(4+µ−2α) 8 + A, 0 o −(1 −ε) β0 ln n × 2π2m 4π2(4 + µ −2α) β0 + O 1 (ln n)2 . early, in this case, the above estimate implies that there exists n large enough such that (3.15) holds. 4 The proof of Theorem 1.1 In this section, we will give the proof of Theorem 1.1. 22 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Lemma 4.1. Assume that (m2) and (f3) hold, then c∗⩽b, where b = infN Φ in (1.7). Lemma 4.1. Assume that (m2) and (f3) hold, then c∗⩽b, where b = infN Φ in (1.7). Proof. For each (u, v) ∈N, then it follows that ⟨Φ′(u, v), (u, v)⟩= 0. Now we deﬁne the continuous map g : (0, +∞) →R such that g(t) = Φ(tu, tv). By using (3.2) we have g′(t) = m ∥(tu, tv)∥2 ∥(u, v)∥2t − Z Ω Iµ ∗F(x, tu, tv) \|x\|α f1(x, tu, tv)u + f2(x, tu, tv)v \|x\|α dx, g′(t) = m ∥(tu, tv)∥2 ∥(u, v)∥2t − Z Ω Iµ ∗F(x, tu, tv) \|x\|α f1(x, tu, tv)u + f2(x, tu, tv)v \|x\|α dx, for all t ∈(0, +∞) and g′(t) = g′(t) −t2θ−1⟨Φ′(u, v), (u, v)⟩ = m ∥(tu, tv)∥2 ∥(u, v)∥2t −t2θ−1m ∥(u, v)∥2 ∥(u, v)∥2 + t2θ−1 Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v \|x\|α dx − Z Ω Iµ ∗F(x, tu, tv) \|x\|α f1(x, tu, tv)u + f2(x, tu, tv)v \|x\|α dx = t2θ−1 " m ∥(tu, tv)∥2 (∥(tu, tv)∥2)θ−1 −m ∥(u, v)∥2 (∥(u, v)∥2)θ−1 # ∥(u, v)∥2θ + t2θ−1 Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v \|x\|α dx −1 t2θ Z Ω Iµ ∗F(x, tu, tv) \|x\|α f1(x, tu, tv)tu + f2(x, tu, tv)tv \|x\|α dx . From Remark 1, we choose p = θ < l, then we derive that From Remark 1, we choose p = θ < l, then we derive that From Remark 1, we choose p = θ < l, then we derive that tf1(x, t, s) −θF(x, t, s) > 0, sf2(x, t, s) −θF(x, t, s) > 0, for all (x, t, s) ∈Ω× R2, along with (f3) which shows that for all (x, t, s) ∈Ω× R2, along with (f3) which shows that t 7→F(x, tu, tv) tθ is nondecreasing for t > 0. (4.1) t 7→F(x, tu, tv) tθ is nondecreasing for t > 0. 4 The proof of Theorem 1.1 (4.1) (4.1) Then for 0 < t ⩽1, x ∈Ωfrom (f3) and (4.1) , we obtain Then for 0 < t ⩽1, x ∈Ωfrom (f3) and (4.1) , we obtain Then for 0 < t ⩽1, x ∈Ωfrom (f3) and (4.1) , we obtain g′(t) ⩾t2θ−1 m ∥(tu, tv)∥2 (∥(tu, tv)∥2)θ−1 −m ∥(u, v)∥2 (∥(u, v)∥2)θ−1 ! ∥(u, v)∥2θ + t2θ−1 Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u uθ −f1(x, tu, tv)tu (tu)θ uθdx + Z Ω Iµ ∗F(x, u, v) \|x\|α f2(x, u, v)v vθ −f2(x, tu, tv)tv (tv)θ vθdx ⩾0. This shows that g′(t) ⩾0 for 0 < t ⩽1 and g′(t) < 0 for t > 1. So we obtain g(1) = maxt⩾0 Φ(tu, tv), thus Φ(u, v) = maxt⩾0 Φ(tu, tv). Now we deﬁne h : [0, 1] →H2 0(Ω, R2) as h(t) = (t0u, t0v)t where t0 > 1 is such that Φ(t0u, t0v) < 0. So, h ∈Γ which implies that This shows that g′(t) ⩾0 for 0 < t ⩽1 and g′(t) < 0 for t > 1. So we obtain g(1) = maxt⩾0 Φ(tu, tv), thus Φ(u, v) = maxt⩾0 Φ(tu, tv). Now we deﬁne h : [0, 1] →H2 0(Ω, R2) as h(t) = (t0u, t0v)t where t0 > 1 is such that Φ(t0u, t0v) < 0. So, h ∈Γ which implies that c∗⩽max t∈[0,1] Φ(h(t)) ⩽max t⩾0 Φ(tu, tv) = Φ(u, v). c∗⩽max t∈[0,1] Φ(h(t)) ⩽max t⩾0 Φ(tu, tv) = Φ(u, v). Since (u, v) ∈N is arbitrary, we get c∗⩽b. 23 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 Lemma 4.2. Assume that (f4) holds and let {(un, vn)} ∈H2 0(Ω, R2) be a Palais-Smale sequence for Φ, i.e. Φ(un, vn) →c∗, Φ′(un, vn) →0 as n →∞. 4 The proof of Theorem 1.1 Then there exists (u, v) ∈H2 0(Ω, R2) such that, up to subaequence, (un, vn) ⇀(u, v) weakly in H2 0(Ω, R2) Then there exists (u, v) ∈H2 0(Ω, R2) such that, up to subaequence, (un, vn) ⇀(u, v) weakly in H2 0(Ω, R2), Z I F(x, un, vn) fi(x, un, vn) d Z I F(x, u, v) fi(x, u, v) d (4 2) Ω Iµ ∗F(x, un, vn) \|x\|α fi(x, un, vn) \|x\|α ϕdx → Z Ω Iµ ∗F(x, u, v) \|x\|α fi(x, u, v) \|x\|α ϕdx (4.2) Z Ω Iµ ∗F(x, un, vn) \|x\|α fi(x, un, vn) \|x\|α ϕdx → Z Ω Iµ ∗F(x, u, v) \|x\|α fi(x, u, v) \|x\|α ϕdx (4.2) for all ϕ ∈C∞ 0 (Ω) and i = 1 2 and (4.2) for all ϕ ∈C∞ 0 (Ω) and i = 1, 2, and Z Ω Iµ ∗F(x, un, vn) \|x\|α F(x, un, vn) \|x\|α dx → Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx. Proof. Similar to [31, Lemma 3.3], so we delete the proof. Proof. Similar to [31, Lemma 3.3], so we delete the proof. ow we are ready to give the proof of our main result. Now we are ready to give the proof of our main result. Proof of Theorem 1.1. Let {(un, vn)} be a Palais Smale sequence at the Mountain pass level c∗. By using Lemma 3.3, we know {(un, vn)} is bounded, then there exists u, v ∈H2 0(Ω) such that, up to subsequence, un ⇀u, vn ⇀v weakly in H2 0(Ω) as n →∞. Next we will make some claims as follows. Claim 1: u, v ̸≡0. , ̸ If u = 0 or v = 0, by using Lemma 4.2 we obtain , ̸ If u = 0 or v = 0, by using Lemma 4.2 we obtain Z Ω Iµ ∗F(x, un, vn) \|x\|α F(x, un, vn) \|x\|α dx →0 as n →∞. (4.3) (4.3) From (3.9) and (4.3) we know limn→∞Φ(un, vn) = 1 2 limn→∞M ∥(un, vn)∥2 = c∗. Now from (3.15) and m(t) > 0 for t ≥0 which implies M(t) is strictly increasing we obtain From (3.9) and (4.3) we know limn→∞Φ(un, vn) = 1 2 limn→∞M ∥(un, vn)∥2 = c∗. 4 The proof of Theorem 1.1 Now from (3.15) and m(t) > 0 for t ≥0 which implies M(t) is strictly increasing we obtain ∥(un, vn)∥2 < 4π2(4 + µ −2α) β0 , ∥(un, vn)∥2 < 4π2(4 + µ −2α) β0 , for n large enough, and this shows that supn R Ω(fi(un, vn))qdx < +∞for some q > 8 4+µ−2α, i = 1, 2. Besides this, from Lemma 2.2, Proposition 2.5, (3.2) and Vitali’s convergence theorem we derive that for n large enough, and this shows that supn R Ω(fi(un, vn))qdx < +∞for some q > 8 4+µ−2α, i = 1, 2. Besides this, from Lemma 2.2, Proposition 2.5, (3.2) and Vitali’s convergence theorem we derive that Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)un + f2(x, un, vn)vn \|x\|α dx →0 as n →∞. Then from (3.8), we know limn→∞⟨Φ′(un, vn), (un, vn)⟩= 0, hence we obtain Then from (3.8), we know limn→∞⟨Φ′(un, vn), (un, vn)⟩= 0, hence we obtain lim n→∞m ∥(un, vn)∥2 ∥(un, vn)∥2 = 0. lim n→∞m ∥(un, vn)∥2 ∥(un, vn)∥2 = 0. From (f1), we know it is obvious that limn→∞∥(un, vn)∥2 = 0. Then (3.9) and (4.3) show that c∗= limn→∞Φ(un, vn) = 0, which contradicts c∗> 0. Hence we claim that u, v ̸≡0, now we assume that ∥(un, vn)∥2 →σ2 as n →∞. From Lemma 4.2 we obtain Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)φ + f2(x, un, vn)ψ \|x\|α dx → Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, as n →∞, and m(σ2) Z Ω (∆u∆φ + ∆v∆ψ)dx = Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, (4.4) m(σ2) Z Ω (∆u∆φ + ∆v∆ψ)dx = Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, (4.4) (4.4) 24 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 for all φ, ψ ∈H2 0(Ω). Indeed, if we take φ = u−and ψ = 0 in (4.4), we have m σ2 ∥u−∥= 0. By using (m1), we get that u−= 0 a.e. in Ω. Therefore u, v ≥0 a.e. in Ω. for all φ, ψ ∈H2 0(Ω). 4 The proof of Theorem 1.1 Indeed, if we take φ = u−and ψ = 0 in (4.4), we have m σ2 ∥u−∥= 0. By using (m1), we get that u−= 0 a.e. in Ω. Therefore u, v ≥0 a.e. in Ω. Claim 2: m ∥(u, v)∥2 ∥(u, v)∥2 ⩾ R Ω h Iµ ∗F (x,u,v) \|x\|α i f1(x,u,v)u+f2(x,u,v)v \|x\|α dx. Claim 2: m ∥(u, v)∥2 ∥(u, v)∥2 ⩾ R Ω h Iµ ∗F (x,u,v) \|x\|α i f1(x,u,v)u+f2(x,u,v)v \|x\|α dx. Supposing by contradiction that m ∥(u, v)∥2 ∥(u, v)∥2 < Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v \|x\|α dx, (4.5) (4.5) let h(t) = ⟨Φ′(tu, tv), (tu, tv)⟩, then from (3.2) and (4.5) we know h(1) = ⟨Φ′(u, v), (u, v)⟩< 0. Then for t > 0 small enough, using (f5) and Remark 1, we obtain that let h(t) = ⟨Φ′(tu, tv), (tu, tv)⟩, then from (3.2) and (4.5) we know h(1) = ⟨Φ′(u, v), (u, v)⟩< 0. Then for t > 0 small enough, using (f5) and Remark 1, we obtain that ⟨Φ′(tu, tv), (tu, tv)⟩ ⩾m0t2∥(u, v)∥2 −1 2 Z Ω Z Ω (f1(x, tu, tv)tu + f2(x, tu, tv)tv) (f1(y, tu, tv)tu + f2(y, tu, tv)tv) \|x −y\|4−µ\|x\|α\|y\|α dxdy ⩾m0t2∥(u, v)∥2 −t2r+2 2 Z Ω Z Ω (ur + vr)u + (ur + vr)v \|x −y\|4−µ\|y\|α dy (ur + vr)u + (ur + vr)v \|x\|α dx > 0 ⟨Φ′(tu, tv), (tu, tv)⟩ > 0. Since r > 0, then the above inequality shows that h(t) > 0 for t small enough. So there exists a t∗∈(0, 1) such that h(t∗) = ⟨Φ′(t∗u, t∗v), (t∗u, t∗v)⟩= 0, i.e. (t∗u, t∗v) ∈N. So using (3.1), (3.2), Lemma 4.1 and the lower semicontinuity we get Since r > 0, then the above inequality shows that h(t) > 0 for t small enough. So there exists a t∗∈(0, 1) such that h(t∗) = ⟨Φ′(t∗u, t∗v), (t∗u, t∗v)⟩= 0, i.e. (t∗u, t∗v) ∈N. 4 The proof of Theorem 1.1 So using (3.1), (3.2), Lemma 4.1 and the lower semicontinuity we get ( ) ⟨ ( , ), ( , )⟩ , ( , ) ng (3.1), (3.2), Lemma 4.1 and the lower semicontinuity we get c∗⩽b ⩽Φ(t∗u, t∗v) −1 2θ ⟨Φ′(t∗u, t∗v), (t∗u, t∗v)⟩ = 1 2M ∥(t∗u, t∗v)∥2 −1 2 Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α F(x, t∗u, t∗v) \|x\|α dx −1 2θ m ∥(t∗u, t∗v)∥2 ∥(t∗u, t∗v)∥2 + 1 2θ Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α f1(x, t∗u, t∗v)t∗u + f2(x, t∗u, t∗v)t∗v \|x\|α dx < 1 2M ∥(u, v)∥2 −1 2θ m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α f1(x, t∗u, t∗v)t∗u + f2(x, t∗u, t∗v)t∗v −θF(x, t∗u, t∗v) \|x\|α dx ⩽1 2M ∥(u, v)∥2 −1 2θ m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v −θF(x, u, v) \|x\|α dx ⩽lim inf n→∞ 1 2M ∥(un, vn)∥2 −1 2θ m ∥(un, vn)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)u + f2(x, un, vn)vn −θF(x, un, vn) \|x\|α dx = lim inf n→∞ Φ(un, vn) −1 2θ ⟨Φ′(un, vn), (un, vn)⟩ = c∗. 4 The proof of Theorem 1.1 c∗⩽b ⩽Φ(t∗u, t∗v) −1 2θ ⟨Φ′(t∗u, t∗v), (t∗u, t∗v)⟩ = 1 2M ∥(t∗u, t∗v)∥2 −1 2 Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α F(x, t∗u, t∗v) \|x\|α dx −1 2θ m ∥(t∗u, t∗v)∥2 ∥(t∗u, t∗v)∥2 2θ + 1 2θ Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α f1(x, t∗u, t∗v)t∗u + f2(x, t∗u, t∗v)t∗v \|x\|α dx < 1 2M ∥(u, v)∥2 −1 2θ m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, t∗u, t∗v) \|x\|α f1(x, t∗u, t∗v)t∗u + f2(x, t∗u, t∗v)t∗v −θF(x, t∗u, t∗v) \|x\|α dx ⩽1 2M ∥(u, v)∥2 −1 2θ m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v −θF(x, u, v) \|x\|α dx ⩽lim inf n→∞ 1 2M ∥(un, vn)∥2 −1 2θ m ∥(un, vn)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)u + f2(x, un, vn)vn −θF(x, un, vn) \|x\|α dx = lim inf n→∞ Φ(un, vn) −1 2θ ⟨Φ′(un, vn), (un, vn)⟩ = c∗. + 1 2θ Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)u + f2(x, un, vn)vn −θF(x, un, vn) \|x\|α dx = lim inf n→∞ Φ(un, vn) −1 2θ ⟨Φ′(un, vn), (un, vn)⟩ = c∗. This gives a contradiction and completes the proof of Claim 2. Claim 3: Φ(u, v) = c∗. ( ) By using the weakly lower semicontinuity of norms we know Φ(u, v) ⩽limn→+∞Φ(un, vn) = c∗. If Φ(u, v) < c∗, By using the weakly lower semicontinuity of norms we know Φ(u, v) ⩽limn→+∞Φ(un, vn) = c∗. If Φ(u, v) < c∗, 25 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 then from (3.1) and Lemma 4.2 we know M(∥(u, v)∥2) < limn→+∞M(∥(un, vn)∥2). Since M(t) is increasing and continuous and limn→∞∥(un, vn)∥2 = σ2, we obtain ∥(u, v)∥2 < σ2. Moreover, from (3.1), (3.8) and Lemma 4.2 we derive that then from (3.1) and Lemma 4.2 we know M(∥(u, v)∥2) < limn→+∞M(∥(un, vn)∥2). Since M(t) is increasing and continuous and limn→∞∥(un, vn)∥2 = σ2, we obtain ∥(u, v)∥2 < σ2. Now let (eun, evn) = un ∥(un, vn)∥, vn ∥(un, vn)∥ , obviously we obtain ∥(eun, evn)∥= 1 and (eun, evn) ⇀(eu, ev) = ( u σ , v σ ) weakly in H2 0(Ω, R2). From Lemma 2.3, we have that Z 32 2 obviously we obtain ∥(eun, evn)∥= 1 and (eun, evn) ⇀(eu, ev) = ( u σ , v σ ) weakly in H2 0(Ω, R2). From Lemma 2.3, we have that Z 32 2 we obtain ∥(eun, evn)∥= 1 and (eun, evn) ⇀(eu, ev) = ( u σ , v σ ) weakly in H2 0(Ω, R2). From Lemma 2.3, we sup n∈N Z Ω exp β(\|eun\|2 + \|evn\|2) dx < +∞, for 1 < β < 32π2 1 −∥(eu, ev)∥2 . Then from (1.3), (3.1) and Claim 2 we obtain Then from (1.3), (3.1) and Claim 2 we obtain Then from (1.3), (3.1) and Claim 2 we obtain Φ(u, v) = 1 2M ∥(u, v)∥2 −1 2 Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx ≥1 2M ∥(u, v)∥2 −1 2 Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx −1 2θ m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v \|x\|α dx = 1 2θ θM ∥(u, v)∥2 −m ∥(u, v)∥2 ∥(u, v)∥2 + 1 2θ Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v −θF(x, u, v) \|x\|α dx ≥0, and from Remak 1, (3.1), Lemma 3.4 and (4.6) we get and from Remak 1, (3.1), Lemma 3.4 and (4.6) we get and from Remak 1, (3.1), Lemma 3.4 and (4.6) we get M σ2 = 2c∗+ Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx = 2c∗−2Φ(u, v) + M ∥(u, v)∥2 < M 4π2(4 + µ −2α) β0 + M ∥(u, v)∥2 ⩽M 4π2(4 + µ −2α) β0 + ∥(u, v)∥2 . For the monotonicity of the function M(t) we know For the monotonicity of the function M(t) we know For the monotonicity of the function M(t) we know σ2 < 1 1 −∥(eu, ev)∥2 4π2(4 + µ −2α) β0 . σ2 < 1 1 −∥(eu, ev)∥2 4π2(4 + µ −2α) β0 . 4 The proof of Theorem 1.1 Moreover, from (3.1), (3.8) and Lemma 4.2 we derive that M σ2 = lim n→∞M ∥(un, vn)∥2 = 2 c∗+ 1 2 Z Ω Iµ ∗F(x, u, v) \|x\|α F(x, u, v) \|x\|α dx . (4.6) (4.6) Now let Now let Thus for n ∈N large enough it is possible p > β0 and p close to β0 such that Thus for n ∈N large enough it is possible p > β0 and p close to β0 such that 8 4 + µ −2αp∥(un, vn)∥2 ⩽ 32π2 1 −∥(eu, ev)∥2 . 8 4 + µ −2αp∥(un, vn)∥2 ⩽ 32π2 1 −∥(eu, ev)∥2 . From Lemma 2.3, there exists C > 0 such that Z Ω exp 8 4 + µ −2αp \|un\|2 + \|vn\|2 ⩽C 26 Wu; Asian J. Math. Comp. Res., vol. 31, no. 2, pp. 8-28, 2024; Article no.AJOMCOR.12012 and Z Ω Iµ ∗F(x, un, vn) \|x\|α f1(x, un, vn)un + f2(x, un, vn)vn \|x\|α dx → Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)u + f2(x, u, v)v \|x\|α dx, as n →∞, which implies (un, vn) →(u, v) strongly in H2 0(Ω, R2). Hence Φ(u, v) = c∗which gives a contradiction and completes the proof of Claim 3. as n →∞, which implies (un, vn) →(u, v) strongly in H2 0(Ω, R2). Hence Φ(u, v) = c∗which gives a contradiction and completes the proof of Claim 3. Finalizing the proof of Theorem 1.1: By Claim 3, we deduce that lim n→∞M ∥(un, vn)∥2 = M ∥(u, v)∥2 which indicates (un, vn) →(u, v) in H2 0(Ω, R2). Then ﬁnally we have m ∥(u, v)∥2 Z Ω (∆u∆φ + ∆v∆ψ)dx = Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, m ∥(u, v)∥2 Z Ω (∆u∆φ + ∆v∆ψ)dx = Z Ω Iµ ∗F(x, u, v) \|x\|α f1(x, u, v)φ + f2(x, u, v)ψ \|x\|α dx, for all φ, ψ ∈H2 0(Ω). 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https://openalex.org/W2134688139	https://europepmc.org/articles/pmc3068115?pdf=render	English	null	Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers	Journal of experimental & clinical cancer research	2,011	cc-by	5,609	© 2011 van Haaften et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract 50 μg ( μg ) Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 μg/mL and 5 μg/ mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects. Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. Conclusions: For the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer. In 2004 the genus Haeckeria was reassessed by Orch- ard as C. amaranthoides and since then C. amar- anthoides belongs to the genus Calomeria of the family Asteraceae (Compositae) [4]. As a biennial plant it can grow to more than three metres high, with flowers as waving plume bushes and wrinkly leaves with an aro- matic scent. It is also called incense plant. Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers Caroline van Haaften1, Colin C Duke2, Arij M Weerheim3, Nico PM Smit4, Paul MM van Haard5, Firouz Darroudi6, Baptist JMZ Trimbos1 Correspondence: carocell@planet.nl 1Department of Gynaecology, Leiden University Medical Center, The Netherlands Full list of author information is available at the end of the article van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Abstract Background: Ovarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen. Methods: The leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 μg/mL (crude extract) and 1-10 μg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue. In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test. In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test. Results: Two compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an a-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an a-methylene carboxylic acid. y y Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 μg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 μg/mL (95% confidence interval 4.3 to 6.5 μg/mL). Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 μg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 μg/mL (95% confidence interval 4.3 to 6.5 μg/mL). Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 μg/mL and 5 μg/ mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects. Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. Cell lines and cell cultures Cells used in the assays were five ovarian cell lines (JV, JG, JC, JoN, NF), which were earlier established [9,10], two cell lines OVCAR3 and SKOV3 from the American Type Culture Collection (ATCC) as well as epithelial cells from the ovary (serous papillary cystadenomas) [11] and human dermal fibroblasts primary cultures [12]. Graphs and Statistics Graphs and Statistics Graphing and statistical evaluations were carried out with GraphPad Prism 5 for Windows. Graphing and statistical evaluations were carried out with GraphPad Prism 5 for Windows. 1H-NMR and 13C-NMR analyses 1 13 1H-NMR and 13C-NMR spectroscopy was performed on those plant fractions with clear cytotoxicity effects. 1H- NMR, 13C-NMR and Correlation Spectroscopy (COSY) were performed using a Varian Gemini 300 MHz instru- ment (Palo Alto, CA, USA). The spectra were measured in parts per million (ppm) and were referenced to tetra- methylsilane (TMS = 0 ppm). For the first time we have studied C. amaranthoides for its possible anti-tumor activity. An SL (EPD) and a structurally related sesquiterpene (EPA) have been found, extracted and purified. Among them EPD has shown in vitro and in vivo (mice) high toxicity in ovar- ian cancers. Electrospray ionisation in positive and negative mode (ESI) mass spectrometry analyses were performed using a TSQ 7000 Liquid Chromatography Mass Spectrometer (LC-MS/MS; Thermo, San Jose, CA, USA), equipped with Xcalibur data acquisition and processing software. Short-Column Vacuum Chromatography (SCVC) was performed using a column packed with TLC-grade silica gel H60 (Merck, Darmstadt, Germany)) and applying a step-wise gradient of solvents with increasing polarity. Substances were detected by TLC performed on silica gel coated TLC plates (H60 F254, Merck, Germany) and by 1H-NMR spectroscopy. Structures of purified compounds were determined by mass spectrometry and 1H-NMR and 13C-NMR spectroscopy. In vitro cytotoxicity tests with different fractions of C. amaranthoides In vitro cytotoxicity tests were performed using a non- fluorescent substrate, Alamar blue (BioSource Invitro- gen, UK), as described by Pagé et al. [13]. Ovary cells (1 × 104 or 5 × 104) were seeded in 24-wells plates (Costar, USA) and grown in RPMI-1640, supplemented with 6 mM L-glutamine, 10% fetal calf serum (FCS) (Gibco, Invitrogen, UK) and penicillin (100 units/mL) and streptomycin (100 μg/mL), while normal fibroblasts were grown in Dulbecco’s modified Eagle medium (DMEM), also supplemented with L-glutamine and FCS. The cultures were maintained in a humidified atmo- sphere of 5% CO2 at 37°C. Fractionation of extracts by column chromatography Fractionation of extracts by column chromatography Dried plant material (350 g), cut in small pieces was soaked in chloroform (CHCl3) at room temperature. After 24-48 hours a crude extract of the leaves was removed and evaporated under reduced pressure (21.3 grams, 6.0%). The residue, re-dissolved in CHCl3 (30 mL) was applied to a column (21 cm × 5 cm i.d.) filled with Silicagel (Lichroprep Si 60, particle size 15-25 μm; Merck, Germany). Elution was carried out with a step- wise gradient consisting of hexane:dioxane, 98:2 (v/v 400 mL); hexane:chloroform:dioxane, 88:10:2 (v/v 600 mL); hexane:chloroform:dioxane:ethyl acetate:2-propa- nol, 80:10:2:6:1, (v/v 600 mL) and hexane:chloroform: acetone:methanol, 56:20:16:8, (v/v 400 mL). A total of 157 fractions (10 mL each) were collected and combined into groups based on HPLC analysis. The combined group of fractions showing the highest toxicity towards ovarian cancer cells was further fractionated by short column vacuum chromatography. Methods A voucher specimen of Calomeria amaranthoides, col- lected near Old Bell’s Line of Road, Mount Tomah NSW 2758, Australia, is held in the John Ray Herbar- ium, University of Sydney, Collection number: Silvester 110118-01. Leaves of C. amaranthoides, gathered in the Blue Mountains (Mount Tomah, NSW, Australia) were air- dried while protected from sunlight. Background Calomeria amaranthoides, described both by Ventenat and Smith in 1804 [1,2] as Humea elegans belonging to the genus Haeckeria in the tribe of Inuleae was grown in France and England from seeds originating from the Blue Mountains, New South Wales (NSW) in Australia. The plant is of a monotypic genus, endemic to NSW and Victoria, Australia [3]. The plant family of Asteraceae are known for their natural products. One type includes sesquiterpene lac- tones (SL) which to date is of great interest for their potential as anti-cancer agents as reviewed by Heinrich et al. and Zhang et al. [5,6]. * Correspondence: carocell@planet.nl 1Department of Gynaecology, Leiden University Medical Center, The Netherlands Full list of author information is available at the end of the article van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Page 2 of 6 Page 2 of 6 Ovarian cancer is the fifth leading cause of death in women and remains the leading cause of death from gynaecological malignancy in many countries, in spite of chemotherapy with Platinum derivates and/or Taxol after surgery. Of the malignant epithelial tumors (>90% of all ovarian cancers), the serous papillary variants form the largest subgroup [7,8]. Due to its dismal prog- nosis there is an urgent need for new treatment strategy for ovarian cancer. dioxane (0.5 mL). The first 10 minutes the column was eluted at a flow rate of 0.5 mL/min with eluent A, fol- lowed by 30 minutes with eluent B: hexane (85 mL)- diethyl ether (10 mL)-ethanol (5 mL). 1H-NMR and 13C-NMR analyses 1 13 In vivo pilot experiment An in vivo pilot experiment was performed with 20 BALB/c nude mice (Charles River Laboratories, France). In order to mimic advanced ovarian cancer the mice were injected intraperitoneally (i.p.) with 107 OVCAR3 cells (ATCC) into the abdominal cavity to form ascites. Three groups of mice were examined: 6 control mice (no treatment), 6 mice treated with Cisplatin and 6 mice treated with EPD after ascites had formed. Cells of ascites of two mice were frozen and stored for future experiments. To study reduction of the swollen abdo- men 5 mg/kg Platosin (Cisplatin, Pharma Chemie, The Netherlands) and the isolated compound EPD at a final concentration of 20 mg/kg were administered i.p. A small sample of freshly dried leaves (1.63 g) was extracted with dichloromethane (100 mL), filtered and the dichloromethane removed under reduced pressure leaving a dark green residue (62.6 mg, yield 3.9%). Quantitative 1H-NMR analysis of a CDCl3 solution showed EPD 44%, EPA 31% and a complex mixture of unidentified constituents 25%. A small sample of dried leaves (10.31 g), that had been stored in the dark under ambient conditions for 3.5 years was extracted with CHCl3 (100 mL, 48 hours) filtered and the CHCl3 removed under reduced pressure leaving a dark green-brown residue (0.62 g, yield 6.0%). Quantitative 1H-NMR analysis of a CDCl3 solution showed that EPD and EPA were almost completely absent and a very complex mixture of unidentified con- stituents made up the bulk of the material. High-performance liquid chromatography (HPLC) HPLC analyses were carried out using the Akta purifier (Amersham Pharmacia Biotech, Sweden) with a HPLC- column (150 mm × 4.6 mm i.d. plus pre-column; Grace, The Netherlands), filled with HS Silica (particle size 3 μm), UV detection at 214 nm, 254 nm and 280 nm. Ten μL of the fractionated extract was injected, after dilution to 100 μL with eluent A: hexane (99.5 mL)- van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Page 3 of 6 Page 3 of 6 Cell cultures, in triplicates, in exponential growth were treated with the different dried fractions of the plant extract, redissolved in dimethyl sulfoxide (DMSO) and added at final concentrations of 1, 10 and 100 μg/mL. The control cultures had 0.02% (1 μg/mL) 0.2% (10 μg/ mL) and 2% (100 μg/mL) DMSO added to the medium. In 2 mL medium/well 10% Alamar blue was added and 100 μl of the supernatants of the 24-well plates after 24, 48 and 72 hrs incubations were pipetted into 96-well plates (Costar, USA). Cell viability was measured with a 96-well plate reader (Molecular Devices Ltd, UK). In a later stage, after identifying fractions with high cytotoxic effects, the final concentrations of extracts tested ranged from 1-10 μg/mL, with final concentrations of 0.02 up to 0.2% DMSO. fractions, see above) as the fraction with most of the cytotoxicity and its main chemical constituent was iden- tified as EPD. A second main non-cytotoxic constituent, present mostly in Fractions 7 to 9 was identified as EPA (137 mg, 91% purity by NMR and MS analyses). Again, fractionation was applied to fraction 4 (enriched in EPD) using normal-phase short-column vacuum chromatography (silica gel H; column dimen- sions 18 mm × 65 mm i.d.), eluting with stepwise sol- vent gradients of hexane:dichloromethane, 2:1 v/v (100 mL); hexane: dichloromethane, 1:1 v/v (2 × 50 mL); hex- ane:dichloromethane, 1:2 v/v (2 × 50 mL); dichloro- methane (2 × 50 mL); dichloromethane: ethyl acetate 4:1 (2 × 50 mL); dichloromethane: ethyl acetate, 1:1 v/v (2 × 50 mL) to give the main chemical constituent, identified as an SL, EPD (93 mg, 90% purity by NMR and MS analyses) and containing lipids and waxes (10% by NMR analyses). Results Fractionation of extracts by column chromatography In total 157 fractions were sampled and, based on HPLC analyses, divided into four groups of combined fractions (fractions: 1-6, 60-70, 90-100 and 120-130) and then tested in vitro against ovarian cancer cell lines and nor- mal cells. Group 2 (fractions: 60-70) showed the stron- gest cytotoxicity, killing all ovarian cancer cells at 10 μg/ mL but not at 1 μg/mL. Other fractions did not show significant activities. This second group of fractions 60-70 (1.30 g, 0.37% yield from crude extract) was further fractionated by normal-phase short-column vacuum chromatography on silica gel H (column dimen- sions 18 mm × 65 mm i.d.), eluted with stepwise solvent gradients of hexane: dichloromethane, 1:1 v/v (100 mL and 50 mL); dichloromethane (2 × 50 mL); dichloro- methane: ethyl acetate, 4:1 v/v (2 × 50 mL); dichloro- methane: ethyl acetate, 1:1 v/v (2 × 50 mL); ethyl acetate (2 × 50 mL). From each fraction (12 in total) solvent was evaporated under reduced pressure and the residue was weighed. 1H-NMR and 13C-NMR analyses Eremophila-1(10)-11(13)-dien-12,8b-olide (EPD) (3aa,4aa,5a,9aa)-3a,4,4a,5,6,7,9,9a-octahydro-4a,5- dimethyl-3-methylenenaphtho[2,3-b]furan-2(3H)-2-one 1H-NMR and 13C-NMR analyses Eremophila-1(10)-11(13)-dien-12,8b-olide (EPD) (3aa,4aa,5a,9aa)-3a,4,4a,5,6,7,9,9a-octahydro-4a,5- dimethyl-3-methylenenaphtho[2,3-b]furan-2(3H)-2-one (3aa,4aa,5a,9aa)-3a,4,4a,5,6,7,9,9a-octahydro-4a,5- dimethyl-3-methylenenaphtho[2,3-b]furan-2(3H)-2-one C15H20O2 colourless liquid; 1H-NMR (CDCl3): δ0.92 (s, H-14), 0.93 (d, J4,15 = 6.8 Hz, H-15), 1.50 (m, H-3), 1.60 (m, H-4), 1.70 (m, H-6), 2.03 (m, H-2), 2.30 (m, H- 9), 2.58 (dd, J9,9’ = 12.6 Hz, J8,9’ = 7.7 Hz, H-9’), 2.92 (m, H-7), 4.53 (dt, J7,8 = 9.6 Hz, J8,9 = 7.4 Hz, H-8), 5.48 (br t, J1,2 = 3.4 Hz, H-1), 5.59 (d, J13,13’ = 2.2 Hz, H-13’), 6.23 (d, J13,13’ = 2.2 Hz, H-13); 13C-NMR (CDCl3): δ16.08, 20.59, 25.03, 26.72, 34.69, 34.91, 36.63, 37.01, 38.73, 79.00, 121.82, 124.57, 138.32, 139.36, 170.65. Posi- tive ion ESI-MS [M+Na]+ 255 (100), [M+H]+ 233 (65). Xanthanodien or EPD is an a-methylene SL [14]. van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Results Eremophila-1(10),11(13)-dien-12-oic acid (EPA) C15H22O2 colourless liquid; 1H-NMR (CDCl3): δ0.85 (d, J4,15 = 6.4 Hz, H-15), 0.91 (s, H-14), 1.45 (m, H-6), 1.50 (m, H-4), 1.55 (m, H-3), 1.60 (m, H-8), 1.85 (m, H-9), C15H20O2 colourless liquid; 1H-NMR (CDCl3): δ0.92 (s, H-14), 0.93 (d, J4,15 = 6.8 Hz, H-15), 1.50 (m, H-3), 1.60 (m, H-4), 1.70 (m, H-6), 2.03 (m, H-2), 2.30 (m, H- 9), 2.58 (dd, J9,9’ = 12.6 Hz, J8,9’ = 7.7 Hz, H-9’), 2.92 (m, H-7), 4.53 (dt, J7,8 = 9.6 Hz, J8,9 = 7.4 Hz, H-8), 5.48 (br t, J1,2 = 3.4 Hz, H-1), 5.59 (d, J13,13’ = 2.2 Hz, H-13’), 6.23 (d, J13,13’ = 2.2 Hz, H-13); 13C-NMR (CDCl3): δ16.08, 20.59, 25.03, 26.72, 34.69, 34.91, 36.63, 37.01, 38.73, 79.00, 121.82, 124.57, 138.32, 139.36, 170.65. Posi- tive ion ESI-MS [M+Na]+ 255 (100), [M+H]+ 233 (65). Xanthanodien or EPD is an a-methylene SL [14]. Eremophila 1(10) 11(13) dien 12 oic acid (EPA) p C15H22O2 colourless liquid; 1H-NMR (CDCl3): δ0.85 (d, J4,15 = 6.4 Hz, H-15), 0.91 (s, H-14), 1.45 (m, H-6), 1.50 (m, H-4), 1.55 (m, H-3), 1.60 (m, H-8), 1.85 (m, H-9), Bioassays with ovarian cancer cells indicated fraction 4 (309 mg, 0.09% of the dried plant; out of the twelve Page 4 of 6 Page 4 of 6 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Table 1 Cell viability with EPD treatment of normal fibroblasts, OVCAR3 and SKOV3 cancer cells (average (AV) and standard deviation (SD)) % cell viability: average and standard deviation EPD Conc 24 hours 48 hours 72 hours μg/mL AV SD AV SD AV SD Normal fibroblasts 1 102 2.5 107 3.9 105 3.3 5 105 6.3 108 1.6 72 2.1 10 101 10.1 112 1.8 47 4.6 OVCAR3 1 96 5.1 101 7.4 109 29.2 5 87 6.7 67 4.5 50 14.4 10 70 7.4 23 0.9 21 6.4 SKOV3 1 103 5.0 123 8.2 119 6.0 5 102 4.0 96 18.2 69 16.5 10 86 11.6 31 36.0 23 1.8 IC50 for OVCAR3 at 24 hours was 13 μg/mL (95% C.I. 10 to 18 μg/mL), at 48 hours 6.4 μg/mL (95% C.I. 5.3 to 7.8 μg/mL) and at 72 hours 5.3 μg/mL (95% C.I. 4.3 to 6.5 μg/mL). IC50 for SKOV3 at 24 hours was 16 μg/mL (95% C.I. for OVCAR3 and SKOV3 cells showed that more than 50% and 80% of cells were killed at doses of 5 and 10 μg/mL, respectively. The screening test for the JC cells with doses of 1, 10 and 100 μg/mL measured for 1 μg/mL: after 24 hours showed cell viability of 98%; after 48 hours 97%; and after 72 hours 70%; for 10 μg/mL: after 24 hours cell viability showed 85%; after 48 hours 84%; and after 72 hours 21%; for 100 μg/mL: after 24 hours cell viabi- lity showed 77%; after 48 hours 84%; and after 72 hours 8%. At the time points 24 and 48 hours IC50 was greater than 100 μg/mL and at 72 hours IC50 was 2.5 μg/mL (95% confidence interval (C.I.) 0.22 to 28 μg/mL). Results 9.4 to 27 μg/mL), at 48 hours 8.4 μg/mL (95% C.I. 6.7 to 11 μg/mL) and at 72 hours 6.5 μg/mL (95% C.I. 5.2 to 8.3 μg/mL). Table 1 Cell viability with EPD treatment of normal fibroblasts, OVCAR3 and SKOV3 cancer cells (average (AV) and standard deviation (SD)) 2.01 (m, H-2), 2.40 (m, H-9’), 2.55 (m, H-7), 5.38 (br t, J1,2 = 3.4 Hz, H-1), 5.66 (br s, H-13’), 6.29 (br s, H-13); 13C-NMR (CDCl3): δ16.08, 20.59, 25.03, 26.72, 34.69, 34.91, 36.63, 37.01, 38.73, 79.00, 121.82, 124.57, 138.32, 139.36, 170.65. Negative ion ESI-MS [M-H]- 233 (100) EPA, is an a-methylene carboxylic acid [15]. 2.01 (m, H-2), 2.40 (m, H-9’), 2.55 (m, H-7), 5.38 (br t, J1,2 = 3.4 Hz, H-1), 5.66 (br s, H-13’), 6.29 (br s, H-13); 13C-NMR (CDCl3): δ16.08, 20.59, 25.03, 26.72, 34.69, 34.91, 36.63, 37.01, 38.73, 79.00, 121.82, 124.57, 138.32, 139.36, 170.65. Negative ion ESI-MS [M-H]- 233 (100) EPA, is an a-methylene carboxylic acid [15]. The remaining impurities in the purified sample of EPD and EPA (Figures 1A and 1B) were identified as waxes and lipids. No other sesquiterpenoid substances of similar structure to EPD and EPA were detected. In vitro cytotoxicity tests Cell viability of normal skin fibroblasts and of cells of the ovarian cell line JC treated with the crude plant extract for 24, 48 and 72 hours at final concentrations of 1, 10 and 100 μg/mL was as follows: The screening test for the fibroblasts with doses of 1, 10 and 100 μg/mL measured for 1 μg/mL: after 24 hours showed cell viability of 104%; after 48 hours 97%; and after 72 hours 98%; for 10 μg/ml: after 24 hours cell viability showed 100%; after 48 hours 96%; and after 72 hours 80%; and for 100 μg/mL: after 24 hours cell viabi- lity showed 98%; after 48 hours 83%; and after 72 hours 65%. At all time points (24, 48 and 72 hours) IC50 was greater than 100 μg/mL. for OVCAR3 and SKOV3 cells showed that more than 50% and 80% of cells were killed at doses of 5 and 10 μg/mL, respectively. van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 Discussion q p g y In 1972 a diastereoisomer of EPD, (3ab,4aa,5a,9ab)- 3a,4,4a,5,6,7,9,9a octahydro4a,5-dimethyl-3-methylene- naphtho[2,3-b]furan-2(3H)-2-one, has been described as “naphthofuranone” by the National Cancer Institute (NCI) in their “in vivo“ anti-tumor screening data, test- ing the drug against P388 Leukemia in CD2F1 mice, however, no final conclusive results were reported [17]. An allergenic sesquiterpene lactone, Alantolactone, found in “Elfdock” Inula helenium has been shown to be toxic to leukocytes. Although with the same molecu- lar weight and molecular formula as EPD it belongs to the eudesmanolide structure sub-type [18]. This SL has a different chemical structure from EPD, with different positions of one methyl and one double bond. The chemical constituents composition of aerial parts of C. amaranthoides have been examined once before by Zdero et al. [16]. None of the constituents reported by them were identified in the C. amaranthoides described in this study. The three constituents reported [16] are isomeric with the two major constituents reported in this study, EDP and EPA. The different constituents reported previously may be due to incomplete isolation and analyses or possibly the result of variation in consti- tuent profiles of plant phenotypes. Another possible explanation is degradation on storage. Our studies have shown that freshly dried plant material is necessary as dried plant material stored for over three years was found to yield less than one-tenth of the normal yield of EDP and EPA. In the present study, EPA, the other sesquiterpene iso- lated and identified, did not show cytotoxic effects on the ovarian cancer at concentrations up to 10 μg/mL of purified compound. For the first time the anti-cancer activity of C. amar- anthoides has been examined. Two major sesquiterpenes with the eremophilanolide structure sub-type were Besides the cytotoxic effects of the crude extract of C. amaranthoides with clear effects at 10 μg/mL (cell reduction >80%), the isolated biologically active com- pound EPD has been shown to have high cytotoxicity (>50%) for ovarian cancer cells at lower concentrations of 5 μg/mL (72 hours) and increased (> 60%) with a dose of 10 μg/mL (at 48 hours; Table 1). Interestingly, both the crude plant extract and EPD did show only a slight cytotoxic effect (20%-30%) on normal fibroblasts in vitro at a concentration of 10 μg/mL (at 72 hours). In vivo pilot experiment Control mice only injected with the OVCAR3 cells, were killed when the ascites became a burden. EPD (at final concentration of 20 mg/kg b.w.) was administered i.p. twice/week for six weeks and Cisplatin (at final concen- tration of 5 mg/kg b.w.) was administered i.p. during 4 weeks, once/week. In general a similar cytotoxic effect was observed between EPD and Cisplatin on the OVCAR3 cells. However, mice treated with EPD could be kept for a much longer period of time than those mice treated with Cisplatin, for the latter the mice had lost weight significantly and had to be sacrificed after the fourth week. Moreover, following EPD treatment for A similar type of biological assay was performed with the purified compound EPD at final concentrations of 1, 5 and 10 μg/mL for 24, 48 and 72 hours (Table 1). Per- cent of cell reduction for normal fibroblasts at 72 hours at the highest dose (10 μg/mL) was approximately 30%, while IC 50 was greater than 10 μg/mL. Screening tests O H H O H COOH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 EPD EPA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B 15 Figure 1 Chemical structures. A. Chemical structure of an a-methylene sesquiterpene lactone, EPD. B. Chemical structure of an a-methylene carboxylic acid, EPA. Page 5 of 6 Page 5 of 6 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 identified by 1H-NMR and 13C-NMR and by mass spec- trometry and by comparison with published 1H-NMR partial spectra as eremophila-1(10)-11(13)-dien-12,8b- olide (EPD or Xanthanodien) and eremophila-1(10),11 (13)-dien-12-oic acid (EPA) [14,15]. Belonging to the family of Asteraceae, this family has contributed a large number of natural products including SL’s. The alpha- methylene gamma-lactone ring is responsible for their bioactivity. Various SL’s have demonstrated their anti- cancer capability in in vitro cell culture and by preven- tion of metastasis in in vivo animal models [6]. Thus, it is not surprising that C. amaranthoides extract can kill cancer cells, given the fact that one of the two isolated sesquiterpenes, EPD, shows high toxicity. 6 weeks, three mice were kept alive for another month to see if the reduced abdomen would stay of normal size. In vivo pilot experiment Two mice kept their normal size abdomen, whereas, after 6 weeks the abdomen of the third mouse started to increase in size (Table 2). The rate of change in abdomen size for the mice was determined by linear regression (Figure 2) and statisti- cally evaluated for significance by the unpaired t test. Control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. 1. Ventenat EP: ’Jardin de la Malmaison’. De Crapelet and Orchard (Paris); 18041,2. 1. Ventenat EP: ’Jardin de la Malmaison’. De Crapelet and Orchard (Paris); 18041,2. 2. Smith JE: ’Exotic botany’. Taylor R & Co. (London); 18041. 2. Smith JE: ’Exotic botany’. Taylor R & Co. (London); 18041. 3. Puttock CF: Calomeria. In Flora of Victoria. Volume 4. Edited by: Walsh NG and Entwistle TJ. Melbourne, Inkata Press; 1993. 4. Orchard AE: A reassessment of the genus Haeckeria (Asteraceae: Gnaphalieae), with definition of new species in Cassinia. Australian Systematic Botany 2004, 17:447-449. Systematic Botany 2004, 17:447-449. 5. Heinrich M, Robles M, West JE, Ortiz de Montellano BR, Rodriguez E: Ethnopharmacology of Mexican Asteraceae (Compositae). Annual Reviews 1998 38:539-565 5. Heinrich M, Robles M, West JE, Ortiz de Montellano BR, Rodriguez E: Ethnopharmacology of Mexican Asteraceae (Compositae). Annual Reviews 1998, 38:539-565. 5. Heinrich M, Robles M, West JE, Ortiz de Montellano BR, Rodriguez E: Ethnopharmacology of Mexican Asteraceae (Compositae). Annual Review 1998, 38:539-565. 6. Zhang S, Won Y-K, Ong C-N, Shen H-M: Anti-Cancer potential of sesquiterpene lactones: Bioactivity and molecular mechanisms. Curr Med Chem-Anti-Cancer Agents 2005, 5:239-249. 6. Zhang S, Won Y-K, Ong C-N, Shen H-M: Anti-Cancer potential of sesquiterpene lactones: Bioactivity and molecular mechanisms. Curr Med Chem-Anti-Cancer Agents 2005, 5:239-249. 7. Scully RE, Young RH, Clement PB: Tumors of the ovary, maldeveloped gonads, fallopian tube, and broad ligament. In Atlas of Tumor Pathology. Volume Third. Edited by: Scully RE, Young RH, Clement PB. Washington, DC, Armed Forces Institute of Pathology; 1998. 7. Scully RE, Young RH, Clement PB: Tumors of the ovary, maldeveloped gonads, fallopian tube, and broad ligament. In Atlas of Tumor Pathology. Volume Third. Edited by: Scully RE, Young RH, Clement PB. Washington, DC, Armed Forces Institute of Pathology; 1998. 8. The Merck Manual of Diagnosis and Therapy, Gynecology And Obstetrics. Gynecol Neoplasms 2006, 241(18). Figure 2 Changes in abdomen size for control and treated mice. 9. Van Haaften-Day C, Russell P, Rugg C, Wills EJ, Tattersall MHN: Flow cytometric and morphological studies of ovarian carcinoma cell lines and xenografts. Cancer Res 1983, 43:3725-3731. In spite of the introduction of new drugs into the management of ovarian cancer there is still need for more novel treatments. 10. Van Haaften-Day C, Russell P, Brammah-Carr S: Two homologous mixed Müllerian tumor lines of the ovary and their characteristics. Cancer 1990, 65:1753-1761. 11. Van Haaften-Day C, Russell P, Davies S, Brammah-Carr S: An in vitro study of ovarian atypical proliferating (borderline) serous tumors. Discussion The in vivo pilot experiment with BALB/c nude mice (Table 2, Figure 2) did show that both EPD and Cispla- tin reduced the size of the abdomen. The difference, however, was that mice treated with Cisplatin were in poor condition and became wasted compared with the EPD treated mice. Table 2 Average abdomen size and standard deviation of BALB/c nude mice Average abdomen size and standard deviation (cm) Control cisplatin EPD Days AV SD AV SD AV SD 1 2.1 0.173 2.567 0.115 2.333 0.115 7 2.4 0.173 8 2.333 0.153 2.525 0.33 12 2.367 0.231 14 2.5 0.258 16 2.767 0.153 19 2.475 0.222 2.267 0.058 21 3 0.346 2.5 0.183 26 3.1 0.141 2.1 0.1 1.967 0.208 33 2 0 36 2.267 0.058 61 2.467 0.289 63 2.533 0.321 68 2.7 0.794 Table 2 Average abdomen size and standard deviation of BALB/c nude mice Ovarian cancer has a poor prognosis. With more than 60% of the patients presenting the disease in stage III or IV, combination chemotherapy with Platinum and Taxol after cytoreductive surgery gives the most tolerated stan- dard regimen [19,20]. Page 6 of 6 Page 6 of 6 Page 6 of 6 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 van Haaften et al. Journal of Experimental & Clinical Cancer Research 2011, 30:29 http://www.jeccr.com/content/30/1/29 control cisplatin EPD -0.02 0.00 0.02 0.04 0.06 change in abdomen size cm/day Figure 2 Changes in abdomen size for control and treated mice. Authors’ contributions Data were extracted by CvH and CCD and analyzed by FD and NPMS. CCD and AWW contributed substantially to data acquisition and analysis. The paper was written by CvH and critically revised by FD and approved by all other authors including BJMZT. Revision of the manuscript was largely performed by CvH and CCD. All authors have read and approved the final manuscript. 1. Ventenat EP: ’Jardin de la Malmaison’. De Crapelet and Orchard (Paris); 18041,2. Int J Gynecol Cancer 1992, 2:41-48. Acknowledgements y , 17. NCI: In Vivo Antitumor Screening Data. Cancer Chemotherapy Reports 1973, 2:3. y , 17. NCI: In Vivo Antitumor Screening Data. Cancer Chemotherapy Reports 1973, 2:3. g We thank Fred Romijn, Wouter Temmink (LUMC, Leiden) and Alma Edelman (RDGG, Delft) for their technical assistance. We thank Fred Romijn, Wouter Temmink (LUMC, Leiden) and Alma Edelman (RDGG, Delft) for their technical assistance. 18. Dupuis G, Brisson J: Toxic effect of alantolactone and dihydroalantolactone in in vitro cultures of leukocytes. Chem Biol Interact 1976, 15:205-217. 18. Dupuis G, Brisson J: Toxic effect of alantolactone and dihydroalantolactone in in vitro cultures of leukocytes. Chem Biol Interact 1976, 15:205-217. A European patent was recently granted for the crude extract of Calomeria amaranthoides: EP 1843759 19. Markman M: Optimizing primary chemotherapy in ovarian cancer. Hematol Oncol Clin N Am 2003, 17:957-968. 19. Markman M: Optimizing primary chemotherapy in ovarian cancer. Hematol Oncol Clin N Am 2003, 17:957-968. Competing interests Th h d l h The authors declare that they have no competing interests. Received: 16 November 2010 Accepted: 14 March 2011 Published: 14 March 2011 Received: 16 November 2010 Accepted: 14 March 2011 Published: 14 March 2011 Conclusion The compound EPD has shown unique cytotoxicity effects on both in vitro (ovarian cancer cell lines) as well as in vivo (mice). Interestingly, it had low cytotoxic effects on normal cells. 12. Brookes S, Rowe J, Ruas M, Llianos S: INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. The EMBO Journal 2002, 21:2936-2945. 13. Pagé B, Pagé M, Noel C: A new fluorometric assay for cytotoxicity measurements in vitro. Int J Oncol 1993, 3:473-476. More studies in vivo are required to verify the mechanisms and mode of action of EPD, and to further validate the potential of EPD as an anti-cancer drug in ovarian cancer and other types of cancer. 14. Tanaka N, Yazawa T, Aoyama K, Murakami T: Chemische untersuchungen der inhaltsstoffe von Xanthium canadense Mill. Chem Pharm Bull 1976, 24:1419-1421. 15. Bohlmann F, Zdero C, Silva M: Two further eremophilane derivatives from Tessaria absynthioides. Phytochem 1977, 16:1302-1303. 16. Zdero C, Bohlmann F, Anderberg A, King RM: Eremophilane derivates and other constituents from Haeckeria species and further Australian Inuleae. Phytochem 1991, 30:2643-2650. 16. Zdero C, Bohlmann F, Anderberg A, King RM: Eremophilane derivates and other constituents from Haeckeria species and further Australian Inuleae. Phytochem 1991, 30:2643-2650. 1Department of Gynaecology, Leiden University Medical Center, The Netherlands. 2Faculty of Pharmacy, University of Sydney, NSW 2006, Australia. 3Skin Research Laboratory, Leiden University Medical Center, Leiden, The Netherlands. 4Department of Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands. 5Department of Clinical Chemistry, Medical Laboratories, Reinier de Graaf Group of Hospitals, Delft, The Netherlands. 6Department of Toxicogenetics, Leiden University, Medical Center Leiden, The Netherlands. References 1. Ventenat EP: ’Jardin de la Malmaison’. De Crapelet and Orchard (Paris); 18041,2. Author details 1 20. Bookman MA, Greer BE, Ozols RF: Optimal therapy of advanced ovarian cancer: carboplatin and placitaxel (GOG158) and an update on GOG0182-ICON5. Int J Gynecol Cancer 2003, 13:149-155. 1Department of Gynaecology, Leiden University Medical Center, The Netherlands. 2Faculty of Pharmacy, University of Sydney, NSW 2006, Australia. 3Skin Research Laboratory, Leiden University Medical Center, Leiden, The Netherlands. 4Department of Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands. 5Department of Clinical Chemistry, Medical Laboratories, Reinier de Graaf Group of Hospitals, Delft, The Netherlands. 6Department of Toxicogenetics, Leiden University, Medical Center Leiden, The Netherlands. doi:10.1186/1756-9966-30-29 Cite this article as: van Haaften et al.: Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers. Journal of Experimental & Clinical Cancer Research 2011 30:29.
https://openalex.org/W4287193850	https://cris.unibo.it/bitstream/11585/845633/1/jimaging-07-00099-v2.pdf	English	null	Directional TGV-based image restoration under Poisson noise	arXiv (Cornell University)	2,021	cc-by	9,923	Citation: di Seraﬁno, D.; Landi, G.; Viola, M. Directional TGV-Based Image Restoration under Poisson Noise. J. Imaging 2021, 7, 99. https:// doi.org/10.3390/jimaging7060099 Journal of Imaging Journal of Imaging Journal of Imaging Journal of Imaging Article Daniela di Seraﬁno 1 , Germana Landi 2,* and Marco Viola 3 Daniela di Seraﬁno 1 1 Department of Mathematics and Applications “R. Caccioppoli”, University of Naples Federico II, 1 Department of Mathematics and Applications “R. Caccioppoli”, University of Naples Federico II, 80126 Naples, Italy; daniela.diseraﬁno@unina.it p , y; 2 Department of Mathematics, University of Bologna, 40126 Bologna, Italy 3 Department of Mathematics and Physics, University of Campania “L. Vanvitelli”, 81100 Caserta, Italy; marco viola@unicampania it p y 2 Department of Mathematics, University of Bologna, 40126 Bologna, Italy * Correspondence: germana.landi@unibo.it Abstract: We are interested in the restoration of noisy and blurry images where the texture mainly follows a single direction (i.e., directional images). Problems of this type arise, for example, in microscopy or computed tomography for carbon or glass ﬁbres. In order to deal with these problems, the Directional Total Generalized Variation (DTGV) was developed by Kongskov et al. in 2017 and 2019, in the case of impulse and Gaussian noise. In this article we focus on images corrupted by Poisson noise, extending the DTGV regularization to image restoration models where the data ﬁtting term is the generalized Kullback–Leibler divergence. We also propose a technique for the identiﬁca- tion of the main texture direction, which improves upon the techniques used in the aforementioned work about DTGV. We solve the problem by an ADMM algorithm with proven convergence and subproblems that can be solved exactly at a low computational cost. Numerical results on both phantom and real images demonstrate the effectiveness of our approach. Keywords: directional image restoration; Poisson noise; DTGV regularization; ADMM method Citation: di Seraﬁno, D.; Landi, G.; Viola, M. Directional TGV-Based Image Restoration under Poisson Noise. J. Imaging 2021, 7, 99. https:// doi.org/10.3390/jimaging7060099 Academic Editor: Nicolas Papadakis Received: 30 April 2021 Accepted: 11 June 2021 Published: 16 June 2021 Article Directional TGV-Based Image Restoration under Poisson Noise Daniela di Seraﬁno 1 , Germana Landi 2,* and Marco Viola 3 1. Introduction Poisson noise appears in processes where digital images are obtained by the count of particles (generally photons). This is the case of X-ray computed tomography, positron emission tomography, confocal and ﬂuorescence microscopy and optical/infrared astro- nomical imaging, to name just a few applications (see, e.g., [1] and the references therein). In this case, the object to be restored can be represented as a vector u ∈Rn and the data can be assumed to be a vector b ∈Nn 0, whose entries bj are sampled from n independent Poisson random variables Bj with probability Academic Editor: Nicolas Papadakis Published: 16 June 2021 P(Bj = bj) = e−(Au+γ)j(Au + γ) bj j bj! . Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. The matrix A = (aij) ∈Rn×n models the observation mechanism of the imaging system and the following standard assumptions are made: aij ≥0 for all i, j, n ∑ i=1 aij = 1 for all j. (1) (1) Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// The vector γ ∈Rn, with γ > 0, models the background radiation detected by the sensors. The vector γ ∈Rn, with γ > 0, models the background radiation detected by the sensors. By applying a maximum-likelihood approach [1,2], we can estimate u by minimizing the Kullback–Leibler (KL) divergence of Au + γ from b: DKL(Au + γ, b) = n ∑ i=1 bi ln bi [Au + γ]i + [Au + γ]i −bi , (2) (2) creativecommons.org/licenses/by/ 4.0/). 60099 https://www.mdpi.com/journal/jimaging https://www.mdpi.com/journal/jimaging J. Imaging 2021, 7, 99. https://doi.org/10.3390/jimaging7060099 2 of 18 J. Imaging 2021, 7, 99 where we set where we set bi ln bi [Au + γ]i = 0 if bi = 0. Regularization is usually introduced in (2) to deal with the ill-conditioning of this problem. The Total Variation (TV) regularization [3] has been widely used in this context, because it preserves edges and is able to smooth ﬂat areas of the image. However, since it may produce staircase artifacts, other TV-based regularizers have been proposed. 1. Introduction For example, the Total Generalized Variation (TGV) has been proposed and applied in [4–7] to overcome the staircasing effect while keeping the ability of identifying edges. On the other hand, to improve the quality of restoration for directional images, the Directional TV (DTV) regularization has been considered in [8], in the discrete setting. In [9,10], a regularizer combining DTV and TGV, named Directional TGV (DTGV), has been successfully applied to directional images affected by impulse and Gaussian noise. g y p Given an image u ∈Rn, the discrete second-order Directional TGV of u is deﬁned as DTGV2(u) = min w∈R2n α0 e∇u −w 2,1\|R2n + α1 eEw 2,1\|R4n, (3) (3) where w ∈R2n, e∇∈R2n×n and eE ∈R4n×2n are the discrete directional gradient operator and the directional symmetrized derivative, respectively, and α0, α1 ∈(0, +∞). For any vector v ∈R2n we set ∥v∥2,1\|R2n = n ∑ j=1 q v2 j + v2 n+j, (4) (4) and for any vector y ∈R4n we set and for any vector y ∈R4n we set and for any vector y ∈R4n we set ∥y∥2,1\|R4n = n ∑ j=1 q y2 j + y2 n+j + y2 2n+j + y2 3n+j. (5) (5) Given an angle θ ∈[−π, π] and a scaling parameter a > 0, we have that the discrete directional gradient operator has the form e∇= Dθ Dθ⊥ = cos(θ)DH + sin(θ)DV a(−sin(θ)DH + cos(θ)DV) , where Dθ, Dθ⊥∈Rn×n represent the forward ﬁnite-difference operators along the di- rections determined by θ and θ⊥= θ + π 2 , respectively, and DH, DV ∈Rn×n represent the forward ﬁnite-difference operators along the horizontal and the vertical direction, respectively. Moreover, the directional symmetrized derivative is deﬁned in block-wise form as where Dθ, Dθ⊥∈Rn×n represent the forward ﬁnite-difference operators along the di- rections determined by θ and θ⊥= θ + π 2 , respectively, and DH, DV ∈Rn×n represent the forward ﬁnite-difference operators along the horizontal and the vertical direction, respectively. Moreover, the directional symmetrized derivative is deﬁned in block-wise form as eE =   Dθ 0 1 2 Dθ⊥ 1 2 Dθ 1 2 Dθ⊥ 1 2 Dθ 0 Dθ⊥  . It is worth noting that, by ﬁxing θ = 0 and a = 1, we have Dθ = DH and Dθ⊥= DV, and the operators e∇and eE deﬁne the TGV2 regularization [4]. p g We observe that the deﬁnition of both the matrix A and the ﬁnite difference operators DH and DV depend on the choice of boundary conditions. We make the following assumption Assumption 1. We assume that periodic boundary conditions are considered for A, DH and DV. Therefore, those matrices are Block Circulant with Circulant Blocks (BCCB). Assumption 1. We assume that periodic boundary conditions are considered for A, DH and DV. Therefore, those matrices are Block Circulant with Circulant Blocks (BCCB). In this work we focus on directional images affected by Poisson noise, with the aim of assessing the behaviour of DTGV in this case. Besides extending the use of DTGV J. Imaging 2021, 7, 99 3 of 18 3 of 18 to Poisson noise, we introduce a novel technique for estimating the main direction of the image, which appears to be more efﬁcient than the techniques applied in [9,10]. We solve the resulting optimization problem by using a customized version of the Alternating Direction Method of Multipliers (ADMM). 2. The KL-DTGV2 Model We brieﬂy describe the KL-DTGV2 model for the restoration of directional images corrupted by Poisson noise. Let b ∈Rn be the observed image. We want to recover the original image by minimizing a combination of the KL divergence (2) and the DTGV2 regularizer (3), i.e., by solving the optimization problem min u,w λ DKL(Au + γ, b) + α0 e∇u −w 2,1\|R2n + α1 eEw 2,1\|R4n s.t. u ≥0, (6) (6) where u ∈Rn, A ∈Rn×n, γ, b ∈Rn, w ∈R2n, and e∇∈R2n×n and eE ∈R4n×2n are the linear operators deﬁning the DTGV2 regularization. The parameters λ ∈(0, +∞) and α0, α1 ∈(0, 1) determine the balance between the KL data ﬁdelity term and the two components of the regularization term. We note that problem (6) is a nonsmooth convex optimization problem because of the properties of the KL divergence (see, e.g., [11]) and the DTGV operator (see, e.g., [10]). and for any vector y ∈R4n we set We note that all the ADMM subproblems can be solved exactly at a low cost, thanks also to the use of FFTs, and that the method has proven convergence. Finally, we show the effectiveness of our approach on a set of test images, corrupted by out-of-focus and Gaussian blurs and noise with different signal-to-noise ratios. In particular, the KL-DTGV model of our problem is described in Section 2 and the technique for estimating the main direction is presented in Section 3. A detailed description of the ADMM version used for the minimization is given in Section 4 and the results of the numerical experiments are discussed in Section 5. Conclusions are given in Section 6. Throughout this work we denote matrices with uppercase lightface letters, vectors with lowercase boldface letters and scalars with lowercase lightface letters. All the vectors are column vectors. Given a vector v, we use vi or (v)i to denote its i-th entry. We use R+ to indicate the set of real nonnegative numbers and ∥· ∥to indicate the two-norm. For brevity, given any vectors v and w we use the notation (v, w) instead of [v⊤w⊤]⊤. Likewise, given any scalars v and w, we use (v, w) to indicate the vector [v w]⊤. We also use the notation ([v]1, [v]2) to highlight the subvectors [v]1 and [v]2 forming the vector v. Finally, by writing v > 0 we mean that all the entries of v are nonnegative and at least one of them is positive. 3. Efﬁcient Estimation of the Image Direction An essential ingredient in the DTGV regularization is the estimation of the angle θ representing the image texture direction. In [10], an estimation algorithm based on the one in [12] is proposed, whose basic idea is to compute a pixelwise direction estimate and then θ as the average of that estimate. In [9], which focuses on impulse noise removal, a more efﬁcient and robust algorithm for estimating the direction is presented, based on the Fourier transform. The main idea behind this algorithm is to exploit the fact that two- dimensional Fourier basis functions can be seen as images with one-directional patterns. However, despite being very efﬁcient from a computational viewpoint, this technique does not appear to be fully reliable in our tests on Poissonian images (see Section 5.1). Therefore, we propose a different approach for estimating the direction, based on classical tools of image processing: the Sobel ﬁlter [13] and the Hough transform [14,15]. Our technique is based on the idea that if an image has a one-directional structure, i.e., its main pattern consists of stripes, then the edges of the image mainly consist of lines going in the direction of the stripes. The ﬁrst stage of the proposed algorithm uses the Sobel ﬁlter to determine the edges of the noisy and blurry image. Then, the Hough transform is applied to the edge image in order to detect the lines. The Hough transform is based on J. Imaging 2021, 7, 99 4 of 18 the idea that each straight line can be identiﬁed by a pair (r, η) where r is the distance of the line from the origin, and η is the angle between the x axis and the segment connecting the origin with its orthogonal projection on the line. The output of the transform is a matrix in which each entry is associated with a pair (r, η), i.e., with a straight line in the image, and its value is the sum of the values in the pixels that are on the line. Hence, the elements with the highest value in the Hough transform indicate the lines that are most likely to be present in the input image. 3. Efﬁcient Estimation of the Image Direction Because of its deﬁnition, the Hough transform tends to overestimate diagonal lines in rectangular images (diagonal lines through the central part of the image contain the largest number of pixels); therefore, before computing the transform we apply a mask to the edge image, considering only the pixels inside the largest circle centered in the center of the image. After the Hough transform has been applied, we compute the square of the two-norm of each column of the matrix resulting from the transform, to determine a score for each angle from −90◦to 90◦. Intuitively, the score for each angle is related to the number of lines with that particular inclination which have been detected in the image. Finally, we set the direction estimate θ ∈[−π, π] as θ =      90 −ηmax 180 π, ηmax ≥0, −90 −ηmax 180 π, ηmax < 0. 180 where ηmax is the value of η corresponding to the maximum score. A pseudocode for the estimation algorithm is provided in Algorithm 1 and an example of the algorithm workﬂow is given in Figure 1. Algorithm 1 Direction estimation. 1: Use the Sobel operator to obtain the image e of the edges of the noisy and blurry image b. A l di k k di l d i b i i d i e (Fi 1b p g g y y 2: Apply a disk mask to cut out some diagonal edges in e, obtaining a new edge image ee 2: Apply a disk mask to cut out some diagonal edges in e, obtaining a new edge image ee (Figure 1b). 3: Compute the Hough transform h(ee) (Figure 1c). 3: Compute the Hough transform h(ee) (Figure 1c). p g ( ) ( g ) 4: Set ηmax as the value of η corresponding to the column of h(ee) with maximum 2-norm. (Figure 1d) p g ( ) g 4: Set ηmax as the value of η corresponding to the column of h(ee) with maximum 2-norm. (Figure 1d) 5: Set θ = ( 90−ηmax 180 π, ηmax ≥0, −90−ηmax 180 π, ηmax < 0. (yellow line in Figure 1a) (a) image (b) edge detection (Sobel ﬁlter + mask) -80 -60 -40 -20 0 20 40 60 80 -600 -400 -200 0 200 400 600 r 0 20 40 60 80 100 120 -80 -60 -40 -20 0 20 40 60 80 2 3 4 5 6 7 8 9 10 11 score 104 (c) Hough transform (d) direction scoring Figure 1. Workﬂow of Algorithm 1 on a random directional image. (b) edge detection (Sobel ﬁlter + mask) (a) image -80 -60 -40 -20 0 20 40 60 80 -600 -400 -200 0 200 400 600 r 0 20 40 60 80 100 120 (c) Hough transform (b) edge detection (Sobel ﬁlter + mask) (a) image -80 -60 -40 -20 0 20 40 60 80 2 3 4 5 6 7 8 9 10 11 score 104 (d) direction scoring (c) Hough transform (d) direction scoring Figure 1. Workﬂow of Algorithm 1 on a random directional image. J. Imaging 2021, 7, 99 5 of 18 4. ADMM for Minimizing the KL-DTGV2 Model Although problem (6) is a bound-constrained convex optimization problem, the nondifferentiability of the DTGV2 regularizer does not allow its solution by classical optimization methods for smooth problems, such as gradient methods (see [16–18] and the references therein). However, the problem can be solved by methods based on splitting techniques, such as [19–23]. Here we solve (6) by the Alternating Direction Method of Multipliers (ADMM) [20]. To this end, we ﬁrst reformulate the problem as follows: min u,w,z1,z2,z3,z4 λ DKL(z1 + γ, b) + α0 ∥z2∥2,1\|R2n + α1 ∥z3∥2,1\|R4n + χRn+(z4) s.t. z1 = A u, z2 = e∇u −w, z3 = eEw, z4 = u, (7) (7) where z1 ∈Rn, z2 ∈R2n, z3 ∈R4n, z4 ∈Rn, and χRn+(z4) is the characteristic function of the nonnegative orthant in Rn. A similar splitting has been used in [24] for TV-based deblurring of Poissonian images. By introducing the auxiliary variables x = (u, w) and z = (z1, z2, z3, z4) we can further reformulate the KL-DTGV2 problem as min x,z F1(x) + F2(z) s.t. H x + G z = 0, (8) min x,z F1(x) + F2(z) s.t. H x + G z = 0, (8) (8) where we set where we set F1(x) = 0, F2(z) = λ DKL(z1 + γ, b) + α0 ∥z2∥2,1\|R2n + α1 ∥z3∥2,1\|R4n + χRn+(z4), (9) F1(x) = 0, F2(z) = λ DKL(z1 + γ, b) + α0 ∥z2∥2,1\|R2n + α1 ∥z3∥2,1\|R4n + χRn+(z4), (9) F1(x) = 0, F2(z) = λ DKL(z1 + γ, b) + α0 ∥z2∥2,1\|R2n + α1 ∥z3∥2,1\|R4n + χRn+(z4), (9) (9) and we deﬁne the matrices H ∈R8n×3n and G ∈R8n×8n as H =   A 0 e∇ −I2n 0 eE In 0  , G =   −In 0 0 0 0 −I2n 0 0 0 0 −I4n 0 0 0 0 −In  . 4. ADMM for Minimizing the KL-DTGV2 Model (10) (10) We consider the Lagrangian function associated with problem (8), L(x, z, ξ) = F1(x) + F2(z) + ξ⊤(H x + G z), (11) L(x, z, ξ) = F1(x) + F2(z) + ξ⊤(H x + G z), (11) where ξ ∈ R8n is a vector of Lagrange multipliers, and then the augmented Lagrangian function L(x, z, ξ) = F1(x) + F2(z) + ξ⊤(H x + G z), (11) where ξ ∈ R8n is a vector of Lagrange multipliers, and then the augmented Lagrangian function (11) where ξ ∈ R8n is a vector of Lagrange multipliers, and then the augmented Lagrangian function LA(x, z, ξ; ρ) = F1(x) + F2(z) + ξ⊤(H x + G z) + ρ 2∥H x + G z∥2 2, (12) 0 LA(x, z, ξ; ρ) = F1(x) + F2(z) + ξ⊤(H x + G z) + ρ 2∥H x + G z∥2 2, (12) (12) Now we are ready to introduce the ADMM method for the solution of problem (8). Let x0 ∈R3n, z0 ∈R8n, ξ0 ∈R8n. At each step k > 0 the ADMM method computes the new iterate xk+1, zk+1, ξk+1 as follows: xk+1 = arg min x∈R3n LA(x, zk, ξk; ρ), zk+1 = arg min z∈R8n LA(xk+1, z, ξk; ρ), ξk+1 = ξk + ρ H xk+1 + G zk+1 . (13) (13) Note that the functions F1(x) and F2(z) in (8) are closed, proper and convex. Moreover, the matrices H and G deﬁned in (10) are such that G = −I8n and H has full rank. Hence, J. Imaging 2021, 7, 99 6 of 18 the convergence of the method deﬁned by (13) can be proved by applying a classical con- vergence result from the seminal paper by Eckstein and Bertsekas [25] (Theorem 8), which we report in a form that can be immediately applied to our reformulation of the problem. the convergence of the method deﬁned by (13) can be proved by applying a classical con- vergence result from the seminal paper by Eckstein and Bertsekas [25] (Theorem 8), which we report in a form that can be immediately applied to our reformulation of the problem. Theorem 1. Let us consider a problem of the form (8) where F1(x) and F2(z) are closed, proper and convex functions and H has full rank. Let x0 ∈R3n, z0 ∈R8n, ξ0 ∈R8n, and ρ > 0. 4. ADMM for Minimizing the KL-DTGV2 Model Suppose {εk}, {νk} ⊂R+ are summable sequences such that for all k xk+1 −arg min x∈R3n LA(x, zk, ξk; ρ) ≤εk, zk+1 −arg min z∈R8n LA(xk+1, z, ξk; ρ) ≤νk, ξk+1 = ξk + ρ H xk+1 + G zk+1 . If there exists a saddle point (x∗, z∗, ξ∗) of L(x, z, ξ), then xk →x∗, zk →z∗and ξk →ξ∗. If such saddle point does not exist, then at least one of the sequences {zk} or {ξk} is unbounded. Since we are dealing with linear constraints, we can recast (13) in a more convenient form, by observing that the linear term in (12) can be included in the quadratic one. By in- troducing the vector of scaled Lagrange multipliers µk = 1 ρξk, the ADMM method becomes xk+1 = arg min x∈R3n ρ 2 H x −zk + µk 2 2, (14) zk+1 = arg min z∈R8n F2(z) + ρ 2 H xk+1 −z + µk 2 2, (15) µk+1 = µk + H xk+1 + G zk+1. (16) (14) (15) (16) In the next sections we show how the solutions to subproblems (14) and (15) can be computed exactly with a small computational effort. 4.1. Solving the Subproblem in x 4.1. Solving the Subproblem in x p p y Let F ∈Cn×n be the matrix representing the two-dimensional DFT operator, and let F ∗denote its inverse, i.e., its adjoint. We can write H⊤H as Let F ∈Cn×n be the matrix representing the two-dimensional DFT operator, and let F ∗denote its inverse i e its adjoint We can write H⊤H as p g p F ∗denote its inverse, i.e., its adjoint. We can write H⊤H as 4.1. Solving the Subproblem in x By (18) and the deﬁnition of x, we can reformulate (17) as   Γ −∆∗ θ −∆∗ θ⊥ −∆θ Φ11 Φ12 −∆θ⊥ Φ21 Φ22     F u F w1 F w2  =   F [H⊤vk x]1 F [H⊤vk x]2 F [H⊤vk x]3  , (19) (19) where we split w and vk x in two and three blocks of size n, respectively. Now we recall a result about the inversion of block matrices. Suppose that a square matrix M is partitioned into four blocks, i.e., Now we recall a result about the inversion of block matrices. Suppose that a square matrix M is partitioned into four blocks, i.e., M = M11 M12 M21 M22 ; then, if M11 and M22 are invertible, we have M−1 = M11 M12 M21 M22 −1 =   M11 −M12M−1 22 M21 −1 0 0 M22 −M21M−1 11 M12 −1   " I −M12M−1 22 −M21M−1 11 I # . (20) 0 M22 −M21M−1 11 M12 −1   " I −M12M−1 22 −M21M−1 11 I # . (20) (20) By applying (20) to the matrix consisting of the second and third block rows and columns of the matrix in (19), which we denote Φ, we get By applying (20) to the matrix consisting of the second and third block rows and columns of the matrix in (19), which we denote Φ, we get Φ−1 = Φ11 Φ12 Φ21 Φ22 −1 =   Φ11 −Φ12Φ−1 22 Φ21 −1 − Φ11 −Φ12Φ−1 22 Φ21 −1 Φ12Φ−1 22 − Φ22 −Φ21Φ−1 11 Φ12 −1 Φ21Φ−1 11 Φ22 −Φ21Φ−1 11 Φ12 −1  . (21) (21) To simplify the notation we set Ψ = Ψ11 Ψ12 Ψ21 Ψ22 = Φ−1, (22) (22) and observe that the matrices Ψij ∈Cn×n are diagonal. 4.1. Solving the Subproblem in x Problem (14) is an overdetermined least squares problem, since H is a tall-and- skinny matrix with full rank. Hence, its solution can be computed by solving the normal equations system H⊤H x = H⊤vk x, (17) (17) where we set vk x = zk −µk. Starting from the deﬁnition of H given in (10), we have where we set vk x = zk −µk. Starting from the deﬁnition of H given in (10), we have H⊤H =  In + A⊤A + e∇⊤e∇ −e∇⊤ −e∇ I2n + eE⊤eE  = =   In + A⊤A + e∇⊤e∇ −D⊤ θ −D⊤ θ⊥ −Dθ In + D⊤ θ Dθ + 1 2 D⊤ θ⊥Dθ⊥ 1 2 D⊤ θ⊥Dθ −Dθ⊥ 1 2 D⊤ θ Dθ⊥ In + 1 2 D⊤ θ Dθ + D⊤ θ⊥Dθ⊥  . System (17) may be quite large and expensive, also for relatively small images. How- ever, as pointed out in Assumption 1, A, Dθ and Dθ⊥have a BCCB structure, hence all the blocks of H⊤H maintain that structure. By recalling that BCCB matrices can be diagonal- ized by means of two-dimensional Discrete Fourier Transforms (DFTs), we show how the solution to (17) can be computed expeditiously. Let F ∈Cn×n be the matrix representing the two-dimensional DFT operator, and let F ∗denote its inverse, i.e., its adjoint. We can write H⊤H as Let F ∈Cn×n be the matrix representing the two-dimensional DFT operator, and let F ∗denote its inverse i e its adjoint We can write H⊤H as J. Imaging 2021, 7, 99 7 of 18 H⊤H =   F ∗ 0 0 0 F ∗ 0 0 0 F ∗     Γ −∆∗ θ −∆∗ θ⊥ −∆θ Φ11 Φ12 −∆θ⊥ Φ21 Φ22     F 0 0 0 F 0 0 0 F  , (18) (18) where each block of the central matrix is the diagonal complex matrix associated with the corresponding block in H⊤H, and ∆∗ θ, ∆∗ θ⊥denote the (diagonal) adjoint matrices of ∆θ, ∆θ⊥. We note that Ξ ∈Cn×n is diagonal (and its inversion is straightforward), while Ω∈C2n×2n has a 2 × 2 block structure with blocks that are diagonal matrices belonging to Cn×n. Thus, we can compute Υ = Ω−1 by applying (20): 4.1. Solving the Subproblem in x Hence, the matrices ∆, Γ, Ψ, Ξ−1, and Υ can be computed only once before the ADMM method starts. This means that the overall cost of the exact solution of (14) at each iteration reduces to six two-dimensional DFTs and two matrix–vector products involving two 3 × 3 block matrices with diagonal blocks of dimension n. 4.2. Solving the Subproblem in z 4.1. Solving the Subproblem in x Letting ∆∗= h ∆∗ θ ∆∗ θ⊥ i , applying the inversion formula (20) to the whole matrix in (19), and using (21) and (22), we get Γ −∆∗ −∆ Φ −1 = Ξ−1 0 0 Ω−1 In −∆∗Ψ −∆Γ−1 I2n , (23) (23) where Ξ = Γ −∆∗Ψ ∆= Γ − ∆∗ θ ∆∗ θ⊥ Ψ11 Ψ12 Ψ21 Ψ22 ∆θ ∆θ∗ , Ω= Φ −∆Γ−1∆∗= Φ11 Φ12 Φ21 Φ22 − ∆θ ∆θ⊥ Γ−1 ∆∗ θ ∆∗ θ⊥ . We note that Ξ ∈Cn×n is diagonal (and its inversion is straightforward), while Ω∈C2n×2n has a 2 × 2 block structure with blocks that are diagonal matrices belonging to Cn×n. Thus, we can compute Υ = Ω−1 by applying (20): J. Imaging 2021, 7, 99 8 of 18 Υ = Υ11 Υ12 Υ21 Υ22 = Ω11 Ω12 Ω21 Ω22 −1 =   Ω11 −Ω12Ω−1 22 Ω21 −1 − Ω11 −Ω12Ω−1 22 Ω21 −1 Ω12Ω−1 22 − Ω22 −Ω21Ω−1 11 Ω12 −1 Ω21Ω−1 11 Ω22 −Ω21Ω−1 11 Ω12 −1   (24) (24) =   Ω11 −Ω12Ω−1 22 Ω21 −1 − Ω11 −Ω12Ω−1 22 Ω21 −1 Ω12Ω−1 22 − Ω22 −Ω21Ω−1 11 Ω12 −1 Ω21Ω−1 11 Ω22 −Ω21Ω−1 11 Ω12 −1   (24) Summing up, by (19), (23) and (24), the solution to (17) can be obtained by computing   y1 y2 y3  = Ξ−1 0 0 Υ In −∆⊤Ψ −∆Γ−1 I2n   F [H⊤vk x]1 F [H⊤vk x]2 F [H⊤vk x]3  , (25) (25) and setting and setting uk+1 = F ∗y1, wk+1 1 = F ∗y2, wk+1 2 = F ∗y3. (26) (26) Remark 1. The only quantity in (25) that varies at each iteration is vk x. Hence, the matrices ∆, Γ, Ψ, Ξ−1, and Υ can be computed only once before the ADMM method starts. This means that the overall cost of the exact solution of (14) at each iteration reduces to six two-dimensional DFTs and two matrix–vector products involving two 3 × 3 block matrices with diagonal blocks of dimension n. Remark 1. The only quantity in (25) that varies at each iteration is vk x. 4.2. Solving the Subproblem in z By looking at the form of F2(z)–see (9)–and by deﬁning the vector vk z = H xk+1 + µk, we see that problem (15) can be split into the four problems zk+1 1 = arg min z1∈Rn λDKL(z1 + γ, b) + ρ 2 z1 −[vk z]1 2 2, (27) zk+1 2 = arg min z2∈R2n α0∥z2∥2,1\|R2n + ρ 2 z2 −[vk z]2 2 2, (28) zk+1 3 = arg min z3∈R4n α1∥z3∥2,1\|R4n + ρ 2 z3 −[vk z]3 2 2, (29) zk+1 4 = arg min z4∈Rn χRn+(z4) + ρ 2 z4 −[vk z]4 2 2, (30) (27) (28) (29) (30) where vk z = ([vk z]1, [vk z]2, [vk z]3, [vk z]4), with [vk z]1 ∈Rn, [vk z]2 ∈R2n,[vk z]3 ∈R4n, and [vk z]4 ∈Rn. Now we focus on the solution of the four subproblems. where vk z = ([vk z]1, [vk z]2, [vk z]3, [vk z]4), with [vk z]1 ∈Rn, [vk z]2 ∈R2n,[vk z]3 ∈R4n, and [vk z]4 ∈Rn. Now we focus on the solution of the four subproblems. Since the objective function of this problem is strictly convex, its solution can be determined by setting the gradient equal to zero, i.e., by solving 4.2.3. Update of z4 It is straightforward to verify that the update of z4 in (30) can be obtained as It is straightforward to verify that the update of z4 in (30) can be obtained as zk+1 4 = ΠRn+ [vk z]4 , zk+1 4 = ΠRn+ [vk z]4 , where ΠRn+ is the Euclidean projection onto the nonnegative orthant in Rn. 4.2.1. Update of z1 4.2.1. Update of z1 By the form of the Kullback–Leibler divergence in (2), the minimization problem (27) is equivalent to min z1∈Rn λ n ∑ i=1 bi ln bi (z1)i + γi + (z1)i + γi −bi + ρ 2 n ∑ i=1 ((z1)i −di)2, (31) (31) where we set d = [vk z]1 to ease the notation. From (31) it is clear that the problem in z1 can be split into n problems of the form min z∈R λ(−b ln(z + γ) + z) + ρ 2(z −d)2. (32) (32) Since the objective function of this problem is strictly convex, its solution can be determined by setting the gradient equal to zero, i.e., by solving J. Imaging 2021, 7, 99 9 of 18 λ − b z + γ + 1 + ρ(z −d) = 0, which leads to the quadratic equation which leads to the quadratic equation z2 + λ ρ + γ −d z −λ ρ ρ λγd −γ + b = 0. (33) (33) Since, by looking at the domain of the objective function in (32), z + γ has to be strictly positive, we set each entry of zk+1 1 as the largest solution of the corresponding quadratic Equation (33). 4.2.2. Update of z2 and z3 The minimization problems (28) and (29) correspond to the computation of the proximal operators of the functions f (z2) = α0 ρ ∥z2∥2,1\|R2n and g(z3) = α1 ρ ∥z3∥2,1\|R4n, respectively. ρ \| ρ \| By the deﬁnitions given in (4) and (5), we see that the two (2,1)-norms correspond to the sum of two-norms of vectors in R2 and R4, respectively. This means that the computation of both the proximal operators can be split into the computation of n proximal operators of functions that are scaled two-norms in either R2 or R4. p The proximal operator of the function f (y) = c∥y∥, c > 0, at a vector d is proxc∥·∥(d) = arg min y c∥y∥+ 1 2∥y −d∥2. proxc∥·∥(d) = arg min y c∥y∥+ 1 2∥y −d∥2. It can be shown (see, e.g., [26] [Chapter 6]) that It can be shown (see, e.g., [26] [Chapter 6]) that proxc∥·∥(d) = 1 − c max{∥d∥, c} d = max ∥d∥−c ∥d∥ , 0 d. (34) (34) Hence, for the update of z2 we proceed as follows. By setting d = [vk z]2 and c = α0 ρ , for each i = 1, . . . , n we have Hence, for the update of z2 we proceed as follows. By setting d = [vk z]2 and c = α0 ρ , for each i = 1, . . . , n we have (zk+1 2 )i, (zk+1 2 )n+i = proxc∥·∥((di, dn+i)). To update z3, we set d = [vk z]3 and c = α1 ρ and compute (zk+1 3 )i, (zk+1 3 )n+i, (zk+1 3 )2n+i, (zk+1 3 )3n+i = proxc∥·∥((di, dn+i, d2n+i, d3n+i)). 4.2.3. Update of z4 Algorithm 2 ADMM for problem (7). Algorithm 2 ADMM for problem (7). 1: Let u0 ∈Rn, w0 1 = Dθu0, w0 2 = Dθ⊥u0, µ0 = 0, z0 = 0, λ, ρ ∈(0, +∞), α0, α1 ∈(0, 1) 2: Compute matrices ∆, Γ, Ψ, Ξ−1, and Υ as speciﬁed in Section 4.1 3: Let k = 0, u1 = u0, w1 = w0, stop = f alse, tol ∈(0, 1), kmax ∈N 4: while not stop and k ≤kmax do 5: Compute zk+1 by solving the four subproblems (27)–(30) 6: Compute µk+1 as in (16) 7: k = k + 1 8: Compute uk+1, wk+1 1 and wk+1 2 by (25) and (26) 9: Set stop = ∥uk+1 −uk∥< tol ∥uk∥ 10: end while g p ( ) 1: Let u0 ∈Rn, w0 1 = Dθu0, w0 2 = Dθ⊥u0, µ0 = 0, z0 = 0, λ, ρ ∈(0, +∞), α0, α1 ∈(0, 1) 2: Compute matrices ∆, Γ, Ψ, Ξ−1, and Υ as speciﬁed in Section 4.1 3: Let k = 0, u1 = u0, w1 = w0, stop = f alse, tol ∈(0, 1), kmax ∈N 4: while not stop and k ≤kmax do 5: Compute zk+1 by solving the four subproblems (27)–(30) 6: Compute µk+1 as in (16) 7: k = k + 1 8: Compute uk+1, wk+1 1 and wk+1 2 by (25) and (26) 9: Set stop = ∥uk+1 −uk∥< tol ∥uk∥ 10: end while g p ( ) 1: Let u0 ∈Rn, w0 1 = Dθu0, w0 2 = Dθ⊥u0, µ0 = 0, z0 = 0, λ, ρ ∈(0, +∞), α0, α1 ∈(0, 1) 2: Compute matrices ∆, Γ, Ψ, Ξ−1, and Υ as speciﬁed in Section 4.1 3: Let k = 0, u1 = u0, w1 = w0, stop = f alse, tol ∈(0, 1), kmax ∈N 4: while not stop and k ≤kmax do 5: Compute zk+1 by solving the four subproblems (27)–(30) 6: Compute µk+1 as in (16) 7: k = k + 1 8: Compute uk+1, wk+1 1 and wk+1 2 by (25) and (26) 9: Set stop = ∥uk+1 −uk∥< tol ∥uk∥ 10: end while 8: Compute uk+1, wk+1 1 and wk+1 2 by (25) and (26) 4.3. Summary of the ADMM Method For the sake of clarity, in Algorithm 2 we sketch the ADMM version for solving problem (7). In many image restoration applications, a reasonably good starting guess for u is often available. For example, if A represents a blur operator, a common choice is to set u0 equal to the the noisy and blurry image. We make this choice for u0. By numerical experiments we also veriﬁed that once x = (u, w) has been initialized, it is convenient to set u1 = u0, w1 1 = w0 1 and w1 2 = w0 2 and to shift the order of the updates in the ADMM scheme (14)–(16), so that a “more effective” initialization of z and µ is performed. We see from line 9 of Algorithm 2 that the algorithm stops when the relative change in the restored J. Imaging 2021, 7, 99 10 of 18 image u goes below a certain threshold tol ∈(0, 1) or a maximum number of iterations kmax is reached. Finally, we note that for the case of the KL-TGV2 model, corresponding to θ = 0 and a = 1, we have that Dθ = DH and Dθ⊥= DV; hence, we use the initialization w0 1 = DHu0 and w0 2 = DVu0. image u goes below a certain threshold tol ∈(0, 1) or a maximum number of iterations kmax is reached. Finally, we note that for the case of the KL-TGV2 model, corresponding to θ = 0 and a = 1, we have that Dθ = DH and Dθ⊥= DV; hence, we use the initialization w0 1 = DHu0 and w0 2 = DVu0. 5. Numerical Results All the experiments were carried out using MATLAB R2018a on a 3.50 GHz Intel Xeon E3 with 16 GB of RAM and Windows operating system. In this section, we ﬁrst illustrate the effectiveness of Algorithm 1 for the estimation of the image direction by comparing it with the one given in [9] and by analysing its sensitivity to the degradation in the image to be restored. Then, we present numerical experiments that demonstrate the improvement of the KL-DTGV2 model upon the KL-TGV2 model for the restoration of directional images corrupted by Poisson noise. p y Four directional images named phantom (512 × 512), grass (375 × 600), leaves (203 × 300) and carbon (247 × 300) were used in the experiments. The ﬁrst image is a piecewise afﬁne ﬁbre phantom image obtained with the fibre_phantom_pa MATLAB function avail- able from http://www2.compute.dtu.dk/~pcha/HDtomo/ (accessed on 20 September 2020). The second and third images represent grass and veins of leaves, respectively, which naturally exhibit a directional structure. The last image is a Scanning Electron Microscope (SEM) image of carbon ﬁbres. The images are shown in Figures 2–5. To simulate experimental data, each reference image was convolved with two PSFs, one corresponding to a Gaussian blur with variance 2, generated by the psfGauss function from [27], and the other corresponding to an out-of-focus blur with radius 5, obtained with the function fspecial from the MATLAB Image Processing Toolbox. To take into account the existence of some background emission, a constant term γ equal to 10−10 was added to all pixels of the blurry image. The resulting image was corrupted by Poisson noise, using the MATLAB function imnoise. The intensities of the original images were pre-scaled to get noisy and blurry images with Signal to Noise Ratio (SNR) equal to 43 and 37 dB. We recall that in the case of Poisson noise, which affects the photon counting process, the SNR is estimated as [28] SNR = 10 log10   Nexact q Nexact + Nbackground  , SNR = 10 log10   Nexact q Nexact + Nbackground  , where Nexact and Nbackground are the total number of photons in the image to be recovered and in the background term, respectively. Finally, the corrupted images were scaled to have their maximum intensity values equal to 1. For each test problem, the noisy and blurry images are shown in Figures 2–5. 5. Numerical Results 11 of 18 11 of 18 J. Imaging 2021, 7, 99 Figure 2. Test problem phantom: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 3. Test problem grass: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 2. Test problem phantom: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 2. Test problem phantom: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 3. Test problem grass: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 4. Test problem leaves: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 3. Test problem grass: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 3. Test problem grass: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. oblem grass: original and corrupted images. The yellow dash-dotted line indicates the direction estimated and the red dashed line the direction estimated by the method in [9]. by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 4. Test problem leaves: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 4. Test problem leaves: original and corrupted images. 5. Numerical Results The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 4. Test problem leaves: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. 12 of 18 J. Imaging 2021, 7, 99 Figure 5. Test problem carbon: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. Figure 5. Test problem carbon: original and corrupted images. The yellow dash-dotted line indicates the direction estimated by Algorithm 1 and the red dashed line the direction estimated by the method in [9]. 5.2. Image Deblurring We compare the quality of the restorations obtained by using the DTGV2 and TGV2 regularizers and ADMM for the solution of both models. In all the tests, the value of the penalty parameter was set as ρ = 10 and the value of the stopping threshold as tol = 10−4. A maximum number of kmax = 500 iterations was allowed. By following [9,10], the weight parameters of DTGV were chosen as α0 = β and α1 = (1 −β) with β = 2/3. For each test problem, the value of the regularization parameter λ was tuned by a trial-and-error strategy. This strategy consisted in running ADMM with initial guess u0 = b several times on each test image, varying the value of λ at each execution. For all the runs the stopping criterion for ADMM and the values of α0, α1 and ρ were the same as described above. The value of λ yielding the smallest Root Mean Square Error (RMSE) at the last iteration was chosen as the “optimal” value. p The numerical results are summarized in Table 1, where the RMSE, the Improved Signal to Noise Ratio (ISNR) [29], and the structural similarity (SSIM) index [30] are used to give a quantitative evaluation of the quality of the restorations. As a measure of the computational cost, the number of iterations and the time in seconds are reported. Table 1 also shows, for each test problem, the values of the regularization parameter λ. The restored images are shown in Figures 8–11. For the carbon test problem, Figure 12 shows the error images, i.e., the images obtained as the absolute difference between the original image and the restored one. The values of the pixels of the error images have been scaled in the range [m, M] where m and M are the minimum and maximum pixel value of the DTGV2 and TGV2 error images. From the results, it is evident that the DTGV2 model outperforms the TGV2 one in terms of quality of the restoration. A visual inspection of the ﬁgures shows that the DTGV2 regularization is very effective in removing the noise, while for high noise levels the TGV2 reconstructions still exhibit noise artifacts. 5.1. Direction Estimation In Figures 2–5 we compare Algorithm 1 with the algorithm proposed in [9], showing that Algorithm 1 always correctly estimates the main direction of the four test images. We also test the robustness of our algorithm with respect to noise and blur. In Figure 6 we show the estimated main direction of the phantom image corrupted by Poisson noise with SNR = 35, 37, 39, 41, 43 dB and out-of-focus blurs with radius R = 5, 7, 9. In only one case (SNR = 35, R = 7) Algorithm 1 fails, returning as estimate the orthogonal direction, i.e., the direction corresponding to the large black line and the background color gradient. Finally, we test Algorithm 1 on a phantom image with vertical, horizontal and diagonal main directions corresponding to θ = 0, 90, 45. The results, in Figure 7, show that our algorithm is not sensitive to the speciﬁc directional structure of the image. Figure 6. Direction estimation for phantom with SNR = 35, 37, 39, 41, 43 dB (from left to right) and out-of-focus blur with radius R = 5, 7, 9 (from top to bottom). Figure 6. Direction estimation for phantom with SNR = 35, 37, 39, 41, 43 dB (from left to right) and out-of-focus blur with radius R = 5, 7, 9 (from top to bottom). 13 of 18 13 of 18 J. Imaging 2021, 7, 99 Figure 7. Direction estimation for phantom with SNR = 37, 43 (top, bottom) and out-of-focus blur with radius R = 5. Figure 7. Direction estimation for phantom with SNR = 37, 43 (top, bottom) and out-of-focus blur with radius R = 5. Figure 7. Direction estimation for phantom with SNR = 37, 43 (top, bottom) and out-of-focus blur with radius R = 5. 5.2. Image Deblurring Finally, by observing the “Iters” column of the table, we can conclude that, on average, the TGV2 regularization requires less ADMM iterations to achieve a relative change in the restoration that is below the ﬁxed threshold. However, the computational time per iteration is very small and also ADMM for the KL-DGTV2 regularization is efﬁcient. Finally, to illustrate the behaviour of ADMM, in Figure 13 we plot the RMSE history for the carbon test problem. A similar RMSE behaviour has been observed in all the numerical experiments. J. Imaging 2021, 7, 99 14 of 18 Table 1. Numerical results for the test problems. 5.2. Image Deblurring Blur SNR Model λ RMSE ISNR MSSIM Iters Time phantom Out-of-focus 43 DTGV 57.5 2.2558 × 10−2 9.5472 9.3007 × 10−1 86 10.95 TGV 275 2.8043 × 10−2 7.6568 8.9887 × 10−1 89 11.33 37 DTGV 3.25 3.7573 × 10−2 7.4431 8.5823 × 10−1 122 15.45 TGV 22.5 4.1719 × 10−2 6.5339 8.4061 × 10−1 52 6.64 Gaussian 43 DTGV 25 1.5530 × 10−2 9.1966 9.7829 × 10−1 56 7.17 TGV 100 1.8100 × 10−2 7.8667 9.7200 × 10−1 45 5.76 37 DTGV 3 2.5498 × 10−2 9.0841 9.2994 × 10−1 90 11.41 TGV 17.5 3.0674 × 10−2 7.4788 9.0199 × 10−1 53 6.76 grass Out-of-focus 43 DTGV 60 3.6313 × 10−2 7.7364 8.7262 × 10−1 136 15.55 TGV 550 3.6575 × 10−2 7.6738 8.7188 × 10−1 179 20.39 37 DTGV 50 5.6164 × 10−2 4.7390 7.6165 × 10−1 160 18.56 TGV 55 5.7604 × 10−2 4.5191 7.4566 × 10−1 72 8.31 Gaussian 43 DTGV 65 2.9883 × 10−2 6.3343 9.2764 × 10−1 106 12.08 TGV 650 3.0814 × 10−2 6.0676 9.2523 × 10−1 136 15.48 37 DTGV 5.5 4.2274 × 10−2 4.7973 8.5615 × 10−1 98 11.13 TGV 35 4.3936 × 10−2 4.4624 8.4795 × 10−1 54 6.18 leaves Out-of-focus 43 DTGV 125 6.2767 × 10−2 7.4978 8.2099 × 10−1 251 31.18 TGV 1100 8.2397 × 10−2 5.1342 7.1557 × 10−1 435 53.74 37 DTGV 12.5 9.5597 × 10−2 4.1497 6.3065 × 10−1 257 31.87 TGV 90 1.1874 × 10−1 2.2665 4.3294 × 10−1 113 14.03 Gaussian 43 DTGV 150 7.3332 × 10−2 4.8675 7.7456 × 10−1 236 29.13 TGV 1750 8.0857 × 10−2 4.0190 7.3001 × 10−1 380 46.77 37 DTGV 12.5 9.0999 × 10−2 3.3907 6.6469 × 10−1 148 18.36 TGV 100 1.0308 × 10−1 2.3081 5.6534 × 10−1 103 12.85 carbon Out-of-focus 43 DTGV 150 1.8360 × 10−2 1.2830 × 101 9.4734 × 10−1 331 13.78 TGV 850 2.3825 × 10−2 1.0567 × 101 9.3671 × 10−1 233 9.73 37 DTGV 20 3.1682 × 10−2 8.2416 8.6294 × 10−1 171 7.07 TGV 150 3.8840 × 10−2 6.4723 8.2237 × 10−1 155 6.55 Gaussian 43 DTGV 250 2.0453 × 10−2 8.6178 9.5974 × 10−1 305 12.53 TGV 950 2.4839 × 10−2 6.9302 9.5698 × 10−1 171 7.12 37 DTGV 15 2.7995 × 10−2 6.2017 9.3007 × 10−1 128 5.36 TGV 150 3.3061 × 10−2 4.7572 8.9690 × 10−1 118 4.73 Table 1. 5.2. Image Deblurring Numerical results for the test problems. 15 of 18 J. Imaging 2021, 7, 99 Figure 8. Test problem phantom: images restored with DTGV2 (top) and TGV2 (bottom). Figure 8. Test problem phantom: images restored with DTGV2 (top) and TGV2 (bottom). Figure 8. Test problem phantom: images restored with DTGV (top) and TGV (bottom). Figure 9. Test problem grass: images restored with DTGV2 (top) and TGV2 (bottom). Figure 10. Test problem leaves: images restored with DTGV2 (top) and TGV2 (bottom). Figure 9. Test problem grass: images restored with DTGV2 (top) and TGV2 (bottom). Figure 10. Test problem leaves: images restored with DTGV2 (top) and TGV2 (bottom). 16 of 18 J. Imaging 2021, 7, 99 Figure 11. Test problem carbon: images restored with DTGV2 (top) and TGV2 (bottom). Figure 11. Test problem carbon: images restored with DTGV2 (top) and TGV2 (bottom). Figure 12. Test problem carbon: difference images with DTGV2 (top) and TGV2 (bottom). Figure 12. Test problem carbon: difference images with DTGV2 (top) and TGV2 (bottom). Figure 13. Test problem carbon: RMSE history for the KL-DGTV2 (continuous line) and KL-TGV2 (dashed line) models. problem carbon: RMSE history for the KL-DGTV2 (continuous line) and KL-TGV2 (dashed line) models. Figure 13. Test problem carbon: RMSE history for the KL-DGTV2 (continuous line) and KL-TGV2 (dashe 6. Conclusions We dealt with the use of the Directional TGV regularization in the case of directional images corrupted by Poisson noise. We presented the KL-DTGV2 model and introduced a two-block ADMM version for its minimization. Finally, we proposed an effective strategy for the estimation of the main direction of the image. Our numerical experiments show that for Poisson noise the DTGV2 regularization provides superior restoration performance compared with the standard TGV2 regularization, thus remarking the importance of taking into account the texture structure of the image. A crucial ingredient for the success of the model was the proposed direction estimation strategy, which proved to be more reliable than those proposed in the literature. J. Imaging 2021, 7, 99 17 of 18 Possible future work includes the use of space-variant regularization terms and the analysis of automatic strategies for the selection of the regularization parameters. Author Contributions: All authors have contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially supported by the Istituto Nazionale di Alta Matematica, Gruppo Nazionale per il Calcolo Scientiﬁco (INdAM-GNCS). D. di Seraﬁno and M. Viola were also supported by the V:ALERE Program of the University of Campania “L. Vanvitelli”. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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https://openalex.org/W2316788698	https://academicjournals.org/journal/AJMR/article-full-text-pdf/1EFD8B446044.pdf	English	null	Characterization of oregano (Origanum vulgare) essential oil and definition of its antimicrobial activity against Listeria monocytogenes and Escherichia coli in vitro system and on foodstuff surfaces	African journal of microbiology research	2,014	cc-by	6,197	Vol. 8(29), pp. 2746-2753, 16 July, 2014 DOI: 10.5897/AJMR2014.6677 Article Number: 1EFD8B446044 ISSN 1996-0808 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR African Journal of Microbiology Research Vol. 8(29), pp. 2746-2753, 16 July, 2014 DOI: 10.5897/AJMR2014.6677 Article Number: 1EFD8B446044 ISSN 1996-0808 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR African Journal of Microbiology Research African Journal of Microbiology Research g p Characterization of oregano (Origanum vulgare) essential oil and definition of its antimicrobial activity against Listeria monocytogenes and Escherichia coli in vitro system and on foodstuff surfaces Lorenzo Siroli1, Francesca Patrignani1, Chiara Montanari2, Giulia Tabanelli2, Eleonora Bargossi1, Fausto Gardini1 and Rosalba Lanciotti1 1Department of Agricultural and Food Sciences, P.zza Goidanich 60, 47521 Cesena (FC), Italy. 2Interdepartmental Centre for Industrial Agri-Food Research, P.zza Goidanich 60, 47521 Cesena (FC), Italy. Received 1 February, 2014; Accepted 23 June, 2014 First aim of this research was to characterize oregano (Origanum vulgare) essential oil and the characterization of its minimum inhibitory concentration against the pathogenic species, Listeria monocytogenes and Escherichia coli. Moreover, the oregano essential oil antimicrobial activity was tested against these pathogenic species, inoculated onto wood and stainless steel surface. The GC/MS profile of oregano essential oil revealed the presence of 34 compounds, principally terpinolene, carvacrol and p-cymene accounting for about 70% of the total area of the identified molecules. Oregano essential oil showed higher antimicrobial activity against L. monocytogenes in comparison with E. coli. In fact, the L. monocytogenes minimum inhibitory concentration ranged between 125 and 200 mg/L while those for E. coli ranged between 250 and 350 mg/L. Regarding the decontamination efficacy, the washing of the two surfaces with oregano fastened the viability decrease of both the inoculated microorganisms over time. This phenomenon was more pronounced for wood as compared to steel. The data obtained suggests the great potential of this essential oil to be employed, as alternative to traditional chemicals, and as sanitizing strategy for surfaces. Key words: Oregano essential oil, GC/MS, surface decontamination, minimum inhibitory concentration. INTRODUCTION Additionally, if certain microorganisms remain on a given surface for a relatively long time, they can continue to replicate and eventually form biofilms (Uhlich et al., 2006). The microbial attachment and the eventual biofilm formation, acting as reservoir of spoilage and pathogenic species, increase significantly the risk for food contamination (Valeriano et al., 2012). In fact, microorganisms can be easily detached from surfaces and/or biofilms and conta- minate foods, causing reduced product shelf-life and disease transmission (Shi and Zhu, 2009). Several studies have shown that various foodborne pathogens including Escherichia coli and Listeria monocytogenes can survive for hours or even days on utensils and equipment surfaces (Humphrey et al., 2001; Wilks et al., 2005, 2006; Martinon et al., 2012). On the other hand, L. monocytogenes and E. coli are among the most frequently involved bacterial species in foodborne diseases (Scallan et al., 2011; Oliveira et al., 2012). Consequently, controlling the longevity of microorga- nisms in surfaces is fundamental in reaching food safety standards and improving food quality and shelf-life (Nitschke et al., 2009). volatile, natural, complex compounds characterized by a strong odor and formed by aromatic plants as secondary metabolites. They have been studied for their antimi- crobial activity against many microorganisms, including several pathogens (Dorman and Deans, 2000; Delaquis et al., 2002). ) The activity of oils from Lamiaceae (Tassou et al., 2000; Gunduz et al., 2010) has been investigated in model and real food systems in order to understand the action of single constituents, their cell targets and to balance their intrinsic variability. Moreover, EOs and their bioactive components have been recently studied also for their antibacterial activity on surface adherent microor- ganisms in order to evaluate their potential as disinfectants in the food industry (Chorianopoulos et al., 2008; Oliveira et al., 2012) and as promising anti-biofilm agents (Amalaradjou and Venkitanarayanan, 2011). Origanum vulgare essen-tial oil has been largely studied for this purpose and its composition, in relation to its geographical origin, dry and extraction methods, has been investigated (Mockute et al., 2001; Teixeira et al., 2013; Figiel et al., 2010). In fact, it is well known that the oil composition, and particularly the presence of phenolic content, can increase its antimicrobial properties. Thus, information regarding the oil composition and the effectiveness of its bioactive components in killing pathogenic species on food contact surfaces is needed to aid in the development of optimal sanitation conditions for food industries. INTRODUCTION The adhesion and persistence of microorganisms in equipment surfaces have the potential to spread pathogens and spoilage microorganisms to foods, influencing their shelf-life and safety (Bae et al., 2012). This is particularly significant in the food processing industry (Giaouris and Nychas, 2006) as well as in the domestic environment (Humphrey et al., 2001; Choi et al., 2012). The surfaces of equipment used for food handling, processing and storage are considered as major sources of microbial contamination (Bae et al., The adhesion and persistence of microorganisms in equipment surfaces have the potential to spread pathogens and spoilage microorganisms to foods, influencing their shelf-life and safety (Bae et al., 2012). This is particularly significant in the food processing Siroli et al. Siroli et al. Siroli et al. 2747 2012). Several studies have shown the ability of microorganisms to attach to surfaces commonly found in the food processing environment, such as stainless steel, polystyrene, hydroxyapatite, rubber, glass and wood (Soares et al., 1992; Barnes et al., 1999). Additionally, if certain microorganisms remain on a given surface for a relatively long time, they can continue to replicate and eventually form biofilms (Uhlich et al., 2006). The microbial attachment and the eventual biofilm formation, acting as reservoir of spoilage and pathogenic species, increase significantly the risk for food contamination (Valeriano et al., 2012). In fact, microorganisms can be easily detached from surfaces and/or biofilms and conta- minate foods, causing reduced product shelf-life and disease transmission (Shi and Zhu, 2009). Several studies have shown that various foodborne pathogens including Escherichia coli and Listeria monocytogenes can survive for hours or even days on utensils and equipment surfaces (Humphrey et al., 2001; Wilks et al., 2005, 2006; Martinon et al., 2012). On the other hand, L. monocytogenes and E. coli are among the most frequently involved bacterial species in foodborne diseases (Scallan et al., 2011; Oliveira et al., 2012). Consequently, controlling the longevity of microorga- nisms in surfaces is fundamental in reaching food safety standards and improving food quality and shelf-life (Nitschke et al., 2009). 2012). Several studies have shown the ability of microorganisms to attach to surfaces commonly found in the food processing environment, such as stainless steel, polystyrene, hydroxyapatite, rubber, glass and wood (Soares et al., 1992; Barnes et al., 1999). Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviation: MIC, Minimum inhibitory concentration; MBC, minimum bactericidal concentration; EO, essential oil; SPME, solid phase micro extraction. GC/MS-SPME characterization oregano essential oil Preliminarily, oregano EO was characterized using GC/MS-SPME. This technique was chosen because it gives a measure of the volatile molecules of the oil and the preliminary condition for the antimicrobial effects of EO is the contact between the antimicrobial molecule and the target cells. The contact is favored if the molecules are in their vapor phase, that corresponds to their most hydrophobic state, because this improves their partition in the cell membranes. In addition, this technique provides a volatile profile fingerprinting fundamental to standardize the EO composition in terms of the most effective molecules and consequently to standardize antimicrobial activity of the essential oils. In fact, the EO composition, and consequently the volatile molecule profile, can notably vary with plant variety and origin, extraction modality, agronomic practices, etc (Nannapaneni et al., 2009). Table 1 shows the total area of the GC peaks and the percentage (on the basis of the relative peak area) of each compound present in the headspace of the oregano Essential oils In this work, the oregano (Origanum vulgare) essential oil was obtained from Flora s.r.l. (Pisa, Italy). INTRODUCTION ( ) Several chemical detergents and disinfectants are commonly used and their application depends on their efficacy, safety and toxicity, corrosive effects, ease of removal and the subsequent sensory impact on the final products (Møretrø et al., 2009). Many of these chemicals are corrosive to equipment and toxic to humans if over exposure occurs (Lee and Pascall, 2012). In addition, conventional cleaning and disinfection regimes may also contribute to antimicrobial resistance dissemination (Lunden et al., 2003; Minei et al., 2008; Ryu and Beuchat et al., 2005; Surdeau et al., 2006; Cruz and Fletcher, 2012). The aims of this study were:(i) to evaluate the efficacy of killing L. monocytogenes and E. coli in solution, calcu- lating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oregano essential oil, reported to have antimicrobial activity against a large variety of microorganisms (Marino et al., 2001; Viuda-Martos et al., 2007) and (ii) to evaluate the oregano EO efficacy in reducing pathogenic cell loads on food contact surfaces such as wood and stainless steel. Most of the food processing industry's surfaces such as machinery, pipelines and working surfaces are made of stainless steel. This material is traditionally selected in the kitchen for food preparation because of its mechanical strength, corrosion resistance and longevity (Carrasco et al., 2012). Wood, although less employed in food industry than in domestic food preparation, is often used as cutting boards (Soares et al., 2012). Different contamination levels and contact times were ) Therefore, new sanitizing strategies based on the use of bio-solutions containing enzymes, phages, inter- species competitions, antimicrobials of microbial origin and natural plant molecules are constantly emerging (Simões et al., 2010; Chorianopoulos et al., 2008). The growing negative consumer perception against synthetic chemical compounds favors the research of such natural alternatives (Davidson, 1997). Essential oils (EOs) are Corresponding author. E-mail: francesca.patrignani@unibo.it. Tel: +39 0547 338133. Fax: +39 0547 382348. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviation: MIC, Minimum inhibitory concentration; MBC, minimum bactericidal concentration; EO, essential oil; SPME, so phase micro extraction. *Corresponding author. E-mail: francesca.patrignani@unibo.it. Tel: +39 0547 338133. Fax: +39 0547 382348. Afr. J. Microbiol. Res. Afr. J. Microbiol. Res. 2748 10 µL spot plated onto BHI agar. assessed for each tested surface. Characterization of oregano essential oil using GC/MS-solid phase micro extraction (SPME) Oregano EO in amount of 0.5 mL was placed into a 10 mL vial and sealed through a PTFE/silicon septum. Three different samples were prepared for each EO. The samples were conditioned for 30 min at 25°C. An SPME ﬁber covered by 50 mm divinylbenzene- carboxen-poly (dimethylsiloxane)- (DVB/CARBOXEN/PDMS StableFlex) (Supelco, Steiheim, Germany) was exposed to each sample at room temperature (25°C) for 20 min, and finally, the adsorbed molecules were desorbed in the GC for 10 min. For peak detection, an Agilent Hewlett-Packard 6890 GC gas-chromatograph equipped with a MS detector 5970 MSD (Hewlett-Packard, Geneva, Switzerland) and a Varian (50 m×320 µm×1.2 µm) fused silica capillary column were used. The temperature program, starting from 50°C, increased to 230°C at 3°C/min, this temperature was maintained for 1 min. Injector, interface, and ion source temperatures were 200, 200 and 230°C, respectively. Injections were performed with a split ratio of 30:1 and helium as carrier gas (1 mL/min). Compounds were identiﬁed by the use of the Agilent Hewlett-Packard NIST 98 mass spectral database. Data processing and statistical analysis The cell load data were analyzed by means of ANOVA one way by using Statistica for Windows. Strains L. monocytogenes Scott A and E. coli 555, used in this work, belong to the strain collection of the Department of Agricultural and Food Sciences, University of Bologna. The strains were maintained at -80°C and cultured in brain heart infusion (BHI) broth (Oxoid, Basingstoke, Humpshire, UK) for 24 h at 37°C. Before experiments, the strains were sub-cultured, on BHI broth for 24 h. y The target microorganisms chosen for this experiment were E. coli and L. monocytogenes. Both target microorganisms were inoculated at a concentration of 6.2 log cfu/cm2 for wood and 7 log cfu/cm2 for stainless steel. The inoculum was prepared from the pre-inoculum by making serial dilution in aphysiological solution, and the surfaces were inoculated with 10 (stainless steel) or 100 µL (wood). The inoculated surfaces were dried at room temperature for 0, 15, 30 and 60 min before treatments with Oregano EO. The treatments were performed by the immersion of the surfaces in 20 mL of Oregano EO solutions used at concentration of 125 mg/L for the treatment of the surfaces inoculated with L. monocytogenes, and 250 mg/L for the surfaces inoculated with E. coli. Oregano EO was delivered through 1% of ethanol. The duration of treatments was 10 min and the surfaces were removed from the solutions and placed into 10 mL of physiological solution, to determine viable bacteria by plate counting. E. coli was determined on Violet Red Bile Agar (VRBA, Oxoid, Basingstoke, Hants, England) with addition of MUG (Oxoid) supplement while Listeria Selective Agar based (Oxford formuladion) (Oxoid, Basingstoke, Hants, England) was used to detect L. monocytogenes. MATERIALS AND METHODS Stainless steel and wood surfaces were used for decontamination experiment with EOs. The sizes of the surfaces were 1 and 2.25 cm2 for stainless steel and wood, respectively. Before use, the surfaces were sterilized by autoclave at 121°C for 15 min. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of oregano EO against L. monocytogenes and E. coli For the determination of MIC values, 150 µL of BHI broth inoculated at three different levels (2, 4 or 6 log cfu/mL) of the tested pathogens (L. monocytogenes and E. coli), were added to 200 µL microtiter wells (Corning Incorporated, NY, USA). Oregano essential oil was properly diluted in ethanol 96% (VWR international, PROLABO, France) and 50 µL of the different dilutions were added in the microtiter wells, in order to obtain oregano EO concentrations ranging between 50 and 400 mg/L. Microtiter plates were incubated at 37°C and checked after 48 h. The MBC were determined by spotting 10 µL of each well after 48 h, onto BHI agar plates. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compound preventing visible growth of the inoculated cells after 48 h (MIC48h). The MBC was defined as the lowest concentration of the compound that caused the death of the inoculated cells and therefore there was no growth after 48 h of incubation at 37°C of a 2749 Siroli et al. Table 1. GC/MS-SPME characterization of oregano (O. vulgare) essential oil. essential oil. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of oregano EO against L. monocytogenes and E. coli Molecule Total peak area Area (%) α-Pinene 29616709 3.43 Camphene 3431254 0.40 β-Pinene 1664439 0.19 3-Carene 1693569 0.20 β-Myrcene 22216747 2.57 α-Phellandrene 2456150 0.28 α-Terpinene 33644575 3.89 Limonene 8547408 0.99 β-Thujene 5079423 0.59 γ-Terpinene 72693569 8.41 p-Cymene 309885246 35.86 Terpinolene 3166331 0.37 Ylangene 1739261 0.20 α-Cubebene 7410171 0.86 β-Bourbonene 3635866 0.42 Linalol 322546 0.04 Caryophyllene 49654406 5.75 (+)-Aromadendrene 1836300 0.21 Carvone 122563 0.01 α-Caryophyllene 1293689 0.15 γ -Muurolene 2910065 0.34 α-Terpineol 140525 0.02 Borneol 1637263 0.19 Copaene 311791 0.04 β-Farnesene 1325112 0.15 α-Muurolene 237200 0.03 δ-Cadinene 3114797 0.36 γ-Cadinene 1189108 0.14 Anetol 529966 0.06 Calamenene 525772 0.06 p-Cymen-8-ol 244657 0.03 p-Timol 1092970 0.13 Thymol 41459717 4.80 Carvacrol 249347302 28.85 Molecule Total peak area Area (%) α-Pinene 29616709 3.43 Camphene 3431254 0.40 β-Pinene 1664439 0.19 3-Carene 1693569 0.20 β-Myrcene 22216747 2.57 α-Phellandrene 2456150 0.28 α-Terpinene 33644575 3.89 Limonene 8547408 0.99 β-Thujene 5079423 0.59 γ-Terpinene 72693569 8.41 p-Cymene 309885246 35.86 Terpinolene 3166331 0.37 Ylangene 1739261 0.20 α-Cubebene 7410171 0.86 β-Bourbonene 3635866 0.42 Linalol 322546 0.04 Caryophyllene 49654406 5.75 (+)-Aromadendrene 1836300 0.21 Carvone 122563 0.01 α-Caryophyllene 1293689 0.15 γ -Muurolene 2910065 0.34 α-Terpineol 140525 0.02 Borneol 1637263 0.19 Copaene 311791 0.04 β-Farnesene 1325112 0.15 α-Muurolene 237200 0.03 δ-Cadinene 3114797 0.36 γ-Cadinene 1189108 0.14 Anetol 529966 0.06 Calamenene 525772 0.06 p-Cymen-8-ol 244657 0.03 p-Timol 1092970 0.13 Thymol 41459717 4.80 Carvacrol 249347302 28.85 EO, as well as the cumulative percentages of the classes of compounds (monoterpenes, sesquiterpenes, oxygenated monoterpenes, aliphatic alcohols, aliphatic aldehydes, esters and ketones). The volatile profiles of the used oregano essential oil was characterized by the presence of 34 identified molecules belonging to different chemical classes. The main components of this type of oregano were terpinolene, carvacrol and p-cymene accounting for about 70% of the total area of the identified molecules. These data are in agreement with the data of Ortega-Nieblas et al. (2011), Russo et al. (1997) and Bisht (2009) who found carvacrol as one of EO, as well as the cumulative percentages of the classes of compounds (monoterpenes, sesquiterpenes, oxygenated monoterpenes, aliphatic alcohols, aliphatic aldehydes, esters and ketones). The volatile profiles of the used oregano essential oil was characterized by the presence of 34 identified molecules belonging to different chemical classes. The main components of this type of oregano were terpinolene, carvacrol and p-cymene accounting for about 70% of the total area of the identified molecules. These data are in agreement with the data of Ortega-Nieblas et al. MIC and MBC determination The MICs and the MBCs of the oregano EO against L. monocytogenes Scott A and E. coli555 were assessed after incubation at 37°C with three levels of the target microorganisms (Table 2). Differences in the MICs and MBCs were observed in relation to species and the inoculum level taken into consideration. In fact, increasing the inoculation level increased the MIC and MCB values for both microorganisms considered. This data are in agreement with literature (Belletti et al., 2010). Oregano EO showed the highest antimicrobial activity against L. monocytogenes with respect to E. coli. In fact, the L. monocytogenes MIC ranged between 125 and 200 mg/L while those for E. coli ranged between 250 and 350 mg/L. This behavior for Gram-negative bacteria can be due to the presence of the outer membrane, which acts as an efficient permeability barrier against macromolecules and hydrophobic substances, as well as to the high content in cyclopropane fatty acids of the inner membrane (Chang and Cronan, 1999). The addition of oregano EO speed up the viability decrease of both microorganisms. The treatment with EO, at the concentration used, reduced, after 10 min of contact, E. coli cell loads of 1.9 and 1.2 log cfu/cm2 in steel and wood, respectively while L. monocytogenes, immediately after the inoculation on steel and wood coupons reduced its counts of about 2 and 1 log cfu/cm2, respectively. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of oregano EO against L. monocytogenes and E. coli (2011), Russo et al. (1997) and Bisht (2009) who found carvacrol as one of the major components. Also, according to Teixeira et al. (2013), who studied the composition of oregano essential oil from Portuguese origin, carvacrol, terpinene and thymol were the main components. This is positive because a wide literature attributed to carvacrol and to monoterpenes the great antibacterial activity of oregano EO (Burt, 2004; Gutierrez et al., 2008; Oussalah et al., 2006). In fact, such molecules can interact with some cellular structures causing the inhibition of cell growth or cell death. However, according to Caccioni et al. (1998), to evaluate the antimicrobial activity of an EO it is fundamental to use a holistic approach due to 2750 Afr. J. Microbiol. Res. Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oregano (Origanum vulgare) essential oil against L. monocytogenes and E. coli in relation to the inoculum level. Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oregano (Origanum vulgare) essential oil against L. monocytogenes and E. coli in relation to the inoculum level. Microorganism Cell concentration (log cfu/mL) 6 log cfu/mL 4 log cfu/mL 2 log cfu/mL MIC 24 h (mg/L) MBC (mg/L) MIC 24 h (mg/L) MBC (mg/L) MIC 24 h (mg/L) MBC (mg/L) L. monocytogenes 175 225 175 225 125 150 E. coli 350 350 300 325 250 250 Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oregano (Origanum vulgare) essential oil against L. monocytogenes and E. coli in relation to the inoculum level. determination of the surviving L. monocytogenes and E. coli cells. In Figures 1 and 2, the results obtained for E. coli and L. monocytogenes, respectively, are shown. A decrease of viability over time was observed indepen- dently of microorganisms and oregano EO supplemen- tation. The viability decreases were more pronounced on wood material than in steel coupons, independently of the treatment time and EO supplementation. 60 min after inoculation, E. coli and L. monocytogenes were present on the control steel coupons (untreated) at cell loads of 6.6 and 5.9 log cfu/cm2, respectively. Significantly lower counts (3.6 and 4.2 log cfu/cm2, for E. coli and L. monocytogenes, respectively) were recorded in the control wood coupons 60 min after inoculation. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of oregano EO against L. monocytogenes and E. coli Earlier research indicate that survival of microorganisms on surfaces is affected by many factors including tempe- rature, microbial species (Rusin et al., 2002), nature of surfaces (Gill and Jones, 2002), time lapsed post-inocu- lation, moisture level and inoculum size (Monville and Schaffner, 2003). synergistic or antagonistic actions among the different EO components. synergistic or antagonistic actions among the different EO components. Effects of oregano EO in decontaminating stainless steel and wood surfaces inoculated with L. monocytogenes and E. coli When the treatment with the EO was performed after 30 and 60 min from the inoculation of the coupons, lower microbial counts were recorded with respect to treatment carried out immediately after the inoculation. This phenomenon was more pronounced in wood in compa- rison with steel. To evaluate the decontamination efficacy of oregano EO, stainless steel and wood coupons previously sterilized were inoculated at level of 7 and 6.2 log cfu/cm2 with L. monocytogenes and E. coli, respectively. Immediately after the inoculation and after 15, 30, 60 min at room temperature (about 25°C), the coupons were treated with 20 ml of oregano EO treatment solutions at concentration of 125 ppm for L. monocytogenes, or 250 ppm for E. coli., corresponding to the MIC values previously determined in antimicrobial assay. After 10 min of contact between the coupons and the EO solution, the surfaces were removed from treatment solutions and were placed into 10 ml of physiological solution, which was used for the This result can be due to the porosity of the wood where the microbial cells might penetrate under the surface of the wood. On the other hand, several authors make remarks on the problem of recovery of microor- ganisms from porous or damaged surfaces (De Vere and Purchase, 2003). Earlier research indicates the decreased number of microorganisms over time delibe- rately inoculated on wood surfaces (Carpentier, 1997). For example Abrishami et al. (1994) observed a reduction of 98% 2 h after inoculation of new wood by E. coli, 2751 Siroli et al. Figure 1. Recovery of Escherichia coli cell loads (log cfu/cm2) inoculated onto stainless steel (washed or not with oregano essential oil) and wood coupons (washed with oregano essential oil or not ). The treatment with oregano essential oil was performed for 10 min after that the recovery of the pathogenic strain was performed immediately after treatment (0), after 15, 30, 60 min. For each group considered, different letter represent significant differences (p<0.005). Figure 1. Recovery of Escherichia coli cell loads (log cfu/cm2) inoculated onto stainless steel (washed or not with oregano essential oil) and wood coupons (washed with oregano essential oil or not ). The treatment with oregano essential oil was performed for 10 min after that the recovery of the pathogenic strain was performed immediately after treatment (0), after 15, 30, 60 min. For each group considered, different letter represent significant differences (p<0.005). Figure 2. Conclusion y Barnes LM, Lo MF, Adams MR, Chamberlain AHL (1999). Effect of milk proteins on adhesion of bacteria to stainless steel surfaces. Appl. Environ. Microbiol. 65: 4543-4548. This research shows the good potential of the used oregano essential oil to inhibit pathogenic microor- ganisms both when tested as planctonic cells and when inoculated onto surfaces of industrial interest. In particular, the trials of surface decontamination have highlighted the ability of this type of oregano essential oil to inactivate L. monocytogenes and E. coli after just 10 min of contact, independently of the surface considered. The reductions obtained, representing more than 90% of the population, are very promising, also taking into account that the inoculation levels tested exceeded significantly those present on industrial surfaces. The American Public Health Association recommends that chemical sanitizers are able to reduce the pathogenic species and mesophilic bacteria of stainless steel surfaces up to 0.3 log cfu/cm2. The trials we performed inoculating L. monocytogenes and E. coli at level of 10- 100 cfu/cm2 of surface and treating with oregano essential oil permitted reaching cell loads under the detection limit after 10 min of contact. According to APHA, the sanification level is acceptable when the coliform cell loads are under 5 cfu/cm2 and acceptable when ranging between 5-100 cfu/cm2. Moreover, according to Lelieveld e al. (2003), an ideal sanitizer should have characteristics such as wide action spectrum, environmental resistance, toxicity and corrosiveness absence. In our opinion, oregano essential oil could be considered as new tool to prevent or delay colonization of food contact surfaces. However, its use at industrial level still requires additional investigations on the ability of removing it and on its organoleptic impact. Belletti N, Sado Kamdem S, Tabanelli G, Lanciotti R, Gardini F (2010). Modeling of combined effects of citral, linalool and β-pinene used against Saccharomyces cerevisiae in citrus-based beverages subjected to a mild heat treatment. Int. J. Food Microbiol. 136:283- 289. Bisht D, Chanotiya CS, Rana M, Semwal M (2009). Variability in essential oil and bioactive chiral monoterpenoid compositions of Indian oregano (Origanum vulgare L.) populations from northwestern Himalaya and their chemotaxonomy. Ind. Crops Prod. 30:422-426. Burt S (2004). Essential oils: Their antibacterial properties and potential applications in foods: A review. Int. J. Food Microbiol. 94: 223-253. Caccioni DRL, Guizzardi M., Biondi DM, Renda A, Ruberto G (1998). Effects of oregano EO in decontaminating stainless steel and wood surfaces inoculated with L. monocytogenes and E. coli Recovery of Listeria monocytogenes cell loads (log cfu/cm2) inoculated onto stainless steel (washed or not with oregano essential oil) and wood coupons (washed with oregano essential oil or not ). The treatment with oregano essential oil was performed for 10 min after that the recovery of the pathogenic strain was performed immediately after treatment (0), after 15, 30, 60 min. For each group considered, different letter represent significant differences (p<0.005). Figure 2. Recovery of Listeria monocytogenes cell loads (log cfu/cm2) inoculated onto stainless steel (washed or not with oregano essential oil) and wood coupons (washed with oregano essential oil or not ). The treatment with oregano essential oil was performed for 10 min after that the recovery of the pathogenic strain was performed immediately after treatment (0), after 15, 30, 60 min. For each group considered, different letter represent significant differences (p<0.005). while Ak et al. (1994) observed a reduction of 99.9% of L. monocytogenes after 2 h. Also Milling et al. (2005) showed a consistent viability loss of the inoculated micro- organism on wood surfaces. These authors showed that the survival of the bacteria on wood was dependent on various factors such as the wood species, the type of the inoculated bacterium, the ambient temperature, and humidity and attributed it to the better hygienic perfor- mances of pine and oak with respect to plastic in combination with the hygroscopic properties of wood and the effect of wood extractives. Similar results were observed by Gehrig et al. (2002) and Schonwalder et al. Afr. J. Microbiol. Res. 2752 Ak NO, Cliver DO, Kaspar CW (1994). 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https://openalex.org/W2611018459	https://nottingham-repository.worktribe.com/preview/897343/Living%201-s2.0-S0003687017301503-main-UX.pdf	English	null	Living with an autonomous spatiotemporal home heating system: Exploration of the user experiences (UX) through a longitudinal technology intervention-based mixed-methods approach	Applied Ergonomics/Applied ergonomics	2,017	cc-by	17,174	a r t i c l e i n f o Article history: Received 22 September 2016 Received in revised form 1 February 2017 Accepted 23 June 2017 Available online 27 July 2017 Keywords: User-experience UX Design Human-computer interaction Hci Spatiotemporal heating Application Interface Longitudinal Home heating Technology intervention Rising energy demands place pressure on domestic energy consumption, but savings can be delivered through home automation and engaging users with their heating and energy behaviours. The aim of this paper is to explore user experiences (UX) of living with an automated heating system regarding expe- riences of control, understanding of the system, emerging thermal behaviours, and interactions with the system as this area is not sufﬁciently researched in the existing homes setting through extended deployment. We present a longitudinal deployment of a quasi-autonomous spatiotemporal home heating system in three homes. Users were provided with a smartphone control application linked to a self-learning heating algorithm. Rich qualitative and quantitative data presented here enabled a holistic exploration of UX. The paper's contribution focuses on highlighting key aspects of UX living with an automated heating systems including (i) adoption of the control interface into the social context, (ii) how users' vigilance in maintaining preferred conditions prevailed as a better indicator of system over-ride than gross deviation from thermal comfort, (iii) limited but motivated proactivity in system-initiated communications as best strategy for soliciting user feedback when inference fails, and (iv) two main motivations for interacting with the interface e managing irregularities when absent from the house and maintaining immediate comfort, latter compromising of a checking behaviour that can transit to a system state alteration behaviour depending on mismatches. We conclude by highlighting the complex socio-technical context in which thermal decisions are made in a situated action manner, and by calling for a more holistic, UX-focused approach in the design of automated home systems involving user experiences. Article history: Received 22 September 2016 Received in revised form 1 February 2017 Accepted 23 June 2017 Available online 27 July 2017 Keywords: User-experience UX Design Human-computer interaction Hci Spatiotemporal heating Application Interface Longitudinal Home heating Technology intervention © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). heavily on fossil fuels in satisfying energy demands and these fuels contribute signiﬁcantly (74%) to global CO2 emissions (Sims et al., 2007). a r t i c l e i n f o In the UK, domestic energy use (26%) is the second largest contributor by sector (Department of Energy and Climate Change, 2014) and the majority of that, at 66%, is required for space heat- ing (Palmer et al., 2011). In order to reduce energy demands for space heating, “occupants need better guidance and vastly improved systems” (Stevenson and Leaman, 2010). This highlights the complexity of tackling domestic energy usage, with building fabric, heating delivery systems and user interfaces (UI) (in this research used to denote any tangible or graphical computer mechanism for users to control the heating system) all playing a role in satisfying users’ comfort requirements. Living with an autonomous spatiotemporal home heating system: Exploration of the user experiences (UX) through a longitudinal technology intervention-based mixed-methods approach Martin Kruusimagi a, , Sarah Sharples a, Darren Robinson b a University of Nottingham, England, United Kingdom b University of Shefﬁeld, England, United Kingdom Martin Kruusimagi a, , Sarah Sharples a, Darren Robinson b a University of Nottingham, England, United Kingdom b University of Shefﬁeld, England, United Kingdom a University of Nottingham, England, United Kingdom b University of Shefﬁeld, England, United Kingdom * Corresponding author. E-mail address: martin@kruusimagi.com (M. Kruusimagi). Applied Ergonomics 65 (2017) 286e308 Applied Ergonomics 65 (2017) 286e308 Contents lists available at ScienceDirect E mail address: martin@kruusimagi.com (M. Kruusimagi). http://dx.doi.org/10.1016/j.apergo.2017.06.017 0003-6870/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Mankind is currently facing one of its greatest ever challenges in climate change, which is primarily caused by human activity resulting in large quantities of pollutants emitted to the atmo- sphere. The Intergovernmental Panel on Climate Change (IPCC) has suggested that in order to maintain global warming below 2 C over 21st century, a reduction of 40e70% of global anthropogenic greenhouse gas (GHG) emissions by 2050 and reduction to near or below zero emission levels by 2100 are required (Pachauri et al., 2014). This poses great challenge as global population relies Research has shown that theoretical domestic energy usage based on the building's and occupants' characteristics does not M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 287 arise. The implications of these factors and the users' experience of them remains poorly explored in a true-to-life setting, limiting our abilities as designers to be able to design systems that engage users and nudge them towards more sustainable behaviour. often align with actual usage (Audenaert et al., 2011) and the poor performance of many advanced buildings has been attributed to poorly designed controls, occupants' inability to understand the building's functionality, and lack of user control (Stevenson and Rijal, 2010; Tuohy and Murphy, 2012). Peffer et al. reviewed a large body of work into thermostat usability and user perceptions, highlighting many misconceptions and usability barriers of regular and programmable thermostats (Peffer et al., 2011). It has been proposed that automation can solve these issues by operating heating controls on the human's behalf and simulations have demonstrated potential energy savings between 7 and 28% (Gao and Whitehouse, 2009; Gupta et al., 2009; Krumm and Brush, 2011; Lu et al., 2010; Scott et al., 2011). These savings were pro- posed to be achieved by minimising heating based on users' loca- tion within and absences from home, as well as different temperature setback settings. These authors share the belief in this technology's promise for solving the complex issue as automation can perform the tasks users are unwilling or unable to perform (Parasuraman and Riley, 1997) as regulating heater settings forms a minute part of a highly complex social, environmental, and tech- nical setting in which occupants perform a multitude of activities. 2. Material and methods However, the notion of ‘ironies of automation’ (Bainbridge, 1983) has been widely recognised, highlighting unintended con- sequences resulting from delegating human tasks to automation. In this context, it has been suggested that if automation chooses on the human's behalf to take environmentally friendly action, the consequences, on top of reduced autonomy for the humans, may include diminished understanding of their actions' impacts on the environment (Jaffari and Matthews, 2009). Such disengagement their energy and heating behaviour is likely to encourage “creative ways of working around the system rather than straightforward, energy-efﬁcient compliance with it” (Jaffari and Matthews, 2009, p. 9). Disengagement poses a real threat as ambient intelligence or ubiquitous computing systems (which include autonomous home heating) fade into the fabric of life and thus aim for little to no interaction with the end user (Borgmann, 1995). These interactions (used here to denote actions that users take in order to control or obtain information from their heating system, not to be confused with interpersonal interactions) and their implications have not been sufﬁciently researched in their correct context. Several ‘lab homes’ have been built incorporating home automation technology to investigate user experiences (AIRE Group MIT, 2012; Amigo Project, 2012; Brown and Wyatt, 2010; Georgia Institute of Technology, 2012; Herkel et al., 2008; Mozer, 2012; Ruyter and Pelgrim, 2007; University of Essex, 2012; University of Florida, 2012), but this approach lacks ecological validity when one con- siders the manner in which this technology penetrates mainstream. Already various home automation products are being commercially introduced into everyday use such as smart thermostats (Ecobee, 2015; Nest, 2012), home security products (Glate, 2015; Kwikset, 2015; Nest, 2015), lighting solutions (Philips, 2015), wiﬁ-enabled plugs to turn standard home appliances ‘smart’, or general home- automation products (Fibaro, 2015; Smartthings, 2015). This has validated Rodden & Benford's argument that ‘smart’ homes (by which we mean homes with computational capability to make decisions on the occupant's behalf and act these out in the envi- ronment) will be an evolution from existing homes, rather than a revolution with new homes being built with the ‘smart’ infra- structure built in (lab-homes). 2. Material and methods Furthermore, commercial devices we see introduced rely heavily on smartphones and tablets as in- terfaces for these systems, which introduces another interesting dynamic e heating systems and their operation becomes more invisible to us, while the control interfaces become more personal, causing potential issues in multi-occupant households where conﬂicts or unawareness as a result of multiple controllers may For reasons presented above, this methodology focuses on a technological intervention approach situated in individuals' homes. This approach is very intensive in terms of technology deployment, recruitment, and data collection, therefore typically involves a small number of participants over an extended period of time. 1. Introduction Therefore, research for home automation systems needs to focus on user experiences in the real world e in their own homes, as that is where these devices will exist and the energy behaviour of their users emerge. In this paper we present the results of a mixed- methods study investigating the user experience of a quasi- autonomous (system utilising sensory input from the environ- ment and minimal occupant input through thermal feedback to automatically create and implement a heating schedule matching heating times to predicted occupant presence, while providing users with input and over-ride capabilities) spatiotemporal heating system in the wild. We present the highly ecologically valid research methodology of a technology intervention utilising a smartphone control application and a mixture of qualitative and quantitative research methods to understand and explain the emerging user experiences and their implications. 2.1. Participants Sampling was done on availability and self-selection basis. However, several requirements were posed to participants to be eligible. Namely, (1) participants had to be responsible for their household heating expenses, (2) preferably their existing heating system was electricity based and not storage heating (electrical heater storing thermal energy during low electricity cost at night and releasing heat during the day), (3) they lived in a house/ﬂat no bigger than 5e6 rooms, (4) apartments had a minimum of 2 rooms, and lastly, (5) to be eligible, participants were required to own and use an iOS or Android operating system smartphone. While electricity-heated households were preferred, (this was only due to smaller differences in potential heating cost in comparison to par- ticipants switching from gas-based to electric heaters), no limita- tions to households with other fuel types were set. Participant recruitment was done using the academic participant recruitment service callforparticpants.com, by distributing the study page from the site on University of Nottingham email mailing lists, and on social media network Facebook. In total three households (see Table 1 for full detail) were recruited out of several who showed interest, but despite qualifying, chose not to take part. 2.3. Data capture The deployed technology acted as primary method for data capture. Table 2 describes the captured data as various measures at different intervals for different reasons were captured. These calculations were performed and acted out almost invis- ibly to the user, except for feedback through the control application interface. In addition, users were probed on using questionnaires, in- terviews and depictive explanation tools. Prior to the experiment's launch, an online-questionnaire was used to obtain algorithm's training data from users. This questionnaire asked users to provide the number and names of all rooms in their dwelling, preferred temperatures for those rooms, and indicate in 1-h slots their assumed presence. Over the course of the experiment interviews were conducted to solicit participants' feedback regarding their UX and ideas regarding the heating system functionality. The open- ended questions of all three interviews can be seen in Tables 3, 4 and 6. Household-speciﬁc questions derived from Google Ana- lytics app usage data for the second and third interview can be seen in Tables 5 and 7. For the ﬁrst interview, the participants were asked to prepare a diagram explaining how they thought the heating system worked. The interface, seen in Fig. 3, had three primary functions e (i) providing users with thermal information about their house and allowing manual over-rides if requested (a on Fig. 3), (ii) soliciting thermal sensation & preference feedback as well as perceived control votes (b on Fig. 3), and (iii) allowing users to create and manage “away” schedules that denoted uncharacteristic absence periods from home (c on Fig. 3). Two conﬁgurations of the application were deployed e the “visible” version displayed a graph of thermal conditions in each room 2 h into the past and 2 h into the future, which the “blind” version did not (see Fig. 4 for comparison). This variation was used to see whether differences in the user's understanding of the heating system functionality, resulted from feedback or feed- forward data provided by the interface. 2.2. Apparatus Participants’ houses were ﬁtted with a spatiotemporal quasi- autonomous heating system that consisted of stand-alone electric convector heaters, Wi-Fi-enabled plugs and a Raspberry Pi com- puter equipped with temperature and motion sensors, highlighted in Fig. 1. Each room was ﬁtted with a kit of these components that all communicated to a central database on a university server that also hosted the control algorithm for heating. Users were presented with a smartphone or tablet application that acted as their interface for communicating with the heating system. The heating algorithm (seen in Fig. 2), which was imple- mented as a server-side script was a combination of existing mathematical expressions and principles. It created a different M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 288 respectively (10 on Fig. 5). These were seen as methods for the user to inform the heating system about irregularities in behaviour and prevent heating when they were not around. Interface design was not a key element in this study, but rather served its part as the wider technological intervention that was used to explore UX. heating schedule for every day of the week and every room in the house. Triggered every midnight and at key user interactions, the algorithm calculated the presence probability in every room based on previous calculations and motion sensor data (1 on Fig. 2) for every 10-min time step that the day was divided into. Subsequently, it assessed if the presence should be heated for (steps 2 and 3 on Fig. 2) and so, a heating period was scheduled. The Raspberry Pi computers retrieved these schedules and performed heating, using an optimum start algorithm that determined the duration of pre- heating prior to users’ predicted arrival in order to ensure suit- able conditions for predicted presence. 2.4. Design Users were free to utilise the smartphone application as they wished and Fig. 5 highlights all possible interactions and use cases. Initially, the app checked if the user was registered (1 on Fig. 5) to keep user data private and differentiate between users. If this was not true, the user was asked to enter information from the exper- imenter to register their app (2 & 3 on Fig. 5). For registered users, their house-speciﬁc data was retrieved and either a feedback screen (used for asking feedback on heating decisions when users were present in a room without the algorithm predicting their presence (5 & 6 on Fig. 5)), or the home screen (5 on Fig. 5) displayed. On the home screen, users could view different rooms in their house (8 on Fig. 5), or alter the temperature in those rooms (7 on Fig. 5). The experiment was a semi-longitudinal experiment lasting Fig. 1. Illustrating system design of ﬁeld study technology. Users also had the option to submit a vote (12 on Fig. 5), which consisted of selecting the room they were providing feedback for, indicating their thermal sensation and preference on the ASHRAE scale (7-point likert scale: cold, cool, slightly cool, neutral, slightly warm, warm, hot) (ASHRAE, 1966), and their perceived level of control over the heating system (scale from 1 to 7 e no control at all to absolute control, respectively). Users were also directed to this screen after every temperature alteration, and an option to dismiss the vote was provided. Lastly, Users could access the Diary screen (9 on Fig. 5) where they could create and delete (11 on Fig. 5) short and long away schedules, which addressed “I am coming home later than usual” and “I will be away for a couple of days” scenarios Fig. 1. Illustrating system design of ﬁeld study technology. Table 1 Table 1 Displaying characteristics of participating households (all names are pseudonyms). Characteristics House 1 House 2 House 3 Occupants Postgraduate student (male) - Carl 1 postgraduate student (male) - Paul, 1 professional (female) - Diane 2 postgraduate students (1 male - John, 1 female - Mildred) Heating strategy Maximise comfort (algorithm aims for ‘neutral’ thermal sensation) Minimise discomfort (algorithm aims for ‘slightly cool’ thermal sensation) Minimise discomfort (algorithm aims for ‘slightly cool’ thermal sensation) App visibility Visible (app displays future temperature predictions) Blind (current temperature snapshot only) Visible (app displays future temperature predictions) Dwelling type Purpose built ﬂat Converted ﬂat Converted ﬂat Rooms deployed with equipment 5 rooms e Lounge, Bedroom, Second bedroom, Bathroom, Kitchen 4 rooms e Lounge/kitchen, Bedroom, Bathroom, Hallway 3 rooms e Lounge/kitchen, Bedroom, Bathroom Existing heating system Gas central heating Electric convector heaters Electric convector heaters 289 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 Fig. 2. Depicting the functional ﬂow of the proposed control algorithm. Fig. 2. Depicting the functional ﬂow of the proposed control algorithm. 5e6 months. The multitude of different collected data facilitated an explorative study design, rather than a strict independent- dependent variable isolation via highly controlled set-up. Regard- less, the experiment can be described as utilising a between mea- sures study design with two independent variables e smartphone application condition (users' ability to see the feedback/feed- forward graph - see Fig. 4 for graphic differentiation), and the heating strategy condition (control algorithm opting to maximise comfort or minimise discomfort). However, due to individual dif- ferences between the usage of the systems and the algorithm's innate quality of adapting itself to its user, the conditions could not be analysed directly and rigorous inferential statistical analysis was impossible. Rather, the conditions were observed individually and descriptive statistics used across conditions. Dependent variables were the thermal experience of living with an automated heating system, and the user experience of the heating system and control interface. Due to the large amount of data collected, results regarding the system technical performance, algorithm perfor- mance, and thermal experience of different heating strategies are omitted. Despite contributing to the home environment, these 290 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 Fig. 3. Illustrating the smartphone application given to study participants. Fig. 3. Illustrating the smartphone application given to study participants. Fig. 3. Illustrating the smartphone application given to study participants. Fig. 3. Table 4 Question number Question 1 Is there anything you would like to add or change about how the system works? 2 How do you as a household use the heating application? 3 How often have you changed the heating settings using the app in comparison to other strategies such as adjusting your clothing or having a hot or cold drink? 4 [Household-speciﬁc application usage questions e please see Table 5 below for full detail] Question number Question 1 Is there anything you would like to add or change about how the system works? 2 How do you as a household use the heating application? 3 How often have you changed the heating settings using the app in comparison to other strategies such as adjusting your clothing or having a hot or cold drink? 4 [Household-speciﬁc application usage questions e please see Table 5 below for full detail] Table 3 factors did not inﬂuence the questions asked of the data in this paper. Out of 4 total conditions, only 3 were used due to the lack of participating households. The maximise comfort e blind applica- tion condition was not deployed (other conditions are detailed above in Table 1). 2.5. Procedure Ethical approval for the technology intervention was gained from the University of Nottingham Faculty of Engineering Ethics Committee prior to commencement. The experiment took place Table 4 Detailing questions for ﬁeld study Interview 2. Table 4 Detailing questions for ﬁeld study Interview 2. Table 1 Illustrating the smartphone application given to study participants. Fig. 4. Comparing the 'visible' (left) and 'blind' (right) versions of the home screen. Fig. 4. Comparing the 'visible' (left) and 'blind' (right) versions of the home screen. M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 291 Fig. 5. Illustrating the user interaction ﬂow and functional logic of the smartphone application. Fig. 5. Illustrating the user interaction ﬂow and functional logic of the smartphone application. M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 292 Table 2 Detailing the quantitative data obtained during the ﬁeld study experiment. Type of data (measure) Method of obtaining Reason of data gathering Temperature (C) Taken by Raspberry Pi every 10 min using temperature sensor Apparatus functioning (temperature set-point calculation by algorithm) Answering questions about thermal comfort Presence (time start, time end) Taken by Raspberry Pi when motion was detected using motion sensor Apparatus functioning (heating schedule creation by algorithm) Answering questions about algorithm functioning and user experience Calculated slopes (number) Calculated & stored in database by system algorithm after every time heating occurred Apparatus functioning (heater optimum start calculation by algorithm) Answering questions about algorithm functioning Thermal sensation votes (number) Obtained from user whenever the user chose to submit value Apparatus functioning (temperature set-point calculation by algorithm) Answering questions about thermal comfort Thermal preference votes (number) Obtained from user whenever the user chose to submit value Answering questions about thermal comfort Control votes (number) Obtained from user whenever the user chose to submit value Answering questions about user experience System set-point alterations (C) Obtained from user whenever the user chose to change the prevailing temperature in the room Apparatus functioning (temperature set-point) Away schedules (time values) Obtained from user whenever the user chose to submit value Apparatus functioning (heating schedule creation by algorithm) Answering questions about user experience Application launches (timed instances) Automatically logged by Google Analytics when user opened the smartphone application Answering questions about user experience Application page views (timed instances) Automatically logged by Google Analytics when user used the smartphone application Answering questions about user experience Application events (timed instances) Automatically logged by Google Analytics when user accepted or dismissed a suggested schedule, provided a vote, changed temperature, provided or delete an away schedule, or viewed a room overview in the smartphone application Answering questions about user experience Table 3 Detailing questions for ﬁeld study Interview 1. Table 1 Question Number Question 1 How would you describe your original heating system, not the one we installed? 2 Could you please explain to me with the help of your diagram, how the heating system works? over 6 months between February 2015 and July 2015 (inclusive). Potential participants were asked questions about the heating and communications infrastructure in their homes to assure their ability to partake. Suitable participants were asked to ﬁll in the pre- study questionnaire and the obtained data used to set up the experimental equipment. Interview 1 was conducted 1e2 weeks after deployment, second interview 2 months after deployment, and third interview during equipment collection. Throughout the experiment, check-up emails were sent to participants ensuring everything was running as expected and to keep a dialogue with participants. The researcher checked the experiment database daily to make sure the system was functioning properly. When errors occurred, the apparatus was restarted using built in remote trou- bleshooting capabilities. If this was not possible, the participant was contacted with a request to manually restart the equipment by removing and replacing power supply. Participants were sent reminder push notiﬁcations as means to prompt thermal comfort vote submission. The rate of push notiﬁcations decayed over the course of the experiment with a notiﬁcation sent every 2 days in February, every 3 days in March and every 4 days until the end of the experiment thereafter. Following experiment completion, par- ticipants were compensated with £20 Amazon shopping voucher per month of participation. Table 3 3. Results The experiment generated a vast amount of quantitative and qualitative data and subsequently, the results are divided into a brief description of the user types that emerged, followed by a more detail look at some of the potential user interactions and experi- ences that emerged. Finally we explore the relationships between some emerged interactions with the smartphone application and M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 293 Table 5 Detailing household-speciﬁc questions for ﬁeld study Interview 2. Household number Question 1 How do you decide when to change the temperature using the App? 1 When the app notiﬁes you that it wasn't expecting you, how do you decide whether to accept or reject the suggestion or ignore the notiﬁcation altogether? 1 Have your habits in doing this changed over time? 1 Has the way you use the app or when you use the app changed over time? 1 Please describe how and when you use the away schedules? 2 Has the way you use the app or when you use the app changed over time? 2 Most of the temperature changes in the house are done from one device, does this mean that it is one user making the decisions, are devices shared, or do you discuss temperature changes before putting them in the app? Could you describe how these changes happen between you as a household? 2 Please describe how either of you use the app - when do you open the app, and what do you do when you have opened it? 2 You have a third device in the household, could you please describe how the app is used on it - who uses it, when etc.? 2 Over time the number of times you change the temperature has decreased a lot. Please describe how these changes have occurred and the reasons behind them. 2 When the app notiﬁes you that it wasn't expecting you, how do you decide whether to accept or reject the suggestion or ignore the notiﬁcation altogether? 3 Has the way you use the app or when you use the app changed over time? 3 Both of you have the heating application on your phones, could you describe how you as a household make any changes - do you consult among each other before submitting anything to the app, is it individual, etc? Table 6 Question number Question Table 7 Detailing household-speciﬁc questions for ﬁeld study Interview 3. Household number Question 1 You have told me over the last few interviews that you had a guest often stay with you. Could you describe the way in which your guest had any control over the heating application? 1 Over time, your use of changing temperature on the app decreased. Was this due to warming weather or did anything else affect this? 2 You have used the long away schedules on occasion throughout the experiment. Could you describe why you have carried on using these while your overall usage has decayed? 3 The one feature that you have used on occasion throughout the experiment was setting a long away schedule. Could you explain why this was a feature that you used so often? 1 You have told me over the last few interviews that you had a guest often stay with you. Could you describe the way in which your guest had any control over the heating application? 1 Over time, your use of changing temperature on the app decreased. Was this due to warming weather or did anything else affect this? 2 You have used the long away schedules on occasion throughout the experiment. Could you describe why you have carried on using these while your overall usage has decayed? 3 The one feature that you have used on occasion throughout the experiment was setting a long away schedule. Could you explain why this was a feature that you used so often? me over the last few interviews that you had a guest often stay with you. Could you describe the way in which your guest had any he heating application? r use of changing temperature on the app decreased. Was this due to warming weather or did anything else affect this? the long away schedules on occasion throughout the experiment. Could you describe why you have carried on using these while your has decayed? has decayed? e that you have used on occasion throughout the experiment was setting a long away schedule. Could you explain why this was a u used so often? factors affecting those. conﬁrmed this and showed distinctly different emerging thermal preferences and thermal adaptation behavioural patterns. Explo- ration of those establishes an understanding of the collected data and sets the scene for exploring UX in more detail. Table 6 The emergent user types of ‘fashion user’, ‘frugal user’ and ‘everything's ﬁne’ user were not attempts to classify all behaviours, but rather to explore some typical and potential behaviours and interactions that may Table 5 12 Could you describe your overall experience in living with this type of a heating system? 13 How in control of the heating did you feel over the course of the experiment? 14 If this heating system was available on the market today, would you buy it for your home? Table 7 Detailing household-speciﬁc questions for ﬁeld study Interview 3. Household number Question 1 You have told me over the last few interviews that you had a guest often stay with you. Could you describe the way in which your guest had any control over the heating application? 1 Over time, your use of changing temperature on the app decreased. Was this due to warming weather or did anything else affect this? 2 You have used the long away schedules on occasion throughout the experiment. Could you describe why you have carried on using these while your overall usage has decayed? 3 The one feature that you have used on occasion throughout the experiment was setting a long away schedule. Could you explain why this was a feature that you used so often? Table 6 Detailing questions for ﬁeld study Interview 3 (Debrief interview). Question number Question 1 For the last time, I would like for you to take a look at the diagram we have been working with and tell me whether you would like to add or change anything about how in your mind the system works? 2 Did the heating system behave the way you expected it to behave? 3 [Household-speciﬁc questions e please see Table 7 below for full detail] 4 What would you say are the most important differences between this type of a system and conventional heating controls? 5 Did you encounter any funny incidents or disagreements over the course of the experiment regarding the heating? 6 If you had a choice, would you prefer to keep this type of a heating system or would you like to revert to your previous system and why? 7 Could you please describe the experience of controlling the heating through your phone rather than a more conventional method? 8 Similarly to the pre-study questionnaire, would you be able to estimate your expenditure on heating per month over the duration of the experiment? 9 You were not the only one controlling your heating. A computer also made decisions about when to turn the heating on or off. 3. Results 3 Over time the number of times you change the temperature has been consistently low. Please describe how you decide when to change temperature or when not to. 3 Please describe how you have used the away schedules? 3 When the app notiﬁes you that it wasn't expecting you, how do you decide whether to accept or reject the suggestion or ignore the notiﬁcation altogether? 3 Please explain your usage of the voting - when do you submit a vote, when do you dismiss it and how do you decide which to do? Table 5 Table 5 Table 5 Detailing household-speciﬁc questions for ﬁeld study Interview 2. es you that it wasn't expecting you, how do you decide whether to accept or reject the suggestion or ignore the notiﬁcation y y pp y pp g u have the heating application on your phones, could you describe how you as a household make any changes - do you consult among before submitting anything to the app, is it individual, etc? the number of times you change the temperature has been consistently low. Please describe how you decide when to change re or when not to. ? lain your usage of the voting - when do you submit a vote, when do you dismiss it and how do you decide which to do? Table 6 Detailing questions for ﬁeld study Interview 3 (Debrief interview). Question number Question 1 For the last time, I would like for you to take a look at the diagram we have been working with and tell me whether you would like to add or change anything about how in your mind the system works? 2 Did the heating system behave the way you expected it to behave? 3 [Household-speciﬁc questions e please see Table 7 below for full detail] 4 What would you say are the most important differences between this type of a system and conventional heating controls? 5 Did you encounter any funny incidents or disagreements over the course of the experiment regarding the heating? 6 If you had a choice, would you prefer to keep this type of a heating system or would you like to revert to your previous system and why? 7 Could you please describe the experience of controlling the heating through your phone rather than a more conventional method? 8 Similarly to the pre-study questionnaire, would you be able to estimate your expenditure on heating per month over the duration of the experiment? 9 You were not the only one controlling your heating. A computer also made decisions about when to turn the heating on or off. What do you think, how did the heating system make these decisions? 10 [Researcher explained what how heating decision were made] How does it make you feel knowing that this was happening? 11 If you knew at the time that this was occurring, would you have done anything differently than you did now? Table 5 What do you think, how did the heating system make these decisions? 10 [Researcher explained what how heating decision were made] How does it make you feel knowing that this was happening? 11 If you knew at the time that this was occurring, would you have done anything differently than you did now? 12 Could you describe your overall experience in living with this type of a heating system? 13 How in control of the heating did you feel over the course of the experiment? 14 If this heating system was available on the market today, would you buy it for your home? 3.1.1. The ‘fashion user’ The ‘fashion user’, Carl, was observed in House 1 and can be characterised by his expectations of the heating system to deliver thermal comfort to him, matching his chosen garment choices: Prevailing temperatures were slightly higher than measured for fashion user, but frugal users had a very narrow range for neutral sensation (Fig. 8), highlighting not only the conﬂicting views re- ported by Diane, but also the manner in which they operated the system e as a novel way to control heating (telling it to turn on when they were cold and subsequently turning it off when they were hot). Such operation also caused users to attribute automatic heating periods to randomness or system errors and often leaving them surprised at the outcome: “I'm very much with the approach that I will get to a comfortable position clothing wise and then get the building to adjust around me.” [Carl] Personal thermal adaptations such as clothing level alterations or hot/cold drinks consumption were rarely utilised and subse- quently, Carl was the heaviest user of both the interface and the heating system. The user reported varying working-from-home behaviour and life patterns greatly around work demands creating an erratic presence proﬁles across rooms (Fig. 6). “And then a couple of times, one time at the start when we came in and it felt like we just went to the centre of the earth. And all of them had been on … and we were like “oh wow”.” [Paul] Long periods in late afternoons can be observed, where the user was recorded in different places. In addition, Carl sometimes had a partner stay over for long weekends, who was often in the house when Carl was in the ofﬁce. These factors caused the control al- gorithm to heat several rooms, which was perceived by the user as having ‘made mistakes’. One user often worked from home while the other left for the ofﬁce on weekdays (Fig. 9 top), causing the algorithm to adapt to various presence proﬁles. On weekends, the users preferred to spend more time at home, which also gave the algorithm different patterns for ‘start of day’ activities such as eating, washing and dressing. Due to this, the heating system often resorted to unex- pected presences, triggering push notiﬁcations to users that pro- voked interesting social nuances regarding personal location data protection as users became aware of each other's location. 3.1.2. The ‘frugal user’ House 2 were labelled ‘frugal users’ for their reported prioriti- sation of avoiding expenditure on heating above other consider- ations. This was reported collectively and retrospective, while during usage, conﬂicts existed between Diane preferring higher temperatures and Paul, who prioritised personal thermal adapta- tion to save cost. Interestingly, this led to thermal feedback from the application being used as justiﬁcation for turning heating on: 3.1. Three behaviour types We expected some difference between our participating households in their use of the automated system, reﬂecting existing knowledge from classical heating system usage. The results M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 294 arise when a sub-set of users live in their natural environment with a spatiotemporal heating system. Furthermore, despite having three devices equipped with the control application (both users had a smart phone and a shared tablet), only Diane ended up engaging with the interface, leading to a dialogue between users regarding thermal behaviour and preference. 3.1.1. The ‘fashion user’ 3.1.1. The ‘fashion user’ These are discussed in more detail below. “The only reason why I have noticed this is because … I tend to work at night. And if I was doing a lot of heavy working, and then stopped, in the lounge it would turn on at like 2 in the morning even though I was not up still.” [Carl] Such noticeable alterations in personal habits made Carl aware of the system's intent to establish a schedule around his presence, making him forgiving towards the system at times, but also frus- trated when these changes occurred. 3.1.3. The ‘everything's ﬁne’ user Users in House 3 were characterised by their lack of necessity to engage with the heating system and control interface. Their ﬂat's building envelope and high heat gains from neighbouring ﬂats ensured their comfort expectations were naturally met and addi- tional heating was rarely required (Fig. 10), but they could poten- tially be re-classiﬁed to one of the other types if building characteristics were different (most likely to frugal type due to reported preference to personal thermal adaptation for cost saving). Manual system state alterations were primarily motivated by user's thermal sensations and wishes to match thermal conditions to clothing choices, which provoked the formulation of a heater state alteration decision prior to engaging with the application. User's responses to system-initiated contact (unexpected presence notiﬁcations) were addressed based on their alignment with ther- mal sensations and pre-existing alteration decisions. These in- teractions delivered suitable conditions in the living quarters (Fig. 7). As environment was matched to clothing choices, Carl experienced different thermal sensations at same temperatures, resulting in a varied thermal sensation distribution (Fig. 7) and causing the heating algorithm to continuously adapt to ensure user's comfort. E.g. the prevailing temperatures in the bathroom (orange line in Fig. 7) showed that 75% of experiment duration, Carl was most likely to experience a ‘cool’ or ‘slightly cool’ sensation. In contrast, while in the lounge (green line) he was most likely to feel a range of sensations between ‘slightly cool’ and ‘warm’. These users displayed a stark difference between weekday and weekend presence (Fig. 11), displaying highly active mornings and afternoons contrasted with absence during working hours on weekdays. On weekends (Fig. 11 bottom), users had different times of waking up, sometimes being out of the house, or spending weekends in. The algorithm had to adapt to these behaviours and subsequent differences in needs for thermal comfort. Long absence and little need for additional heating meant the control interface was primarily used individually, often leaving users unaware of each other's changes. Heating behaviour was rarely discussed, with some conversations occurring when personal thermal adaptations failed to deliver comfort. Initial excitement of novel technology and testing of all features was replaced by diminished interest in organic and system-initiated interaction. 3.1.4. Implications of emerged behaviours These results from a highly ecologically valid setting demon- strate that given virtually identical equipment, three households displayed vastly different system use strategies, thermal behaviour, and thermal preference; each adjusting the manner of use to their existing social, occupational, presence, and thermal adaptation habits. Furthermore, the emerged behaviours did by no means represent a full set of possible behaviours, highlighting that do- mestic heating behaviour is indeed complex and highly personal. “Occasionally I use it to prove a point. Especially when it was really cold and I would be like “Paul, it's really cold in here” and he'd be like “No, it's ﬁne, put a jumper on” and I would check the tem- perature and use it that way.” [Diane] M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 295 Fig. 6. fashion user average measured presence proﬁles for all weekdays (top) and weekends (bottom). M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 295 Fig. 6. fashion user average measured presence proﬁles for all weekdays (top) and weekends (bottom). Subsequently we explore the user experiences further across the three behaviour types, answering questions formulated around speciﬁc themes. automation. The indifference was attributed to the subtlety of the graph as an explanatory tool within the interface. Furthermore, the graph's forward planning based on predictions was not explicitly communicated anywhere within the application, which meant that users relied on other indicators to explain system's behaviour. This ﬁltered through to the occupants' use strategies of the system and users' perceptions of the heating system and their use of it. These were analysed through user-generated diagrams of how the system worked and interview data from all three interviews, which was used to extract their interaction strategy (Table 8). 3.2. Potential user experiences emerging from a spatiotemporal home heating smartphone control app 3.2.1. Explaining heating system's operation & use strategies 3.2.1. Explaining heating system s operation & use strategies The authors expected users to anticipate automation capabil- ities from what little explanation was provided and for this to come through in users' explanations of the system. Users of the ‘visible’ interface conﬁguration were expected to provide more accurate and extensive descriptions due to the forward-planning nature of the graph. However, our results contradicted this and showed little difference between ‘blind’ and ‘visible’ conditions in explaining the The explanations provided in Table 8 were reportedly based on the hardware that users could observe. In addition, the ‘frugal ’users said the unexpected presence notiﬁcations made them realise the system knew their location, and the ‘fashion’ user noticed learning behaviour upon changing their daily routine. Fig. 7. fashion user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 296 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 296 Fig. 7. fashion user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative te ity distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. Fig. 7. fashion user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. Fig. 7. fashion user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. Fig. 8. frugal user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. Fig. 8. frugal user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. ability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. Fig. 8. frugal user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative tem Because of the users’ low awareness of automation capabilities of the heating system, the heating system was used as a temporal solution. Meaning that users saw the control application as a novel way to tell the heaters to turn on or turn off. 3.2. Potential user experiences emerging from a spatiotemporal home heating smartphone control app Little interaction prevailed regarding planning ahead, especially within the context of a single day. All users except one noted not obtaining feedback of the system activities or thermal conditions from the application before any personal or system-related heating decisions were made and all decisions were reached based on their sensation. model of the system functioning that was often inaccurate or incomplete. The results above highlight that users who were pre- sented with the thermal feedback-feedforward graph (Fig. 4) did not explain system functioning through it, illustrating that pre- vailing environmental conditions and heater system functionality are not innately linked in the users’ perceptions. As the interface type seemed to have no effect on users' un- derstanding of the system's capabilities, the conditions will not be isolated in the rest of this article and all participants will be treated as a single group. This data showed that even when users are not explicitly aware of the capabilities of the automation, they can deduce its behaviour. However, lack of explanations regarding system functionality meant that initially, users relied heavily on guess-work made possible by opportunistic audible feedback from the Wi-Fi-plugs switching on, or through delayed thermal feedback from the environment. These elements allowed users to build a mental 3.2.2. Experience of control over the heating system through the control application / Applied Ergonomics 65 (2017) 286e308 298 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 298 Fig. 10. Everything's ﬁne user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. “All I got at the start was... you sitting on this sofa and asking me there “is the heater on?” and you'd put it on 3 minutes ago and it wasn't on... “Has it gone off?” was another one so...” [Paul] autonomously and deviating from their expectations, causing distrust in them towards the system: “…the few times when it came on when we weren't expecting it to... The ﬁrst thing was to go on the app, try to turn it down from there, vote that I didn't feel in control. I don't know why I did that, maybe I thought that would have some immediate effect…” [Diane] “On the downside, it gives you a feeling of less direct control. So when you are using the conventional you are cold, you just... [does a ﬂicking motion] whereas with this you are relying on a system that you haven't actually...[prompt from researcher] It feels less immediate. You are not in control of each immediately.” [John] “Yeah generally I felt in control. Every now and again there was the odd random increase. And every now and again I would be sitting there and be like “why have you turned the heating on”.” [Carl] These descriptions highlight how users used the interface, environment, as well as lights and sounds from heaters to establish an understanding of system state and how changes to it either involving them or not, caused them to experience loss of control when the system state didn't match their expectation. “For example I haven't ever put the heating on before I have come back. I don't know whether that is out of not being aware of it, not thinking about it or sort of hesitation that it might come on or might not come on. 3.2.2. Experience of control over the heating system through the control application It was expected based on automation literature that users experienced diminished sense of control due to increased auto- mation capability, which would be compensated by the interface's M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 297 Fig. 9. frugal user measured presence proﬁles for all weekdays (top) and weekends (bottom). M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286 308 297 Fig. 9. frugal user measured presence proﬁles for all weekdays (top) and weekends (bottom). Fig. 9. frugal user measured presence proﬁles for all weekdays (top) and weekends (bottom). explanations. However, the results showed this dynamic to be more complex. “…when it did what I wanted it to do, straight away I was like “Yeah very in control” and then again when it took a few minutes to do it I was like “Not in control at all, I have no control.” But over time that kind of steadied out and usually felt pretty in control” [Diane] UX of control was analysed using interview data and user- submitted control votes. Fig. 12 depicts an even distribution of given control votes, highlighting that users experienced various levels of control over the course of the experiment, with average rating across participants at 4.3 (4.4 fashion user, 4.5 frugal user, 2.3 everything's ﬁne user). “Because it was slow in the beginning I was getting angry at it. So at ﬁrst my scores were very low and I think somewhere along if you look they would randomly ﬂip to high. Because I realised I was giving it low scores, because I had been giving it low scores. And then I realised that most of the time it was alright.” [Carl] The users explained their control experience and voting reasons as diverse, ranging from habit to system functionality or responsiveness: “…because we were not really using it for turning on or turning off the heaters, so control over the heaters was like not really control because I am not doing anything.” [Mildred] Retrospectively, the users reported to experience a satisfactory level of control, with all houses making reference to speciﬁc in- stances during the deployment when the system acting Fig. 10. Everything's ﬁne user thermal sensation probability distribution based on user-given votes, with positive and negative accumulative temperature distributions ﬁtted for all rooms. M. Kruusimagi et al. 3.2.2. Experience of control over the heating system through the control application Or just paranoid that it would turn the heater on when you are not there and it would start a ﬁre … I would only put it on in the bedroom once I actually go to that room.” [Paul] Users' thermal sensation was neither a reliable indicator for loss of control, despite feedback votes often being motivated by thermal discomfort e i.e. discomfort caused alteration of system state, which was followed by providing a vote. Fig. 14 highlights users’ thermal sensation during given votes and shows that discomforting sensations dominated both high and low control experiences. These quotes highlight situations in which users’ expectations did not match system functionality, causing experienced loss of control. However, over the course of the experiment participants did not consistently submit lower control votes during automati- cally initiated system-planned heating periods (Fig. 13), meaning loss of control was not solely a result of automation. In fact, during automatically planned heating users were more likely to have a slightly higher perceived level of control in comparison to non- automatic heating periods (Fig. 13) suggesting other factors in addition to system state inﬂuencing perception of control. It was also noted that certain interface features allowed users to increase the level of control they felt, notably setting away sched- ules when users were absent for longer periods of time: “I think it was just that security just to make sure it didn't come on when you were away, because you weren't there to react and turn it down. You could use the app to turn it down but I think it was just that double security.” [Paul] “I think it was just that security just to make sure it didn't come on when you were away, because you weren't there to react and turn it down. You could use the app to turn it down but I think it was just that double security.” [Paul] Interviews highlighted system responsiveness in combination with feedback playing an important role. The relationship of which was further complicated by the multiple channels of obtaining in- formation for users. The interface gave them feedback on their actions, but users additionally used environmental feedback, often prompting multiple interactions with the interface and high- lighting an added intricacy in the aspect of control for quasi- autonomous heating: “Telling the system that you were not there was something that gave us the feeling of control. 3.2.2. Experience of control over the heating system through the control application Like, turning it off.” [John] “Telling the system that you were not there was something that gave us the feeling of control. Like, turning it off.” [John] Furthermore, several users speculated that increased familiarity with the automation could have inspired more trust in them, allowing more autonomy for the without loss of experienced control: M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 299 Fig. 11. Everything's ﬁne user measured presence proﬁles for all weekdays (top) and weekends (bottom). Fig. 11. Everything's ﬁne user measured presence proﬁles for all weekdays (top) and weekends (bottom). Fig. 11. Everything's ﬁne user measured presence proﬁles for all weekdays (top) and weekends (bottom). “…maybe if you had it longer... like a year or two years, you would trust the system more and trust the actual heaters more, then you would be more inclined to then put it on like: “I'm going to be home in 10 minutes and it is in the middle of the winter you knew it was going to be cold”.” [Paul] experience of control, or the lack of, was the result of several concurrent factors. Experiences of control can be most enhanced by reducing mismatches between system state, thermal preference, and feedback on user actions; and the user's expectations of these factors, which supports existing knowledge (Bunt et al., 2012; Kulesza, 2012; Lim et al., 2009) highlighting the value of system rationale communication. In addition, we suggest explaining ther- mal behaviour, as well as responsiveness in delivering feedback throughout the observable environment in order to limit mis- matches in expectations from occurring. “If I had known exactly how the system works, like time intervals and things like that?… I don't know I would have trusted the sys- tem instead of, for instance having tried to turn off the system at some point, maybe would have just trusted that the system will know that it needs to be turned on. Maybe knowing how the system works would have given me more trust in it.” [John] 3.2.3. Social context of use and effects of introducing a smartphone heating interface to the social environment 3.2.3. Social context of use and effects of introducing a smartphone heating interface to the social environment These results show that increased autonomy alone does not promote a UX low in control. 3.2.2. Experience of control over the heating system through the control application “And then some level of variance depending on whether it could see me or not. Based on the motion” [Carl] Heating system as primary strategy to achieve thermal comfort Heating system as primary strategy to achieve thermal comfort Personal adaptation (clothing changes, hot/cold drinks) as primary adaptation, heating system as secondary Personal adaptation (clothing changes, hot/cold drinks) as primary adaptation, heating system as secondary for users, but poses questions regarding the appropriateness of notiﬁcations for other user changes and whether this should be conﬁgurable at the send or receive stage: better understanding of the social element of autonomous heating systems as a whole. better understanding of the social element of autonomous heating systems as a whole. In general, participants noted that there was a social element to the control application use and some common traits to regular heating system operation were reported. For example, in multi- occupant households, participants reported conversing about de- cisions to turn the heating on, which is assumed to be also the case in a ‘standard’ heating control. The ‘frugal’ household installed the application on 3 devices for 2 people, but only Diane ended up performing bulk of the interactions. When Paul wished for alter- ations, he usually asked Diane to perform them. This was reported to be due to ‘being faster’ or simpler if one person performed the actions. In the ‘everything's ﬁne’ household, users had a more in- dividual approach, but still noted making decisions jointly when the social situation facilitated it: “Except for one night that I turned the heating on.” [John] “By the app?” [Mildred] “Yeah!” [John] … “So it turned on?” [Mildred] “Yeah. I had to do it twice.” [John] Furthermore, in some cases, the interface became a critical part in discussions when disagreements occurred and was used to settle arguments or justify heating behaviour: “Occasionally I use it to prove a point. Explanations of automation after told a computer also made decisions 3.2.2. Experience of control over the heating system through the control application It has been demonstrated how the No speciﬁc elements of the social context were being observed in isolation and the experiment was used as a way to establish a M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 300 Table 8 Everything's ﬁne user am Diagram started with user thermal discomfort, proceeding to explanations how their interactions are translated into environmental change. Researcher's explanation of diagram More focused on the technical set-up of the system as interacting with it was less common in their home, making references to all the different functionality the phone application offered and communications links between components. Diagram started with user thermal discomfort, proceeding to explanations how their interactions are translated into environmental change, referencing communications between components. “The way it comes across to me is just trying to work out when I am generally in that room. … generally in the afternoon all the rooms turn on. So I guess this is generally when I come home. … the bedroom for example, the master bedroom, is off most of the time. … but I have noticed that it has become quite good at predicting vaguely when I am going to be in my bedroom, but during the day it seems to be just off, almost like a timer system that it's trying to work out for me.” [Carl] “The heaters, after a while they'll go. For example if we put the temperature in the bedroom to 18 degrees, it will come on for a few minutes and then the heater will click off. And then maybe a little while later it will click on again. I guess it kind of maintains the temperature that you've asked.” [Diane] None provided, heating system was described as a subservient only to their commands through the application “It probably either learned our behaviour, so maybe if we were in and if it was below 16 degrees it would maybe learned that we were maybe would turn the heating on in that instance.” [Diane] “…the only thing I can guess, is that from the temperature and the answers that we give to the app. I can't remember now, but it was like if you feel warm cold... So I am guessing that the system could try to ﬁt our ideal temperature, that's all I could say.” [John] Suggested system was replicating their input. 3.2.2. Experience of control over the heating system through the control application Especially when it was really cold and I would be like “Paul, it's really cold in here” and he'd be like “No, it's ﬁne, put a jumper on” and I would check the tem- perature and use it that way.” [Diane] “for instance if we are watching TV and we are like with the blankets and really-really cold, we talk to each other and say “okay we need to do something because we are not like this” [John] Overall, users were inclined to think a smartphone control application was a more social, yet personal experience for control- ling heating, that may be particularly useful in shared households: However, these users also noted that generally they were very individual in their actions as a result of not being together when making these decisions. This even lead to unawareness of the other's alterations, which could mean diminished understanding “I think it's more of a collaborative thing than normally if you turn the heater on, it would be one person walking to the heater and turn the heater on, but with this if you have different people M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 301 Fig. 12. Distribution of control votes for all houses (top-left), fashion user (bottom-left), frugal user (top right), and everything's ﬁne user (bottom-right) from 1 e none to 7 e complete control, with cumulative distribution function in both directions. Fig. 12. Distribution of control votes for all houses (top-left), fashion user (bottom-left), frugal user (top right), and everything's ﬁne user (bottom-right) from 1 e none to 7 e complete control, with cumulative distribution function in both directions. houses (top-left), fashion user (bottom-left), frugal user (top right), and everything's ﬁne user (bottom-right) from 1 e none to 7 e ion function in both directions. Fig. 13. Distribution of control votes, broken down by system state at the time of vote & cumulative distribution functions in both directions for either system state. tes, broken down by system state at the time of vote & cumulative distribution functions in both directions for either system state. Fig. 13. Distribution of control votes, broken down by system state at the time of vote & cumulative distribution functions in both d accessing the same thing on their own devices. 3.2.2. Experience of control over the heating system through the control application And I was basically as a safety blanket going - “Is that room the same temperature than the other rooms, because if the temperature changed signiﬁcantly … between this room and the other rooms, something may be up. Because a window now has been opened and there is no reason for a window to be opened. And this was particularly true before I got my security system ﬁxed.” [Carl] 3.2.2. Experience of control over the heating system through the control application Or you know the thing where you can give a vote, although we never really did that because it was just me and Paul and we either wanted it on or we didn't. But say in a shared housing if you had like 5 people I can see it being used that way like “Okay, we will vote to have the heater on or not.” or like the workplace or something, I guess that's more … like a shared element.” [Diane] stay over for long weekends, which sometimes meant the user with the control application was not home, when the guest was. Removing control from a physical location in the home meant the user had to make a decision whether to involve the guest as a member of the household and give them access to the house data: accessing the same thing on their own devices. Or you know the thing where you can give a vote, although we never really did that because it was just me and Paul and we either wanted it on or we didn't. But say in a shared housing if you had like 5 people I can see it being used that way like “Okay, we will vote to have the heater on or not.” or like the workplace or something, I guess that's more … like a shared element.” [Diane] “Generally, because it was my other half, I just said to her, if it is too cold, just text me and I will turn it up. Just because it was easier than to get her to install the app. Because it is just like, short periods of time, it never seemed worth for her to get the app. Looking back now, it probably would have been worth [it]” [Carl] However, this shared element created an interesting dynamic for houseguests. The ‘fashion’ user occasionally had their partner M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 302 Fig. 14. Distribution of control votes, broken down by user's thermal sensation at the time of vote, size of node indicating probability sensation being felt and intensity of colour indicating the probability perceived control vote given. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 14. 3.2.2. Experience of control over the heating system through the control application Distribution of control votes, broken down by user's thermal sensation at the time of vote, size of node indicating probability sensation being felt and intensity of colour indicating the probability perceived control vote given. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) These results highlight important problems caused by the data that this technology innately holds, as well as the privacy concerns it raises. In contrast, remote control also provoked interesting beneﬁts as Carl reported utilising temperature readings as a home security surveillance method: These results show that the control interface is used in various social situations and subject to social and privacy dynamics. Mov- ing the control interface from a shared physical location to personal digital device means the user experience design needs to consider the implications of dividing and distributing control over a shared space in individual domains. Furthermore, the results highlight how the interface can inﬂuence both heating behaviour and the social interactions surrounding it. “… because of the way my house is laid out - the front-facing windows to my lounge are road-side and the temperature sensor for the lounge was semi-near a window. And so when I was away I would check the temperature, because before [the weather] got extremely hot, I was keeping all the doors shut so I was getting almost complete separation between rooms. And I was basically as a safety blanket going - “Is that room the same temperature than the other rooms, because if the temperature changed signiﬁcantly … between this room and the other rooms, something may be up. Because a window now has been opened and there is no reason for a window to be opened. And this was particularly true before I got my security system ﬁxed.” [Carl] “… because of the way my house is laid out - the front-facing windows to my lounge are road-side and the temperature sensor for the lounge was semi-near a window. And so when I was away I would check the temperature, because before [the weather] got extremely hot, I was keeping all the doors shut so I was getting almost complete separation between rooms. 3.2.4. Unforeseen interactions that emerged from the application use 16, which indicates the number of ‘view room’ that occurred within a 10 min time step and the volumes of these that translated into ‘change temperature’, and ‘submit vote’ events. From the graph, it is evident that two event ﬂows occurred: (1) there were many occasions when users viewed one room and altered one room e they acted to make their im- mediate surroundings comfortable. And (2) when users viewed several rooms and altered one or more rooms e users acting to establish an overview and potentially guide the system's overall behaviour. Few interactions led to a submitted vote, indicating that explicit feedback submission was not part of a natural interaction. “But I used it when I could remember to basically. So if I knew we were going away for more than say 3e4 days, I used the away feature then because I wanted to make sure the heating deﬁnitely didn't come on basically.” [Diane] “…last week I went to London and then I programmed it and then when I went to Spain I did it again. So at the beginning I wasn't using it that much and within the last week I used it 3 times which is more than usual.” [John] Analysis of the view room and change temperature events over time (Fig. 17) showed that the checking behaviour was dominant during the ﬁrst months of the experiment with a high number of view room events per change temperature event, followed by a decay to similar levels. This was consistent with the users’ expla- nations demonstrating initial learning period substituting with more goal-orientated, controlling interactions. Fig. 15 left highlights that users primarily interacted with the rooms and temperatures visible on the home screen, sometimes managing away schedules, and almost never providing a vote without being provoked for it. Fig. 15 right further illustrates this and depicts the events that were logged on these screens e a large majority of all events regarded clicking through rooms, which sometimes led to a change temperature event. Interestingly, the ‘create long away schedule’ event was second lowest by occurrence, yet all three households mentioned its importance in the in- terviews. 3.4. Were speciﬁc interactions with the system dependent on prevailing conditions? All users described discomfort as the catalyst for interaction, but the ‘fashion’ and ‘frugal’ users also referred to the checking behaviour as a key part of their interactions, while the ‘everything's ﬁne’ users experienced fatigue in this behaviour due to lack of discomfort: In order to understand reasons behind users’ interactions with the heating system, the prevailing conditions e both regarding the environment and system functionality were mapped against the most predominant interaction that altered system state e users changing room temperature. “I am in a given room and I ﬁnd the temperatures either too hot or too cold. Which then proceeds to me checking the app. To adjust the temperature in that given room. … So say I am sitting in the living room, I think it is too cold, I go into the app to turn the heating up in the living room and I will then instinctively go through all the other rooms in the house. Just to see what the heating scenarios in those rooms are. Just because I get very irritated if the heating is on in a room that I am not in. … And then it should kind of, wait for a small period of time to see if it adjusts or not.” [Carl] Firstly, data from different loggers (sensor data and user feed- back data logged directly in experiment database, app interactions logged via Google Analytics) was cleaned and thermal feedback votes matched to existing change temperature events in the same 10-min time step. Only data with both matching entries (174 pairs in total) were used. Interestingly, the temperature distribution (black lines) in Fig.18 highlight that there was around 70% probability that change tem- perature events took place while the prevailing temperature in the room was most likely to make the user feel sensations between “slightly cool” and “slightly warm”. “…beyond actually like activating the heating or deactivating depending on the temperature, I do ﬁnd it quite interesting just to monitor the temperature, just occasionally see what the tempera- ture is. And I keep meaning to use it for the diary function.” [Carl] However, there was no signiﬁcant change in the temperature between the overall and temperature change-speciﬁc tempera- tures, which meant that prevailing temperature was not solely a useful indicator of an impending temperature change event. “So I just choose the room I want to look at. 3.2.4. Unforeseen interactions that emerged from the application use Several unforeseen behaviours emerged, highlighting the un- predictable nature in which users may adapt their use of a ‘con- nected’ or ‘smart’ home. In the ‘frugal’ household, Paul often worked from home, meaning the heating system experienced variation in presence patterns and used push-notiﬁcations to solicit users' feedback. This provoked interesting social nuances regarding privacy: This highlights additional beneﬁts for users merely stemming from a technology intervention that they did not have available to them before and how this enriched their UX adding value above the system's function. “That's something quite funny because quite a lot of the time when I am at work and Paul is at home, I know when he gets up, because that notiﬁcation come on. Like Paul goes in the bathroom and it's like “Hey, should your heating be on?” and it is like half past ten in the morning and I know he has just moved.” [Diane] The results demonstrate potential problems and opportunities arising from increased sensory technology in people's homes, suggesting that successful designs must navigate personal privacy retention without compromising ability to explain system's func- tional logic. This even prompted conﬂicts between users because of the system disclosing presence data: “But like I know when Paul is like... you've said to me before that oh “I will leave uni[versity] at 4 o'clock” and then I will get a notiﬁ- cation from home at half 3 and I know that you've left work early...” [Diane] 3.3. Interactions with the smartphone control application The researchers were interested in gaining an insight into the M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 303 “…at ﬁrst I always looked at it because it was so funny to see the temperature but then at some point I stopped looking at it.” [Mildred] dominant interactions with a smartphone heater control interface that prevailed over long-term in-situ use. Three major use cases prevailed e a checking behaviour (users would go through the different rooms to monitor temperature and system state), a con- trol behaviour (users would use the application as a control device to change the temperature to eliminate discomfort), and pro- gramming behaviour (this prevailed most dominantly for long away schedules, motivated by a wish to ensure the heating stayed off during absence). These results point to a common use case of checking and alteration e better highlighted in Fig. 3.2.4. Unforeseen interactions that emerged from the application use This reinforces an interesting dynamic in UI design where usage frequency and importance and not synonymous and providing a meaningful user experience needs to observe a wider array of interactions that may not prevail in a snapshot UI test (households would not have required this feature if the system was deployed for 1e4 weeks). Therefore, it has been demonstrated that users have two main motivations for interacting with the interface e managing irregu- larities when absent from the house and maintaining immediate comfort. The latter compromises of a checking behaviour (domi- nant during novelty or unfamiliarity with the system) that can transit to a system state alteration behaviour depending on mismatches. 3.4. Were speciﬁc interactions with the system dependent on prevailing conditions? Number of times an event occurred in a 10-min time step, arranged in the designed dominant use case of viewing a room e changing the temperature e providing a thermal feedback vote thereafter Fig. 17. Total number of View Room and Change Temperature events weekly over the course of the experiment. interaction than “restoring” comfort and “correcting” automation, also highlighting occupants’ willingness to behave proactively alongside the system. 3.5. Dialogues with the system User interactions throughout the duration of the deployment revealed interesting dynamics in the types of responses users are willing to give to system-initiated dialogues, as well as the timing of those dialogues. Throughout the experiment, users were prompted continuously to submit thermal sensation feedback through a push notiﬁcation. Despite this, only two instances were recorded where the users viewed the vote screen without being directed there from a temperature alteration event. In total, over 400 votes were sub- mitted, meaning users were much likelier to perform this action when they initiated the interaction and required alteration on system state than if the system asked for feedback on its performance. Fig. 17. Total number of View Room and Change Temperature events weekly over the course of the experiment. 3.4. Were speciﬁc interactions with the system dependent on prevailing conditions? I normally just scroll through the rooms and see what it is like anyway. … So if we are in the bedroom, I select bedroom and just raise it by a few degrees normally and make sure that the message comes through that says “okay I will do that” or whatever it is. And then sometimes I would do the vote and that's it. And then generally then once the heaters get to a certain point, then they will be off anyway, and they heat up quite quickly. I think they are more efﬁcient than the ones we have now. Like it gets really warm and then I will go back into the app and just lower it by a few degrees and that's it really.” [Diane] These are interesting ﬁndings since logic would dictate that users are most likely to perform system state alterations when thermal output was near the extremes of their discomfort. When the thermal sensations during votes were isolated (Fig. 19), it emerged that the highest number occurred at “slightly warm” and “cool” sensations. These results tell an intriguing user experience story of proac- tivity. The data suggests that users acted not only to maintain comfort, but also in anticipation to pre-empt system ‘overshoot’ and curtail heating functionality as soon as they felt a warmer M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 304 Fig. 15. Illustrating the interactions for viewed screens (left) and logged events (right) from all participants. Fig. 15. Illustrating the interactions for viewed screens (left) and logged events (right) from all participants. Fig. 15. Illustrating the interactions for viewed screens (left) and logged events (right) from all participants. Fig. 16. Number of times an event occurred in a 10-min time step, arranged in the designed dominant use case of viewing a room e changing the temperature e providing a thermal feedback vote thereafter. Fig. 16. Number of times an event occurred in a 10-min time step, arranged in the designed dominant use case of viewing a room e changing the temperature e providing a thermal feedback vote thereafter. Fig. 16. Number of times an event occurred in a 10-min time step, arranged in the designed dominant use case of viewing a room e chan feedback vote thereafter. Fig. 16. sensation. “Probably I did at the beginning when I was trying everything but then I think you forget. Like you don't want to be thinking about it right.” [John] These results suggest suggested “maintaining” comfort and “managing” automation output to be better predictors of Fig. 18. Cumulative distribution functions for change temperature events plotted against thermal sensation probability distribution functions for all submitted votes (top) and votes given during temperature set-point changes (bottom). M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 305 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 305 Fig. 18. Cumulative distribution functions for change temperature events plotted against thermal sensation probability distribution functions for all submitted votes (top) and votes given during temperature set-point changes (bottom). Fig. 18. Cumulative distribution functions for change temperature events plotted against thermal sensation probability distribution func given during temperature set-point changes (bottom). “…your default thinking is just to ignore it you know like when you get a lot of notiﬁcations on your phone you just cross them off or whatever” [Diane] “…your default thinking is just to ignore it you know like when you get a lot of notiﬁcations on your phone you just cross them off or whatever” [Diane] system push-notiﬁcations. An average of 6.8 push notiﬁcations per household per day were triggered by the system, which included prompt notiﬁcation sent to solicit thermal feedback. Users opened just 1.8% of all sent notiﬁcations (3059 in total): system push-notiﬁcations. An average of 6.8 push notiﬁcations per household per day were triggered by the system, which included prompt notiﬁcation sent to solicit thermal feedback. Users opened just 1.8% of all sent notiﬁcations (3059 in total): “The only thing I get is “hey, should your heating be on?” Ah, it's the same. I ignore it. They are all... the ***** things. And I stop it. I mean when I see it, it is probably that I am never thinking about it, so you don't want it.” [John] “The only thing I get is “hey, should your heating be on?” Ah, it's the same. I ignore it. They are all... the ***** things. And I stop it. sensation. I mean when I see it, it is probably that I am never thinking about it, so you don't want it.” [John] Similarly, users were very unlikely to respond to system prompts to give feedback on whether to heat or not when it was not predicting them to be in a space. 84.3% of responses declined proposed strategy and only a total of 38 responses were received despite often there being more than one notiﬁcation per day. Furthermore, users experienced a high level of fatigue from the As noted above, users enjoyed using the away schedules as it Fig. 19. Distribution of “change temperature” interactions by thermal sensation and predicted presence. Fig. 19. Distribution of “change temperature” interactions by thermal sensation and predicted presence. M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 306 gave them an easy way to establish enhanced feeling of control, however, it was noted by some users that this was not a natural interaction for them: Personal data concerns were only mentioned by one user, who noted they didn't feel like they were being recorded or watched, but would feel uncomfortable if their energy company started “bombarding” them with savings because of this. Interestingly, this was one of the users most concerned with minimising the cost of heating, highlighting that attitudes and behaviours may not align. All households agreed they would buy this type of a system if it was on the market and particular conditions such as cost (both purchase and savings delivered) were met. In addition, living in rented ac- commodation was a barrier to several participants, as well as home type e several users noted that relatively small number of rooms would mitigate the system's potential. Interestingly, the ‘fashion’ user noted they would miss the system as it had become a part of their home: “It is the kind of thing where … I had been out the house like 3e4 hours, before I went: “ oh yeah, I should probably tell it that I am out.” And then I get like half the weekends away ... And I went: “Oh yeah, I should tell it that I am not there.” [Carl] This highlights interesting elements about the types of di- alogues the system should be proactive about and which not. Furthermore, system proactivity in interaction should be tied to user motivations. sensation. It has been highlighted how exercising override on heater state generates feelings of control in the user, suggesting the system shouldn't rely on user proactivity in highlighting ab- sences, but should rather inform the user when it makes absence- related changes to the environment e for example, when it turns heating on to pre-heat the house, when the user is not present. At that moment, the user is motivated to administer over-ride if they will be home later, as they would not want heating to turn on without them there. Similarly, notiﬁcations at moments of confu- sion for the system i.e. ‘should I be heating or not because the user is here and I didn't predict them to be’ should be limited, alongside with proactive action. Instead, the system should learn from user- initiated interaction, relying on the fact that the user will alter the system state if the proposed strategy is not suitable. “Just as a whole, it was nice having the system in. It was a nice little system to have, it is going to be weird not having it here. Because I realised the other day that I have lived here longer now with the system than without the system.” [Carl] 4. Discussion Our study involved participants living with a quasi-autonomous home heating system, the capabilities of which they were not aware of. Qualitative and quantitative data obtained gave an insight to UX of living with and controlling such a system. It has therefore been highlighted how the system should aim to limit proactivity (which needs to be aligned to user motivations) for interactions and aim to maximise learning from user-initiated interactions. Firstly, our methodological approach highlights the attainability of context-speciﬁc long-term research required to fully understand the manner in which human beings interact with home automation systems. Existing body of research commonly overlooks UX exploration in a highly ecologically valid setting over extended periods, preventing emergence of potential use strategies and in- teractions resulting from the rich use context. This was here demonstrated through the emergence of unexpected home- security and inter-occupant ‘spying’ behaviours, observed manner in which interactions changed over time, heating behaviour change from initial excitement by a new technology through learning stages to knowledgeable usage, and usage of speciﬁc UI functions (including scheduling long periods away from home), all of which would not have emerged during a short deployment and users' subsequent lack of thorough familiarity with system behaviour. Furthermore, the ‘spying’ behaviour further demonstrated the importance of data privacy. While a commonly acknowledged issue, we have shown how even keeping data within the household can cause social issues within the user group. This poses interesting questions regarding the extent to which one's personal life really is personal or whether certain personal privacy limitations such as a parental awareness of their child's presence in the house are acceptable. Furthermore, whether such instances are the users' problems, or whether it is the responsibility of the autonomous system interface to protect privacy at the potential cost of frag- menting the collective awareness and engagement with the heat- ing decisions and behaviours. 3.6. Overall experience of living with the heating system/control application and whether users would prefer it over their existing systems / Applied Ergonomics 65 (2017) 286e308 307 the system and subsequent need to ‘keep an eye on it’. Regardless of the origin of increased engagement, this initial monitoring behav- iour not only facilitated users' understanding of and experience of over-riding control over the system, but also educated them of their thermal preference, which subsequently affected the actions they performed to maintain their thermal comfort (item 3 in Fig. 20). environment with a spatiotemporal heating system. Our results highlighted the complexities of this context within which energy behaviour decisions are taken and the differences, as well as sim- ilarities, in factors affecting those decisions between different users. Furthermore, we have shown how these factors are not permanent, meaning that users primarily motivated cost, can at times act solely motivated by comfort and vice a versa. p ( g ) We have also provided insights into qualities of dialogues users have with the heating system. As interfaces transfer into our smartphones, technology makes it easy for automated systems to trigger communication with users through push notiﬁcations at times of uncertainty or when system state changes are broadcasted. We have highlighted the need for assessing the essence of these dialogues in order to limit noise and prevent disengagement of users. We propose system-initiated dialogues to be aligned closely with critically perceived utility of the communication and the user's motivations for engaging with it. In other words, users need to be prompted when otherwise unnatural interaction (such as telling your home you will be away) would result in user-desired goals such as energy saving, while aiming to minimise all com- munications. Notiﬁcation settings should be utilised to allow users to deﬁne the varying level of system-initiative in dialogues, as any system initiated dialogue can be a barrier to user engagement. Thirdly, we have presented several pieces of evidence for users making sense of the alterations in the environment that the heating system acts out. This type of behaviour is consistent with the ten- dency of novice users to construct mental models of the system to explain its functionality and guide their actions in operating the device. Alignment of the system's behaviour according to the constructed model to users' expectations emerged to be an indi- cation of the user's acceptance of the system and trust in this. Misalignment to user's thermal preferences inspired a lack of trust and doubt in the system's health. 3.6. Overall experience of living with the heating system/control application and whether users would prefer it over their existing systems All study participants reported an enjoyable experience using the deployed system as a result of different reasons. Carl, the ‘fashion’ user beneﬁtted from individual room control, which allowed him not to heat spare rooms while maintaining comfort- able temperatures in the rooms he occupied. He described a high lack of control with his existing central heating solution, which eventually pushed him to taking part of the study. Carl and ‘frugal’ user Diane noted that taking part in the experiment allowed them to think of heating more as a ‘system’ rather than individual heaters on the wall. Both of these households reported to be more engaged with their heating behaviour because of the system, as well as the UI. Several households highlighted the beneﬁt of remote access for both monitoring and control purposes. Despite loss of some direct control as discussed above, it was noted that using a smartphone as an interaction device was regarded completely acceptable as “you use your phone more and more for … everyday things like online banking and everything like” [Diane]. In fact it was noted that the medium facilitates ease of operation for more complex and out-of- the ordinary operations such as irregularities in behaviour: Secondly, we have described the emergence of three distinct user behaviour types that contrast signiﬁcantly and are motivated by various factors including thermal preference, heating system control strategies and perceived co-operation with the autonomous system. These user types were not generalizable to the whole population and were not intended to be so. Humans are funda- mentally stochastic in their nature and vary highly in their behaviour. Therefore, we do not attempt to classify behaviours, but to explore some typical and potential behaviours and interactions that may arise when a sub-set of users live in their natural “For example whenever we go away, my dad would be in the cupboard for ten minutes to make sure everything was 'just so'. Whereas with this system, once you know how to use it, it's very simple to say whenever you are away for a week and it adjusts it quite quickly because you can check on the temperature if you wanted to. So I think user friendliness … especially for people maybe who aren't very mobile or who don't know how the boiler works or how the heating system works…” [Paul] M. Kruusimagi et al. 5. Conclusions & future work Glate, 2015. DefenDoor: a Home Security System that Syncs with Your Phone [WWW Document]. http://www.glate.in/ (accessed 1.22.15). Gupta, M., Intille, S., Larson, K., 2009. Adding gps-control to traditional thermostats: an exploration of potential energy savings and design challenges. Pervasive Comput. 95e114. In this paper we have demonstrated the ability to achieve a ﬁne degree of spatiotemporal heating control in the domestic setting and the socio-thermo-technical complexity of the setting by deploying a quasi-autonomous heating system. 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Soc. 39, 230e253. Acknowledgments Peffer, T., Pritoni, M., Meier, A., Aragon, C., Perry, D., 2011. How people use ther- mostats in homes: a review. Build. Environ. 46, 2529e2541. Philips, 2015. Meet Hue [WWW Document]. http://www2.meethue.com/en-gb/ (accessed 1.22.15). The authors acknowledge Jacob Chapman and Daniel Ratzinger for their invaluable input in the realisation of this project. The study was approved by University of Nottingham Engineering ethics committee. Martin Kruusimagi is supported by the Horizon Doctoral Training Centre at the University of Nottingham (RCUK Grant No. EP/G037574/1). Project, Amigo, 2012. Amigo Project [WWW Document]. http://www.hitech- projects.com/euprojects/amigo/ (accessed 5.30.12). Ruyter, B. de, Pelgrim, E., 2007. Ambient assisted-living research in carelab. in- teractions 14, 30e33. http://dx.doi.org/10.1145/1273961.1273981. Scott, J., Bernheim Brush, A.J., Krumm, J., Meyers, B., Hazas, M., Hodges, S., Villar, N., 2011. PreHeat: controlling home heating using occupancy prediction. In: Pro- ceedings of the 13th International Conference on Ubiquitous Computing, Ubi- Comp ’11. ACM, New York, NY, USA, pp. 281e290. http://dx.doi.org/10.1145/ 2030112.2030151. References Sims, R.E.H., Schock, R.N., Adegbululgbe, A., Fenhann, J., Konstantinaviciute, I., Moomaw, W., Nimir, H.B., Schlamadinger, B., Torres-Martínez, J., Turner, C., 2007. Energy supply. Clim. Chang. 251e322. AIRE Group MIT, 2012. MIT Project AIRE [WWW Document]. http://aire.csail.mit. edu/ (accessed 5.30.12). Smartthings, 2015. SmartThings j Life like Never before [WWW Document]. http:// www.smartthings.com/ (accessed 1.22.15). ASHRAE, 1966. ASHRAE Standard 55.66: Thermal Comfort Conditions. Audenaert, A., Briffaerts, K., Engels, L., 2011. Practical versus theoretical domestic energy consumption for space heating. Energy Policy 39, 5219e5227. B i b id L 1983 I i f t ti A t ti 19 775 779 Stevenson, F., Leaman, A., 2010. Evaluating housing performance in relation to human behaviour: new challenges. Build. Res. Inf. 38, 437e441. http:// dx.doi.org/10.1080/09613218.2010.497282. Bainbridge, L., 1983. Ironies of automation. Automatica 19, 775e779. B A 1995 Th d h f d i I B h R M li V (Ed ) g Borgmann, A., 1995. The depth of design. In: Buchanan, R., Margolin, V. (Eds.), Discovering Design. University of Chicago Press, Chicago, USA, pp. 13e22. Stevenson, F., Rijal, H.B., 2010. Developing occupancy feedback from a prototype to improve housing production. Build. Res. Inf. 38, 549e563. http://dx.doi.org/ 10.1080/09613218.2010.496182. Brown, T., Wyatt, J., 2010. Design thinking for social innovation. Stanf. Soc. Innov. Rev. 8, 30e35. http://dx.doi.org/10.1063/1.430867. Suchman, L., 1986. Plans and Situated Actions. Cambridge Univ, New York. Tuohy, P.G., Murphy, G.B., 2012. Why advanced buildings Don't work? Windsor, UK. Bunt, A., Lount, M., Lauzon, C., 2012. Are explanations always important?: a study of deployed, low-cost intelligent interactive systems. In: Proceedings of the 2012 ACM International Conference on Intelligent User Interfaces, IUI ’12. ACM, New York, NY, USA, pp. 169e178. http://dx.doi.org/10.1145/2166966.2166996. Suchman, L., 1986. Plans and Situated Actions. Cambridge Univ, New York. Tuohy, P.G., Murphy, G.B., 2012. Why advanced buildings Don't work? Windsor, UK. In: Proceedings of 7 Th Windsor Conference: the Changing Context of Comfort in an Unpredictable World, pp. 12e15. Tuohy, P.G., Murphy, G.B., 2012. Why advanced buildings Don t work? Windsor, UK. In: Proceedings of 7 Th Windsor Conference: the Changing Context of Comfort in an Unpredictable World, pp. 12e15. University of Essex, 2012. Intelligent Environments Group [WWW Document]. http://ieg.essex.ac.uk/ (accessed 5.30.12). Department of Energy & Climate Change, 2014. Energy Consumption in the UK - Chapter 1: Overall Energy Consumption in the UK since 1970, 14D/208. University of Florida, 2012. Gator Tech Smart House [WWW Document]. http:// www.icta.uﬂ.edu/gt.htm (accessed 5.30.12). Ecobee, 2015. 3.6. Overall experience of living with the heating system/control application and whether users would prefer it over their existing systems Similarly, we have highlighted how applying a mobile interface can cause disarray through con- current system alterations by multiple users. This disarray is sub- ject to further complication by personal thermal preference, as well as the personal habits, economic and comfort priorities, and communication dynamics, which all affect thermal behaviours. Our users' display of unexpected behaviours regarding interpersonal dialogue highlighted how availability of information and engage- ment with the system can alter not only heating decisions, but the communication process leading to the decisions. All of the aforementioned in combination with our examples of the manner in which users utilised sounds, thermal, and visual cues from the various technological components to monitor and make sense of the system's behaviour highlights the need for a holistic design approach if a successful implementation is required. We can observe that our users displayed a situated action pattern of behaviour (Suchman, 1986) when making decisions regarding the system functionality and their thermal behaviour. While they were able to outline broad strategies and goals for their decisions (such as curtailing expenditure for frugal users), their decision-making in natural situations displayed the quality of reacting to prevailing conditions in order to fulﬁl a number of goals within various con- straints. We, therefore, suggest an entirely holistic approach focused on the interactions of users embedded within the domestic context (context affected by architectural factors, dynamics of the Fourthly, our results highlight several implications regarding the user experience of quasi-autonomous home heating systems, which are arranged according to our conceptual model (Fig. 20). As indicated under item 1 in Fig. 20, the user interface becomes part of social environment, inﬂuencing the social interactions and subsequent energy decisions. Furthermore, (item 3 in Fig. 20) transferring the control interface from a cumbersome interaction in a physical location in the home to a convenient interaction in the smartphone that the user has constant access to, promoted more frequent heating system monitoring behaviour. Arguably, this in- crease could instead be attributed to users' lack of familiarity with Fig. 20. Conceptual contributions & implications of ﬁeld study results. Fig. 20. Conceptual contributions & implications of ﬁeld study results. M. Kruusimagi et al. / Applied Ergonomics 65 (2017) 286e308 308 References Smart WiFi Thermostats by Ecobee j [WWW Document]. https:// www.ecobee.com/ (accessed 1.22.15). Fibaro, 2015. Fibaro UK j Home Intelligence [WWW Document]. http://www.
https://openalex.org/W4289819768	https://zenodo.org/records/6320043/files/158-160.pdf	English	null	First Dissociation Constant of o-Phthalic Acid from 283.15 to 323.15 K	Zenodo (CERN European Organization for Nuclear Research)	1,985	cc-by	2,845	Refereaces In this note an attempt has been made to test indirectly the accuracy of the pK1 values of oxalic acid obtained by them. Simultaneously it has been shown that modified Davies equation gives the values of activity coefficient fairly accurately at least upto p==O.l mol kg-1 • For this, the cell (C-2) was set up. I I 1. K. J. LAIDLJ&R and P. A. LANDSXR.O!CNRR, Pt-afts. .Fareld1111 Soc., 1956, 52, 1100. , , , 11. D. D. RoBJ&R'tS, J, 01-g. Chem., 1965, 30, 8516. , g , 8. D. D. BoBJCR'rS, J, 01-g. Chem., 1966, 31, 4087. 8. D. D. BoBJCR rS, J, 01 g. Chem., 1966, 31, 4087. 4. A. J. PARXBB., Chem. &tl., 1969, 69, 1. g 4. A. J. PARXBB., Chem. &tl., 1969, 69, 1. , , IS. P. S. BADBAKRISHNAMUR'ti and P. 0. PA'tao, Proc. lndtt~11 .Acad. Bet.., 1970, 69. 181. , , 6. P. B. BADHAXIliSHNAMUR'l'I and P. 0. PATao, 'l'Btra- kfJdroft,1974,26,51108. p H. I H.Ph, KCl, AgCl I Ag (Pd) m1 m. (C-2) p H. I H.Ph, KCl, AgCl I Ag (Pd) m1 m. (C-2) (C-2) 7. B. 0. Ju, L. SINGH and S. N. DA'9, J, India11 Chem. Soc., 19'14, 5:1, 65'1. 9 , 5 , 65 . B. L. SINGH, B. T. SINGH and B. 0. JsA, J. I11dian Oh4m. 8oo.,1980, 57, 1089. First Dissociation Constant of o-Phthalic Acid from 183.15 to 323.15 K A. K. SINGH and J, C. GHOSH• Department or Chemistry, Patna University, Patna-800 005 Manuscript r~~t:Bivetl16 .A.ugusl 1983, reWed S9 Ju11s 1984, IJCCB~tlld 16 .Febi'UM1J 1985 Thermodynamic parameters : The thermodynamic activation parameters, ~H•, ~s· and ~G• were calculated by usual methods. They have been listed in Table 3. It is to be noted that ~G• increases very slowly as the proportion of acetone is increased. This shows that the stability of the transition state is very little affected by the addition of organic cosolvent. The variation in ~S• and ~H• obeys the Barclay-Butler ruleu resulting a straight line plot with slope equal to 750. This slope, also known as Glunwald11 solvent stabilisation operator, predicts, the absence of the strong interaction when the value is near about 800. This suggests that only little interaction when acetone is taking place. RECENTLY we have shown that the use of modi- fied Davies equation gives as accurate values of pK1 and pK1 of o-phthalic acid as is obtained by the use of full Debye-HUckel equation1 • Modified Davies equation (1) and the independence of /1, RE RECENTLY we have shown that the use of modi- fied Davies equation gives as accurate values of q g pK1 and pK1 of o-phthalic acid as is obtained by the use of full Debye-HUckel equation1 • Modified Davies equation (1) and the independence of /1, AZ~ .V~ (1) log Y,=- l+ ../p +fJ,p (1) therein on the composition of ionic atmosphere, at least upto p==O.l mol kg- 1 was applied by Sinha et a/. 1 to determine pK1 of oxalic acid from 283.15 to 323.15 Kin steps of 5 K and for this, cell (C-1) was used. Effect of ionic strength : The small effect of ionic strength suggests that the reaction is not of ion-ion but ion-molecule type. s used. H 1 j H 10x, NaCI, AgCl / Ag (Pd) m1 m1 (C-1) H 1 j H 10x, NaCI, AgCl / Ag (Pd) m1 m1 (C-1) (C-1) , , f , , , I. M. BARCI,AY and J, A. V. Bu't:r,Ba, Trans. B'Maday Soc., 1988, 34, 14411. 14. , , , 111. J. E. LJ&PPI,JCR and E. GaONWALD, "Bates and Equili- bria or Organic Beao"ons", Wiley, New York, 1988. J. INDIAN CHEM. SOC., VOL. LXII, FEBRUARY 1985 J. INDIAN CHEM. SOC., VOL. LXII, FEBRUARY 1985 TABLJ& 8-Ts:aaMODYNAMIC ACTIVATION PAaAMl&'tJ&:as IN WATJ&a-Ac:aToN:a 4H, 4G" in k cal mol-• , AS in cal mol-• deg-• 16" 20" 26° so• TABLJ& 8-Ts:aaMODYNAMIC ACTIVATION PAaAMl&'tJ&:as IN WATJ&a-Ac:aToN:a 4H, 4G" in k cal mol-• , AS in cal mol-• deg-• Acetone% 16" 20" 26° so• 36° (vfv) AH* dG* 48" 4G" 48" AG" AS" 4G" 48" AG" 48* 10 22.11 20.6 '10 20,4 70 IICU 7.0 2M 7.0 20,4 7.0 20 211.0 2o.6 6.0 2o.6 5.0 20.6 4.9 205 5.0 20.5 5.1 30 216 2o.7 3.1 2o.7 8.1 llo.7 8.1 206 8.2 206 8.2 40 21.1 20.8 0.8 20.8 0.7 20.S 08 !10.8 0.8 IIO.S 0.8 110 20.9 2o.9 -0.!1 2o.9 -0.8 !10.9 -0.2 20.9 -02 2D.9 -0.1 60 !lo.G !11.0 -1.8 21.0 -18 111.0 -u 21.0 -1.8 !11.0 -u 70 !10.11 1!1.0 -8.0 !U.O -!1.9 111.1 -8.0 llll.l -8.1 111.1 -8.0 M. L. BJCNDBa, J, Am. OhfJm. Soc., 1958, 73, 5986. 18. percentage of acetone. If the decrease is attributed to the solvation change then one of the states (reactant or transition state) is more prone to solvation than the other. , f , , I. M. BARCI,AY and J, A. V. Bu't:r,Ba, Trans. B'Maday Soc., 1988, 34, 14411. 14. , , , 111. J. E. LJ&PPI,JCR and E. GaONWALD, "Bates and Equili- bria or Organic Beao"ons", Wiley, New York, 1988. Since the transition state is a large dipolar anion with two units of negative charge on it, its solvation will mcrease with increasing percentage of acetone compared to the initial state. Naturally E0 value will decrease. However, it is important to note that the rate is not increasing. This may be on account of the fact that the reaction is entropy dependent. First Dissociation Constant of o-Phthalic Acid from 183.15 to 323.15 K A. K. SINGH and J, C. GHOSH• Department or Chemistry, Patna University, Patna-800 005 Manuscript r~~t:Bivetl16 .A.ugusl 1983, reWed S9 Ju11s 1984, IJCCB~tlld 16 .Febi'UM1J 1985 M. L. BJCNDBa, J, Am. OhfJm. Soc., 1958, 73, 5986. 18. Results and Discussion and (6) gives values of (mHPh+2mp11) and mp11 and ffiHPh and (6) gives values of (mHPh+2mp11) and mp11 and ffiHPh The e.m.f. of cell (C-2) is given by equation (2). E=E0 -k log mu.mcJ-k log '>'u.Yc1 (2) It reduces to equation (3) on applying modified Davies equation. hence values of mHp11 and mp11, respectively. The values of mu and m8 p11 when fed in equation (5) gives a fresh value of p. The interaction is continued till we get constant and consistent values of 1£, IDH and mHPII correct to a micro-mol kg-1. Then mH,Ph is obtained from equation (7). I E-E0 2AJt. - og mu =-k-+log mc~------=+P.u l+.J,, where, k- 2·30~6RT, E0 =E0A11A1ChCI- (3) (3) (7) mH,P11=m1 -muph-mPh (7) Now, log K1 =log mH.mHPh+log YH.YHPh, which ffiH 1Pb YH.Ph and fJ =(PH +PC!). . (8) . K mH.ffiHPh reduces to equat10n on putting A(ll==-m --, H 1 Pb For the cell solution, since mK = mc~o mH=ffiHPh+2mph mH=ffiHPh+2mph (4) H 1 Pb assuming YH,Ph = 1 and introducing modified Davies equation. (4) 1 assuming YH,Ph = 1 and introducing modified Davies equation. and the ionic strength, p is and the ionic strength, p is (5) 2Av'- logKAC1l-~=log K1-/J1/A (8) l+v'p 3 1 P=2ma-flHPh+m. 3 1 P=2ma-flHPh+m. (5) 2Av'- logKAC1l-~=log K1-/J1/A l+v'p 3 1 P=2ma-flHPh+m. 3 1 P=2ma-flHPh+m. (5) 2Av'- logKAC1l-~=log K1-/J1/A (8) l+v'p (8) From second dissociation equilibrium, we have Values of th From second dissociation equilibrium, w From second dissociation equilibrium, we have logmp11 =logK1 -logma+ 4Av'~-fJsP. (6) mHPb 1+ Vp Values of the molalities of various ionic species, p ( 2A v'-) and log KAc1 > - 1 + v'~ are recorded in Table 2. From second dissociation equilibrium, we have logmp11 =logK1 -logma+ 4Av'~-fJsP. (6) mHPb 1+ Vp Values of the molalities of various ionic species, p ( 2A v'-) and log KAc1 > - 1 + v'~ are recorded in Table 2. where, P1 =(f1a+PPh-tJHPh)· The values at 303.15 K only have been given for brevity. From the e.m.f. values of Hamer, Pinching and Acree's cell1 values of {11 could be calculated and are given in Table 1. Plots of L.H.S. of equation (8) against pat all the temperatures studied were linear, whose intercept TABL.-2 ·remp.=S03.15 K m, X10' m1 Xl01 E m11 xl01 m 8 p11 x 101 mp11 Xl01 ma 1Pb Jl X 101 u.r logKA(l)- J•q &bS. V )( 101 1+ ,. Experimental , , , 9. L. Sums, B. T. SINGH and B. 0. JHA, ,f. Indiafl C'hem. Boo., 1981, 58, 966. KCl (G.R.) free from Br- and o-phthalic acid (G.R.) on analysis by standard methods were found to be respectively 99.99 and 99.95% pure. All weigh- ings were corrected for buoyency. The stoichio- , , , 10. B. V. Al'I'ANTKRXSBNAN and P.B. BADBAKiliSBNAMUil'ti, Indtt~ft J. Ohem., 1965, 3, 886. , , , 11. A. K. Guru, Ph. D. thesis, Patna UniversUy, 1988. , , y, li. 0. X. INGOI,D, "BtruotQre and Mechanism In Orpnio Chemistry", Bell. London, 1968. lSI NOTES NOTES metric molalities of solutions prepared in double- distilled water were accurate upto a micro-mol kg-1. The cell and its filling up with experimental solution, the thermostat and the potentiometric assembly and the measurement of the e.m.f. of the cells were the same as reported earlier8 • Palladised platinum electrodes used as hydrogen electrodes were pre- pared according to Britton'. Silver/silver chloride electrodes were prepared by thermal electrolytic method 11 • Cells were set up in duplicate and the e.m.f. readings of the two cells agreed with one another within 0.06 mV. The recorded e.m.f. values in abs. volt are the mean of the two duplicate readings after making correction for the barometric pressure, vapour pressure and bubbler depth'. The molalities of the various ionic species were found as described below. metric molalities of solutions prepared in double- distilled water were accurate upto a micro-mol kg-1. The cell and its filling up with experimental solution, the thermostat and the potentiometric assembly and the measurement of the e.m.f. of the cells were the same as reported earlier8 • Palladised platinum electrodes used as hydrogen electrodes were pre- pared according to Britton'. Silver/silver chloride electrodes were prepared by thermal electrolytic method 11 • Cells were set up in duplicate and the e.m.f. readings of the two cells agreed with one another within 0.06 mV. The recorded e.m.f. values in abs. volt are the mean of the two duplicate readings after making correction for the barometric pressure, vapour pressure and bubbler depth'. The molalities of the various ionic species were found as described below. Temp. Experimental K 278.15 283.15 288.15 293.15 298.15 303.15 308.15 313.15 318.15 923.15 TABJ.J$1-VALUJ$9 Oi' p, (3, kg mol-• 0.67±0.02 0,64±0.05 0.6~±0.D'l 0.68±0.001 0.70±0.02 069±0.02 0.67±0.0~ 0.65+0.08 0.64±0.09 0.64±0.(2 The values of k, E0 and A are known from literature '- 0 • The values of fJ to be used in equa- tion (3) have been recorded previously10• Now assuming an arbitrary value of ,u in equation (3) we get a value of m8 , which when fed in equations (4) Kinetics of Oxygen Transfer from Triphenyl- arsine Oxide to Triphenylphosphine in Molten State Monitored by Infrared Spectroscopy S. S. SANDHU, T. S. LOBANA and S. S. DEOL Department of Chemistry, Guru Nana.k Dev University, Amtitsar-148 005 Manu1cnpt rBCetved 10 Ma1J198~. rB'-ts6d B9 Octob•r 198~. accepted 15 Ii'ebruarr 1985 at 1'=0 gave log K1 and the slope /J1 • The values of pK1 thus found and those recalculated from Hamer, Pinching and Acree's data are given in Table 3. at 1'=0 gave log K1 and the slope /J1 • The values of pK1 thus found and those recalculated from Hamer, Pinching and Acree's data are given in Table 3. Temp. K, K Recalculated From cell (ll-2) from H.-P.-A. 283.15 2.930 ± 0.0007 2.930±0.002 288.15 2.934±0.0003 !1.987±0 0007 299.15 2.942±0.0004 2.940±0.002 298.115 !1.956±0.0025 2.955±0.002 803.16 2.962±0.0009 2.965 ± 0.002 80815 2.967±0.0021 2.967±0001 918.15 2.979±0.0008 11975±0.0011 818.15 11.986±0.0004 11.987 ± 0.002 928.115 II. 996 ± 0.0005 II. 997 ± 0.002 TERTIARYPHOSPHINES/arsines as well as their chalcogenides are known to form vast number of coordination compounds with different metal ions1 • 8 • However, there is no attempt in studying atom transfer reactions between organophosphorus aod organoarsenic compounds based on difference in bond strengths of P=X and As=X (X=O, S, Se). Since these types of ligand~ (as such or in complexed form) find great use in different catalytic processes, it may be useful to know how a tertiaryphospbine/ arsine is undergoing change. In case there is oxidation of phosphine say, how will one monitor oxygen transfer to phosphorus kinetically ? For example, the reaction of manganese(II) chloride with triphenylphosphine yields Mn(Ph 8P0) 9Cl1 and not Mn(PPh 8) 11Cll, mechanism of the oxidation is not known. In order to understand such atom transfer reactions, the system consisting of triphenyl- phosphine and triphenyl arsine oxide has been arbitrarily selected. The reaction It will be seen from Table 3, that the two sets of pK1 values at all the temperatures closely agree with each other, and only at 313.15 K the difference goes upto 0.004. The linearity of the plots and the agreement in pK1 values shown in Table 3 indicate that modified Davies equation fairly correctly represents the acti- vity coefficient of ions at least upto 1'=0.1 mol kg- 1 • Close agreement among the pK1 values recorded in Table 3 may be taken to indicate that pK1 value of oxalic acid reported by Sinha et al. 1 are fairly accurate. Ph 8P+Ph8 As0-+Ph 8PO+Ph 8As Ph 8P+Ph8 As0-+Ph 8PO+Ph 8As Acknowledgement bas been studied under nitrogen atmosphere (PO group absorbs at 1180 while AsO group at 880cm-1), This work reports our initial results. The authors are thankful to Dr. B. Prasad for his interest. Thanks are also due to U.G.C., New Delhi for the grant of a Teacher Fellowship to one of the authors (A.K.S.). In order to know the concentration of PO group, the area method has been employed. The intensity height method is not normally useful in infrared spectroscopy because the bands are usually broad. For conducting the experiment, a known weight, say 75 mg of 1 : 1 mixture of Ph,P and Ph 8As0 was added to each vessel of a set of six or seven glass vessels immersed in oil-bath at temperatures of 215, 225 and 235" under nitrogen atmosphere. At regular intervals of 0,10, . 50 min, a sample tube was taken out and the reaction contents cooled immediately to stop the reaction. The ir spectrum of each sample was recorded in KBr pellets with KSCN as the internal!standard (sample amount : 15 mg; KBr 160 mg and KSCN 8 mg). The ir spectra were recorded with a Spectromom-2000 instrument. The areas of PO groups as a function of time, tempera- ture and mole ratio were corrected with respect to the unit area of CN group and converted into the molar concentration with the help of a calibration curve prepared from Ph9PO. Results and Discussion 0.1652 0.9361 0.115941 0.946 0.935 0.51 0.712 0.4'311 3.0306 0.8419 0.6850 0.112285 1.569 1.557 O.li6 1.857 0.8424 30329 0.6031 1.2095 0.49947 2.281 2.269 0.61 8.756 ].4382 301!94 0.9059 1.8168 0.48~09 2.964 2.950 0.67 6.10l !1.1138 30260 1.2057 !U180 0.47169 8.643 3.1i28 D.72 8.521 2.7790 3.0199 1.7110 3.4BU 0.45771& !1.429 4.418 0.78 12.689 8.8751 30189 2.5011! 5.0!l49 0.44277 5.688 5.571 0.87 19.493 5.60<10 30082 Concentrations and ionio strengths iu mol kg-•. 159 J, INDIAN CHBM. SOC,, VOL. LXII, FEBRUARY 1985 Kinetics of Oxygen Transfer from Triphenyl- arsine Oxide to Triphenylphosphine in Molten State Monitored by Infrared Spectroscopy S. S. SANDHU, T. S. LOBANA and S. S. DEOL Department of Chemistry, Guru Nana.k Dev University, Amtitsar-148 005 Manu1cnpt rBCetved 10 Ma1J198~. rB'-ts6d B9 Octob•r 198~. accepted 15 Ii'ebruarr 1985 Kinetics of Oxygen Transfer from Triphenyl- arsine Oxide to Triphenylphosphine in Molten State Monitored by Infrared Spectroscopy S. S. SANDHU, T. S. LOBANA and S. S. DEOL Department of Chemistry, Guru Nana.k Dev University, Amtitsar-148 005 Manu1cnpt rBCetved 10 Ma1J198~. rB'-ts6d B9 Octob•r 198~. accepted 15 Ii'ebruarr 1985 References 1. W. J, HAM.JlR, G. D. PINCHING and B. F. ACRJlK, J, Bu. Not. Bur. Stand.,l9411, 35,1189 ; W. ;r, HAMER a.nd B. F. ACRKB, J, BA Nat. Bur. Stand, 1944, 33, 87 ; A. K. BUIGB and J. C. Guosu, J, Indian Ch•m. Soc., 1988, 60,'10!1. !1. A. K. SINHA, ;r, C. Guosu and B. PRASAD, Indi11n J, Ch.m., Sect • .A,1981, 20, 89. S. A. K. Gs.oss, J. 0. Guosu and B. PRASAD, J, Indtata Ch.m. Soc., 1980, 57, 1194. 4. H.!. S. BB.IT'l'ON, "Hydrogen Ion", 8rd ed., Chapman and Hall, London, 194!1, Vol. I, p. 70. 15. D. ;r, G. IvJts and G. J. JANZ, "Reference Electrode", Academia Press, New York, 1961, p. 9ll. 6. G. J. HII,I,S and D. J, G. IvJts, J. Chm. Soc., 19151, 165, 918. '1. B. G. BA'l'ltS, "Electrometrio JIH Deierminatioo", Wiley, New York, 19114, p. 319. 8. H. B. HAB.NltD and B. W. EBLJlRS, J, .Am. Chem. Soc., 1989, 55, 11179. The rate of second order reaction was calculated using the relation 9. G. G. MANOV, B. G. B.a.'t:as, W. 1. HAMJlR and B. F. AcltBB, J, Am. Chm. Boo., 1948, 65, 1'1611. t-..!....(2-) at a-x (1) (1) 10. A. K. StNBA, J. 0. Gs.osu ana B. PRASAD, J, Indt41t Chtm. Boo., 19741 51, 586. 160
https://openalex.org/W4361285398	https://zenodo.org/records/8300348/files/openreseurope-3-16872.pdf	English	null	Children learn ergative case marking in Hindi using statistical preemption and clause-level semantics (intentionality): evidence from acceptability judgment and elicited production studies with children and adults	Open research Europe	2,023	cc-by	18,440	Ramya Maitreyee1, Gaurav Saxena 2, Bhuvana Narasimhan3, Dipti Misra Sharma4, Pruthwik Mishra4, Rukmini Bhaya Nair 5,6, Soumitra Samanta7, Ben Ambridge 8,9 Ramya Maitreyee1, Gaurav Saxena 2, Bhuvana Narasimhan3, Dipti Misra Sharma4, Pruthwik Mishra4, Rukmini Bhaya Nair 5,6, Soumitra Samanta7, Ben Ambridge 8,9 1School of Health and Social Care,, University of Essex, Colchester, Essex, CO4 3SQ, UK 2School of Psychological Science, University of Bristol, Bristol, BS8 1TU, UK y g y 3Department of Linguistics,, University of Colorado, Boulder, Boulder, Colorado, 80309, USA p g ,, y , , , , , 4Language Technologies Research Centre, International Institute of Information Technology-Hyderabad, Gachibowli, Hyderbabad, 500032, India 4Language Technologies Research Centre, International Institute of Information Technology-Hyderabad, Gachibowli, Hyderbabad, 500032, India 5School of Languages, Linguistics and Film, Queen Mary University of London, London, E1 4NS, UK 6Department of Humanities and Social Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India 7Department of Computer Science Ramakrishna Mission Vivekananda Educational and Research Institute Belur Math Howrah 5School of Languages, Linguistics and Film, Queen Mary University of London, London, E1 4NS, UK 6 f f 5School of Languages, Linguistics and Film, Queen Mary University of London, London, E1 4NS, UK 6Department of Humanities and Social Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India 7Department of Computer Science, Ramakrishna Mission Vivekananda Educational and Research Institute, Belur Math, Howrah, West Bengal, 711202, India g 8Division of Psychology, Communication and Human Neuroscience, University of Manchester, Manchester, Greater Manchester, M13 9PL, UK , 9ESRC International Centre for Language and Communicative Development (LuCiD), International, UK First published: 29 Mar 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 Latest published: 29 Mar 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 v1 Open Research Europe Open Research Europe Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 RESEARCH ARTICLE Children learn ergative case marking in Hindi using statistical preemption and clause-level semantics (intentionality): evidence from acceptability judgment and elicited production studies with children and adults [version 1; peer review: 1 approved, 2 approved with reservations] Ramya Maitreyee1, Gaurav Saxena 2, Bhuvana Narasimhan3, Dipti Misra Sharma4, Pruthwik Mishra4, Rukmini Bhaya Nair 5,6, Soumitra Samanta7, Ben Ambridge 8,9 Ramya Maitreyee1, Gaurav Saxena 2, Bhuvana Narasimhan3, Dipti Misra Sharma4, Pruthwik Mishra4, Rukmini Bhaya Nair 5,6, Soumitra Samanta7, Ben Ambridge 8,9 Abstract Abstract Page 1 of 31 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 greater the frequency with which a particular verb appears with versus without ne marking on the subject – relative to other verbs – the greater the extent to which participants (a) accepted and (b) produced ne over zero-marked subjects. Both children and adults also showed effects of clause-level semantics, showing greater acceptance of ne over zero-marked subjects for intentional than unintentional actions. Some evidence of semantic effects at the level of the verb was observed in the elicited production task for children and the judgment task for adults. Any reports and responses or comments on the article can be found at the end of the article. Conclusions: participants mainly learn ergative marking on an input- based verb-by-verb basis (i.e., via statistical preemption; verb-level semantics), but are also sensitive to clause-level semantic considerations (i.e., the intentionality of the action). These findings add to a growing body of work which suggests that children learn semi-regular, exception-filled systems using both statistics and semantics. Keywords child language acquisition, preemption, semantics, ergative marking, Hi di child language acquisition, preemption, semantics, ergative marking, Hindi This article is included in the European Research Council (ERC) gateway. This article is included in the Horizon 2020 gateway. This article is included in the Psychology gateway. This article is included in the Linguistic Diversity collection. This article is included in the European Research Council (ERC) gateway. This article is included in the Horizon 2020 gateway. This article is included in the Psychology gateway. This article is included in the Linguistic Diversity collection. This article is included in the European Research Council (ERC) gateway. This article is included in the Horizon 2020 gateway. Page 2 of 31 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Corresponding author: Ben Ambridge (Ben.Ambridge@Manchester.ac.uk) Author roles: Maitreyee R: Data Curation, Investigation, Software, Writing – Original Draft Preparation, Writing – Review & Editing; Saxena G: Investigation, Software; Narasimhan B: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Misra Sharma D: Methodology; Mishra P: Formal Analysis, Software; Bhaya Nair R: Methodology; Samanta S: Formal Analysis, Software; Ambridge B: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: This research was financially supported by the European Union’s Horizon 2020 research and innovation programme under the grant agreement No. [681296] (Cross Linguistic Acquisition of Sentence Structure [CLASS]). Ben Ambridge is Professor in the International Centre for Language and Communicative Development (LuCiD) at The University of Manchester. The support of the Economic and Social Research Council [ES/L008955/1] is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2023 Maitreyee R 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 work is properly cited. How to cite this article: Maitreyee R, Saxena G, Narasimhan B et al. Children learn ergative case marking in Hindi using statistical preemption and clause-level semantics (intentionality): evidence from acceptability judgment and elicited production studies with children and adults [version 1; peer review: 1 approved, 2 approved with reservations] Open Research Europe 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 First published: 29 Mar 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 1 Also, the verb agrees with the highest null-marked argument in the clause and if all arguments receive overt case, the verb exhibits masculine singular default agreement Mohanan (1994) in order to maintain uniformity in verb agreement, the present study used masculine singular nouns for the subject and masculine nouns for the object arguments of transitive verbs. Introduction Ram=Erg glass=Acc break-Msc.Sg.Prf As anyone who has attempted to learn a new language as an adult will know, languages are full of semi-regular patterns, i.e., rules with exceptions. For example, most English nouns form a plural with -s (e.g., cats, dogs etc.), which makes it difficult for children and second-language learners alike to learn exceptions such as feet and men. At the sentence level, English speakers can say both The boy moved and Some- body moved the boy, but not both The boy danced and Some- body danced the boy; and again, learning these exceptions proves difficult for first- and second-language learners alike (e.g., Bowerman, 1988; Robenalt & Goldberg, 2016). Ram broke the glass. Ram broke the glass. Plain language summary Ambridge et al., 2015; Ambridge et al., 2018; Barak et al., 2016; Bidgood et al., 2014; Blything et al., 2014; Boyd & Goldberg, 2011; Brooks & Zizak, 2002; Brooks et al., 1999; Goldberg, 2011; Gropen et al., 1991; Harmon & Kapatsinski, 2017; Hsu & Chater, 2010; Irani, 2009; Li & MacWhinney, 1996; Perek & Goldberg, 2017; Perfors et al., 2010; Robenalt & Goldberg, 2015; Robenalt & Goldberg, 2016; Stefanowitsch, 2008; Theakston, 2004; Twomey et al., 2014; Twomey et al., 2016; Wonnacott et al., 2008), including two book-length treatments (Goldberg, 2019; Pinker, 1989). Native language speakers of Hindi often produce sentences such as “Ram ne cup todaa (Ram broke the cup)”. However, not all subjects (i.e., Ram) of verbs in sentences as such receive ne marking (e.g., Ram kitab laayaa (Ram brought the book). How, then, do children learn when ne marking is required, when it is optional, and when it is ungrammatical? The present study investigated the use of ne marking using two tasks: a) asking children (aged 4–5, 5–6 and 9–10 years) and adults to indicate the extent to which sentence they heard was accept- able or unacceptable (“Ram kitab laayaa”, “Ram-ne kitab laayaa”); b) asking children (aged 4–5, 5–6 years) to pro- duce sentences with verbs where the subject may or may not be marked with a ne marker. Both children and adults accepted or produced the ne marker with the subject of a verb if they frequently heard the subject of the verb associated with the ne marker in the input. Further, children and adults accepted the ne marker on subjects when the action was intentional versus unintentional. Thus, these findings suggest that chil- dren may learn using partial regularities in the language sys- tem based on the input they hear and the properties of the action being performed. This focus on English is unfortunate given that most – and quite possibly all – languages exhibit this phenomenon of gen- eralizations with exceptions somewhere in the system. Our goal in the present study is therefore to investigate this prob- lem in a domain i.e., ergative marking in Hindi. Corresponding author: Ben Ambridge (Ben.Ambridge@Manchester.ac.uk) Corresponding author: Ben Ambridge (Ben.Ambridge@Manchester.ac.uk) Author roles: Maitreyee R: Data Curation, Investigation, Software, Writing – Original Draft Preparation, Writing – Review & Editing; Saxena G: Investigation, Software; Narasimhan B: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Misra Sharma D: Methodology; Mishra P: Formal Analysis, Software; Bhaya Nair R: Methodology; Samanta S: Formal Analysis, Software; Ambridge B: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: This research was financially supported by the European Union’s Horizon 2020 research and innovation programme under the grant agreement No. [681296] (Cross Linguistic Acquisition of Sentence Structure [CLASS]). Ben Ambridge is Professor in the International Centre for Language and Communicative Development (LuCiD) at The University of Manchester. The support of the Economic and Social Research Council [ES/L008955/1] is gratefully acknowledged. [ ] g y g The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. How to cite this article: Maitreyee R, Saxena G, Narasimhan B et al. Children learn ergative case marking in Hindi using statistical preemption and clause-level semantics (intentionality): evidence from acceptability judgment and elicited production studies with children and adults [version 1; peer review: 1 approved, 2 approved with reservations] Open Research Europe 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 First published: 29 Mar 2023, 3:49 https://doi.org/10.12688/openreseurope.15611.1 Page 3 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Plain language summary The phenom- enon is that, for active (i.e., not passive) sentences in the past tense/perfective aspect (i.e., denoting a completed action, but see Singh, 1998), the subject of two-participant transitive sentences (1) – but not single-participant intransitive sentences (2) – is typically marked by the clitic marker ne 1 (1) Raam=ne gilaas=ko toR-aa The boy feared the demon. The boy feared the demon. Complicating the picture still further, in some cases ne mark- ing seems to be optional, in that adult native speakers accept both the version with ne (5) and without ne (6). Unsurprisingly, given its broad linguistic and non-linguistic implications, the question of how to acquire exception-filled generalizations has inspired a great deal of theoretical debate and empirical research. However, with just a handful of excep- tions (e.g., Ambridge et al., 2020; Ambridge et al., 2022) research on child language has mainly been restricted to English (e.g., Alishahi & Stevenson, 2008; Ambridge et al., 2008; Ambridge et al., 2009; Ambridge, 2013; Ambridge & Ambridge, 2020; Ambridge & Blything, 2016; Ambridge & Brandt, 2013; Ambridge et al., 2011; Ambridge et al., 2012a; Ambridge et al., 2012b; Ambridge et al., 2013; Ambridge et al., 2014; (2) Raam-0 hãs-aa Ram-Nom laugh-Msc.Sg.Prf Ram-Nom laugh-Msc.Sg.Prf Ram laughed. However, some sentences that meet all of the other crite- ria (i.e., active, past tense/perfective aspect, transitive) still do not show ne marking (3); indeed, most native-speaking adults would regard such marking as ungrammatical (4). The question of exactly how learners acquire these exception- filled generalizations has implications not just for language, but for human cognition more generally (e.g., Pinker, 1999). Are our cognitive categories and processes best thought of as formal rules with memorized exceptions, or as fuzzier, more probabilistic generalizations? This debate has some surpris- ingly broad implications; for example, how best to design self- driving cars (e.g., Marcus & Davis, 2019). Should such cars be hard-wired with formal rules to follow in particular situ- ations, or simply provided with huge amounts of training data and left to form their own probabilistic generalizations? (3) laRkaa-0 raakshas=se Dar-aa (3) laRkaa-0 raakshas=se Dar-aa boy-Nom demon=Inst fear-Msc.Sg.Prf boy-Nom demon=Inst fear-Msc.Sg.Prf The boy feared the demon. The boy feared the demon. (4) laRke=ne raakshas=se Dar-aa (4) laRke=ne raakshas=se Dar-aa boy=Erg demon=Inst fear-Msc.Sg.Prf boy=Erg demon=Inst fear-Msc.Sg.Prf Mohan=Dat book-Nom want/need Mohan=Dat book-Nom want/need ‘Mohan wants/needs a book. ‘Mohan wants/needs a book. (9) niinaa=mE apnii mausii=keliye baRii mamtaa-0 hae. Nina=Loc self aunt=for much affection-Nom be.3.Sg. Pres ‘Nina has a lot of affection for her aunt.’ (Mohanan, 1994:181) ‘Nina has a lot of affection for her aunt.’ (Mohanan, 1994:181) ‘Nina has a lot of affection for her aunt.’ (Mohanan, 1994:181) In terms of the retreat from overgeneralization, then, the verb- level semantics hypothesis holds that children set up a seman- tically-restricted generalization: that only verbs that are high on this cline of transitivity trigger ergative ne- marking (in otherwise suitable contexts). In the present study, we operationalize this notion of transitiv- ity in verb-level semantics in terms of the degree to which the patient is affected by the action denoted by the verb (Hopper & Thompson, 1980). We ask adult participants to rate sen- tences describing various actions for “the extent to which the [THING] gets affected or changed by the event in some way”. The prediction of a verb-level semantics account is that the higher the rated transitivity, the greater the extent to which ne versus zero-marked subjects of the verb, as depicted by the cor- responding action in the videos, will be judged as acceptable (Study 1) and produced (Study 2). The second possibility – again supported by previous stud- ies of English and a handful of other languages (e.g., Ambridge et al., 2020; Ambridge et al., 2022; Gropen et al., 1989; Gropen et al., 1991) – is that children might be guided by semantics. For example, at least part of the reason that English speakers prefer Somebody made the boy dance over Somebody danced the boy is that “dancing” is not really an action that is ame- nable to outside causation: someone can ask a boy to dance, but not force him, he retains some agency. In contrast, English speakers accept both Somebody made the water boil and Some- body boiled the water because this type of direct, outside cau- sation is possible (Ambridge et al., 2020; Ambridge et al., 2022; Pinker, 1989; Shibatani & Pardeshi, 2002). In the domain of Hindi ergative ne marking, effects of semantics might be observed at either or both of two levels: the verb (lexical) and the clause (clausal). (5) Raam=ne samaachaar bolaa Ram=erg news tell-Msc.Sg.Prf Page 4 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Ram told the news. subjects (e.g., break, squash, eat > fear, escape, find; Hopper & Thompson, 1980; Mohanan, 1994; Montaut, 2013). Intention- ality (or volitionality) at the clausal level can also contribute to higher transitivity (Hopper & Thompson, 1980): events can be presented as intentional versus unintentional by the use of adverbs that modify the action denoted by the verb (7). (6) Raam samaachaar bolaa Ram news tell-Msc.Sg.Prf Ram told the news. (6) Raam samaachaar bolaa (6) Raam samaachaar bolaa Ram told the news. (7) Mohan=ne rassii-0 khiinc-ii galtii se. (7) Mohan=ne rassii-0 khiinc-ii galtii se. Mohan=Erg rope-Nom pull-Fem.Sg.Prf mistake by. ‘Mohan pulled the rope by mistake’ Although previous naturalistic and experimental studies of Hindi children’s ergative marking have been conducted (e.g., Narasimhan, 2005; Pareek et al., 2016) both focussed on errors of omission, rather than the question of how children learn exceptions to general patterns of ergative marking; the key question from a learnability perspective. Mohan=Erg rope-Nom pull-Fem.Sg.Prf mistake by. Mohan=Erg rope-Nom pull-Fem.Sg.Prf mistake by. ‘Mohan pulled the rope by mistake’ On the other hand, verbs that are lower on the transitivity cline mark subject arguments with non-canonical case-marking (Montaut, 2013). For example, two-participant verbs that denote states (versus actions) typically have subject arguments that are low in agency (e.g., experiencers, recipients, posses- sors) and object arguments that are not highly affected. In such cases, subjects receive cases such as dative, locative, or genitive (Mohanan, 1994; Montaut, 2013). How, then, do children learn this partial regularity? The pre- vious studies discussed above (mainly focussing on English) have provided evidence for two complementary possibilities. Under the first – statistical preemption (e.g., Goldberg, 1995; Goldberg, 2006; Goldberg, 2011; Goldberg, 2019) – chil- dren are sensitive to the probabilistic competition between forms with the same meaning. For example, the overgeneral- ized forms foots and mans are outcompeted by repeatedly hear- ing feet and men, while the overgeneralized form [A] danced [B] (e.g., Somebody danced the boy) is outcompeted by repeatedly hearing [A] made [B] dance (e.g., Somebody made the boy dance). When applied to Hindi ergative marking, preemption holds that via repeatedly hearing (active, past tense/perfective aspect, transitive) sentences containing a particular verb but without ne marking (example 3), these “zero-marked” sub- jects come to outcompete ne marked subjects for that verb (example 4). Thus the predictions that we test in the present study are that the greater the frequency with which a particu- lar verb appears with versus without ne marking on the sub- ject – relative to other verbs – the greater the extent to which (a) -ne marked forms will be preferred over zero-marked forms in a judgment task (Study 1) and (b) -ne marked forms will be produced over zero-marked forms in an elicited production task (Study 2). (8) Mohan=ko kitaab-0 caahiye Participants For the grammaticality judgment task, 48 participants from each of three age groups, 5;6–6;6 (M: 5.90, SD: 0.30), 9;6–10;6 (M: 10.06, SD: 0.33)2 and adults (M: 22.01, SD: 2.60), took part in the study. A further 20 adult speakers completed the semantic ratings task (M: 29.81, SD: 9.15). These sample sizes were chosen based on similar research conducted previously (Ambridge et al., 2020) and because of time and financial con- straints (as specified on the relevant grant application). For recruitment, eight schools and two universities were approached in Jabalpur, Bhopal and Delhi in India. Five schools and both the universities agreed to take part in the main study. In total, including online data – see below – data was collected from 100 children aged 9;6–10;6 and 5;6–6;6 (9;6–10;6: 48 data points used, 2 excluded (incorrect lists was administered for one and one child withdrew; 5:6–6;6: 48 data points used, 2 excluded (due to collecting more than our preregistered total)) and 73 adults (Grammatical judgment task: 48 data points used, 4 excluded (4 data points were not saved due to tech- nical issues; Semantics ratings study: 20 data points used, 1 excluded (participant withdrew)). Sentences. For each verb, four sentence structures were gener- ated which were manipulated for the (a) presence or absence of the ergative ne marker on the subject (7a & 7b), and (b) indicating intentionality of the agent performing the action (7a and 7b show the more intentional situations, 7c & 7d the less intentional situations by use of an adverbial phrase galtii se). All sentences were in the form of [AGENT] [PATIENT] [VERB], where the agent was always The boy and the patient an inanimate3 masculine noun (to ensure uniform verb agreement marking: masculine, singular perfective). 7a. laRke=ne khel-0 jiit-aa 7a. laRke=ne khel-0 jiit-aa boy=Erg game-Nom win-Msc.Sg.Prf The boy won the game boy=Erg game-Nom win-Msc.Sg.Prf COVID-19 pandemic restrictions came into force (GOV.UK, 2022; UK Parliament, 2021) before face-to-face testing could be completed and therefore, some participants were recruited via the online recruitment portals Prolific and Amazon Mechanical Turk: For the judgment task, data for all 48 adults and 45/48 children aged 9;6–10;6 were collected in face-to- face sessions. Data from 3/48 children aged 9;6–10;6 and 48 /48 children aged 5;6–6;6 were collected in online sessions. All the data for the adult semantics ratings task (20/20 partici- pants) were collected in online sessions. 3 There was one exception, i.e. for the verb ‘fear from’, we used an animate noun (‘demon’) as the patient. Mohan=Dat book-Nom want/need At the lexical level, linguistic analyses of Hindi suggest that the higher a verb on a cline of seman- tic transitivity based on the type of action it denotes, the greater the likelihood that it triggers ergative ne marking on the However, it is far from clear that a verb-level semantics effect exists for Hindi. Indeed, although ergative case-marking is predominantly found with two-participant verbs, it can also optionally occur on the single argument of a small set of intran- sitive verbs (e.g., chiikh ‘scream’), apparently marking a voli- tional action (e.g., Butt & King, 1991; Butt & King, 2003; de Hoop & Narasimhan, 2005; Mohanan, 1994). This raises the possibility that semantic effects on Hindi ergative ne marking, Page 5 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 task (all face-to-face). These data are not included in the main analyses. task (all face-to-face). These data are not included in the main analyses. if they exist at all, may occur not at the verb level, but at the clausal level: whether the action denoted by a verb is pre- sented as intentional or unintentional. In terms of the retreat from overgeneralization, then, the clausal-level semantics hypothesis holds that children set up a semantically-restricted generalization: that only actions that are clearly intentional trigger ergative ne-marking (in otherwise suitable contexts). In terms of the present study then, the prediction is that pre- senting an action as intentional rather than unintentional will increase the extent to which ne marked forms are – relative to zero-marked forms –rated as acceptable. Note that since none of the theoretical proposals tested make any claims regarding effects of participant sex and/or gender, this information was not recorded. Children and adults with Hindi as their first language and no known history of speech and language impairments were eligible to take part in the study. All participants had Hindi as their first language and knew one or more additional lan- guage. No monetary incentives were provided to schools, though children received stickers and pencils as rewards when data were collected in face-to-face sessions. Parents/caregivers who assisted their children during online data collection received £5 (Indian Rupee equivalent) as compensation for their time and effort. For the grammaticality judgment and the seman- tic ratings tasks, adult participants received 200 INR and £7.50 GBP compensation respectively. 2 The age data for 2 children were missing because of technical faults, so the mean age was calculated from the data obtained for 46 children. Materials b Verbs. Forty action verbs were chosen from the Action/Process category of Concepticon (List et al., 2016), a database of con- cepts that are commonly lexicalized as words across lan- guages (e.g., run, jump, dance) and two published sources on Hindi: Piepers (2016) and Mohanan (1994). Eligible verbs had to be (a) transitive verbs that took an ergative or nomina- tive subject (b) familiar to young children, and (c) easily depict- able in animations. In total, according to the intuitions of two native speakers of Hindi, of the 40 verbs, 25 verbs occur with the ergative ne marker on the subject, 5 verbs occur with zero- marking and 10 verbs exhibited optional ne marking (versus zero-marking). Mohan=Dat book-Nom want/need Study 1: Grammatical acceptability judgments The sample size, methods and data-analysis plan were regis- tered on the Open Science Framework prior to data collection (Rosenthal, 1979). Procedure Trials were presented in a random order. Prior to the main task, all participants completed a training session during which they received feedback for seven sentences with varying degrees of acceptability (which were Hindi translations of the sentences used in a study with English-acquiring children in Ambridge et al., 2008). Grammaticality judgment task. The face-to-face grammatical- ity judgment task was administered in line with the procedure outlined in Ambridge et al. (2008: 105–107), using the free Open Source platform PsychoPy2 (Peirce et al., 2019). In brief, the participant played a game with a talking dog (who pro- duces the sentences via a loudspeaker) and were asked to assist the dog in “learning Hindi”. On each trial the participant and the talking dog watched a video together and the dog pro- vided a description of the action. The participant gave feedback to the dog on the acceptability of the sentence produced (grammatical/ungrammatical) by selecting either a red coun- ter (to indicate ungrammatical) or a green counter (to indicate grammatical), then placing this counter on a five-point smiley face scale ranging from sad (red) to happy (green) to indicate the degree of (un)grammaticality. Due to the COVID-19 pan- demic, part of the data (48 children aged 5;6–6;6 & 3 children aged 9;6–10;6) was collected online using Gorilla, a platform where experiments can be built for free and fees are paid for data collection. There is no free alternative. For the online version, parents/caregivers were asked to assist the child in under- standing the task, completing the practice trials, and inputting children’s answers. However, they were asked to refrain from prompting or assisting their children when completing the main trials. Semantic ratings task. As mentioned in the pre-registration, for the semantic ratings task, participants viewed all 80 anima- tions (the same animations as in the judgment task; both inten- tional and unintentional) along with the corresponding verb and the patient argument that could be used to describe the action and provided ratings for patient affectedness. However, the results that we obtained did not seem, based on our native intui- tions, to really capture the relevant notion of affectedness and hence a revised, simplified version of the task was used instead. The modified semantic ratings task (N=20, adults only) was also completely within-subjects. Participants were given 40 sentences in the passive form describing an action (e.g. Participants As part of a pilot study to refine the tasks, ten adults completed the grammaticality judgment task and two adults completed the semantics rating The boy won the game 7b. laRkaa-0 khel-0 jiit-aa The boy-Nom game-0 win-Msc.Sg.Prf The boy won the game 7c. laRke=ne galtii=se khel=0 jiit-aa 7c. laRke=ne galtii=se khel=0 jiit-aa The boy=Erg mistake=Inst game-Nom win-Msc.Sg.Prf g j The boy=Erg mistake=Inst game-Nom win-Msc.Sg.Prf The boy won the game by mistake Page 6 of 31 Page 6 of 31 Page 6 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 7d. laRkaa-0 galtii=se khel-0 jiit-aa The boy-Nom mistake=Inst game-Nom win-Msc.Sg.Prf The boy won the game by mistake 7d. laRkaa-0 galtii=se khel-0 jiit-aa The boy-Nom mistake=Inst game-Nom win-Msc.Sg.Prf The boy won the game by mistake zero marked verbs, and 10 optional verbs, according to the intuition of two native-speakers) was chosen from the larger set of 40. Since even 80 trials (20 verbs x 4 sentence types) would have been too many for young children, two coun- terbalance lists - each containing 15 verbs were created. The 10 verbs for which ne marking is somewhat “optional” (know, leap-over, lose, find, talk nonsense, sing, smell, speak, under- stand and win) were included in both lists because they are the verbs that are most likely to show sensitivity to our preemption and semantics predictors, as well as the inten- tionality manipulation. The 10 verbs which occur with ne or zero marking (according to native speaker intuitions) were considered to be less sensitive to the experimental manipula- tions, so were split across the two lists. i.e., List 1 comprised 2 obligatory verbs and 3 zero verbs (as well as the 10 optional verbs); list 2 comprised 3 obligatory verbs and 2 zero verbs (as well as the 10 optional verbs). Thus, each child provided rat- ings for all four sentence structures for 15 verbs (60 trials in total). For the full verb lists, see Underlying data (Ambridge et al., 2023a). Children completed the trials over two sessions which took place either on the same day after a break or on two separate days. The boy won the game by mistake Animations. The animations were created using Moho Debut 12. This is proprietary software, and no free alternative is available. However, a free trial version is available, and the animations themselves are encoded as mp4 files, which can be viewed using a wide range of free software packages. For each verb, two different animations were created, one depict- ing the intentional and the other the unintentional event (all performed by the same boy character). For the animations, see Underlying data (Ambridge et al., 2023a). For the grammaticality judgment task (Study 1), the anima- tions were accompanied by the relevant sentence structure as described above. The sentences were recorded by a male Hindi speaker using the freeware package Audacity 2.1.2 (Audacity Team, 2016). Seven different animations were used in the practice trials of the grammaticality judgment task (Ambridge et al., 2023a). 4 In fact, the model syntax in the preregistration contained a typo such that the Verb-semanticsIntentionality interaction was not included, although it is clear from the main text of the pre-registration that it was intended to be. Thus the model did include this analysis. Procedure “The potato was cooked”) and rated the extent to which the object was affected or changed in the action, according to the following instructions (translated into Hindi). They did not view animations along with the sentences. Thanks for taking part in this experiment. In total, you will see 40 sentences. In total, you will see 40 sentences. For adults (N=48), the design was completely within subjects. For each of 40 verbs, adults rated the grammatical acceptabil- ity of four sentences (and the accompanying videos), crossing ergative subject marking (with ne/without ne) and intentionality (intentional/unintentional), for a total of 160 trials in one ses- sion which took approximately 30 minutes to complete (see examples 7a–d). Each sentence will describe an action that has been carried out. Please rate the extent to which the [THING] gets affected or changed by the event in some way. Participants were asked to provide their ratings using a visual analogue scale which ranged from Not at all-----------------------------------------------------------Very much so As children would have found it difficult to complete 160 trials, a reduced set of 20 verbs (5 obligatory ne verbs, 5 obligatory Page 7 of 31 Page 7 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Prior to the main trials, participants completed three practice trials. The materials for the main and practice trials can be accessed on the OSF project link in the folder titled ‘Ergative_ SemanticsTaskMaterials’. were run using the lme4 package (Bates et al., 2015); p values were obtained using lmerTest (Kuznetsova et al., 2017). Sta- tistical analyses were conducted for each age group separately (5;6–6;6; 9;6–10;6; Adults). We did not compare directly across groups since the tasks are not strictly comparable for the adults and children who completed different verb sets. were run using the lme4 package (Bates et al., 2015); p values were obtained using lmerTest (Kuznetsova et al., 2017). Sta- tistical analyses were conducted for each age group separately (5;6–6;6; 9;6–10;6; Adults). We did not compare directly across groups since the tasks are not strictly comparable for the adults and children who completed different verb sets. Predictors i Preemption. The prediction that follows from preemption is that the greater the frequency with which a particular verb appears with versus without ne marking on the subject – rela- tive to other verbs in the test set – the greater the extent to which ne marked forms will be preferred over zero-marked forms in the judgment task. As in previous studies (Ambridge et al., 2018; Ambridge et al., 2020) we operationalized this meas- ure using a scaled and centred chi-square statistic which meas- ures how often, in past tense/perfective aspect active sentences, a particular verb triggers ergative case marking as compared with all other verbs in the test set of 40, e.g., (example figures only): All analyses were conducted according to our pre-registered analysis plan4. Two aspects of this plan are particularly impor- tant to highlight. First, the main set of analysis uses difference scores: preference for the ne over zero marked form of the subject argument of each verb, within each intentionality pair, within each participant. These scores control for general (dis-)preferences that participants may show for particular verbs and/or videos. However, for the sake of completeness, we also report analyses conducted on the raw ne and zero-marked forms. Second, we report both (a) simultaneous models, in which the preemption, verb-level semantics (patient affected- ness) and clause-level semantics (intentionality) are all included and (b) single-predictor models, each of which includes only preemption OR verb-level semantics (along with intentional- ity). This is necessary because we expected a high degree of collinearity between these predictors: Indeed, a by-verb cor- relation analysis revealed moderate correlations between the verb-semantics and preemption predictors: r=.40 for the child set, and r=.26 for the larger adult set. Subject with ne Subject without ne Target verb All other verbs 2 10,000 10 1,500 The frequency counts were obtained from the Hindi monolin- gual corpus, Indic NLP Suite: Monolingual Corpora (Kakwani et al., 2020) which consists of 1 million sentences. The corpus was parsed using the Hindi parser (Bhat et al., 2017) and the counts for subjects marked with the ne ergative and zero mark- ers were extracted. The chi-square method for calculating the preemption predictor was exactly the same as in Ambridge et al. (2018). Results Table 1–Table 9 show, for each age-group, the simultane- ous and single-predictor models for (Table 1–Table 3) Dif- ference scores, (Table 4–Table 6) raw ne marked forms and (Table 7–Table 9) raw zero-marked forms. The ratings are plotted in Figure 1–Figure 4. Semantics. Verb and clause-level semantics were measured using patient affectedness ratings and the intentionality manipu- lation respectively. To create the patient affectedness predic- tor for each verb, we took the mean rating (on the 100-point visual-analogue scale) on the semantics ratings task described above across all 20 participants, then scaled and centred the mean ratings. The binary intentionality manipulation was instan- tiated by having participants rate intentional and unintentional versions of the sentences (see examples 7a–d) on the judg- ment task. We did not investigate the effect of intentionality on the production of ne marking (the subsequent Study 2) since children might not be able to infer the intentionality of an action from the animations alone (recall that in the judg- ment task, the relevant sentences included the term “uninten- tionally”, see examples 7a–d). It is important to also emphasize that the intentional and unintentional versions of each sentence were paired with different videos. For example, the intentional version of The boy soaked the cloth was illustrated by the boy dipping the cloth into water, while the unintentional version was illustrated by a girl knocking the boy, forcing him to unintentionally drop the cloth into water. Children aged 5;6–6;6i g Focussing first on the most informative difference-score analyses (Table 1), the main effect of Preemption (p < .01) was sig- nificant in both the simultaneous and single-predictor models. Intentionality (clause-level semantics) was a significant pre- dictor in the simultaneous -predictor model (p =0.03) though not the single-predictor models (p=0.06 in both cases; though this would not seem to be a meaningful difference, since the estimate is the same, and the p values straddle the 0.05 bound- ary). This significant effect of Preemption was also observed for raw ne marked sentences (p < .05), again in both the simul- taneous and single-predictor models, but not for zero-marked sentences. Main effects of Intentionality (clause-level seman- tics) were also observed for raw ratings of ne (as expected) and zero-marked sentences. This latter finding is unexpected, but may reflect a general preference for intentional actions and the unintentional actions being less typical. In summary, the 5–6 year-olds showed clear evidence of Preemption and Intentionality. Statistical analyses The data were analysed using R version 3.6.3 (R Studio Version 1.2.5042) (R Core Team, 2020). Mixed effects models Statistical analyses The data were analysed using R version 3.6.3 (R Studio Version 1.2.5042) (R Core Team, 2020). Mixed effects models Page 8 of 31 Page 8 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Table 1. Difference Scores: Simultaneous and Single-predictor Models for 5–6 year olds. Est SE df t p Simultaneousa Intercept 0.19 0.12 25.07 1.51 0.14 Preemption 0.43 0.13 22.02 3.30 0.00 Semantics -0.08 0.12 22.07 -0.68 0.50 Intentionality 0.17 0.08 1369.10 2.13 0.03 SemanticsIntentionality 0.06 0.09 1369.10 0.73 0.46 IntentionalityPreemption -0.01 0.09 1369.10 -0.08 0.94 Single-predictor (Preemption) Intercept 0.18 0.12 21.67 1.52 0.14 Preemption 0.39 0.11 20.00 3.42 0.00 Intentionality 0.17 0.08 17.33 2.05 0.06 PreemptionIntentionality 0.02 0.08 18.94 0.28 0.79 Single-predictor (Semantics) Intercept 0.18 0.15 19.77 1.16 0.26 Semantics 0.11 0.14 22.38 0.76 0.46 Intentionality 0.17 0.08 17.80 2.04 0.06 SemanticsIntentionality 0.07 0.09 26.67 0.71 0.48 aThe fully maximal simultaneous model failed to converge and so, the model was run excluding random effects of intentionality. Table 1. Difference Scores: Simultaneous and Single-predictor Models for 5–6 year olds. Table 2. Difference Scores: Simultaneous and Single-predictor Models for 9–10 year olds. Children aged 5;6–6;6i Est SE df t p Simultaneous Intercept 0.24 0.09 30.95 2.79 0.01 Preemption 0.13 0.08 21.24 1.59 0.13 Semantics 0.07 0.08 25.16 0.85 0.40 Intentionality -0.07 0.10 25.06 -0.75 0.46 SemanticsIntentionality -0.07 0.10 28.62 -0.67 0.51 IntentionalityPreemption 0.20 0.11 22.61 1.89 0.07 Single-predictora (Preemption) Intercept 0.24 0.10 41.06 2.31 0.03 Preemption 0.18 0.09 28.00 2.04 0.05 Intentionality -0.05 0.08 1371.13 -0.70 0.49 PreemptionIntentionality 0.13 0.08 1371.13 1.68 0.09 Table 2. Difference Scores: Simultaneous and Single-predictor Models for 9–10 year olds. Table 2. Difference Scores: Simultaneous and Single-predictor Models for 9–10 year olds. Page 9 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Table 3. Difference Scores: Simultaneous and Single-predictor Models for Adults. Est SE df t p Simultaneous Intercept 0.36 0.08 62.84 4.64 0.00 Preemption 0.24 0.06 53.32 4.09 0.00 Semantics 0.08 0.05 43.27 1.53 0.13 Intentionality 0.23 0.07 45.41 3.42 0.00 SemanticsIntentionality 0.01 0.06 36.77 0.15 0.88 IntentionalityPreemption 0.08 0.06 38.45 1.48 0.15 Single-predictor (Preemption) Intercept 0.36 0.08 63.86 4.60 0.00 Preemption 0.26 0.06 56.18 4.36 0.00 Intentionality 0.23 0.06 44.82 3.47 0.00 PreemptionIntentionality 0.09 0.05 39.29 1.58 0.12 Single-predictor (Semantics) Intercept 0.36 0.09 68.64 4.19 0.00 Semantics 0.14 0.07 48.15 2.17 0.03 Intentionality 0.23 0.07 44.05 3.43 0.00 SemanticsIntentionality 0.03 0.06 39.24 0.53 0.60 Table 4. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for 5-6 year olds. Est SE df t p Single-predictora (Semantics) Intercept 0.23 0.11 33.77 2.04 0.05 Semantics 0.15 0.09 25.29 1.58 0.13 Intentionality -0.05 0.08 1370.11 -0.69 0.49 SemanticsIntentionality -0.02 0.08 1370.11 -0.25 0.80 aThe fully maximal single-predictor models failed to converge and so, the model was run excluding random effects of intentionality. Est SE df t p Single-predictora (Semantics) Intercept 0.23 0.11 33.77 2.04 0.05 Semantics 0.15 0.09 25.29 1.58 0.13 Intentionality -0.05 0.08 1370.11 -0.69 0.49 SemanticsIntentionality -0.02 0.08 1370.11 -0.25 0.80 aThe fully maximal single-predictor models failed to converge and so, the model was run excluding random effects of intentionality. Table 3. Difference Scores: Simultaneous and Single-predictor Models for Adults. Children aged 5;6–6;6i Est SE df t p Simultaneous Intercept 0.36 0.08 62.84 4.64 0.00 Preemption 0.24 0.06 53.32 4.09 0.00 Semantics 0.08 0.05 43.27 1.53 0.13 Intentionality 0.23 0.07 45.41 3.42 0.00 SemanticsIntentionality 0.01 0.06 36.77 0.15 0.88 IntentionalityPreemption 0.08 0.06 38.45 1.48 0.15 Single-predictor (Preemption) Intercept 0.36 0.08 63.86 4.60 0.00 Preemption 0.26 0.06 56.18 4.36 0.00 Intentionality 0.23 0.06 44.82 3.47 0.00 PreemptionIntentionality 0.09 0.05 39.29 1.58 0.12 Single-predictor (Semantics) Intercept 0.36 0.09 68.64 4.19 0.00 Semantics 0.14 0.07 48.15 2.17 0.03 Intentionality 0.23 0.07 44.05 3.43 0.00 SemanticsIntentionality 0.03 0.06 39.24 0.53 0.60 Table 3. Difference Scores: Simultaneous and Single-predictor Models for Adults. Table 4. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for 5-6 year olds. Est SE df t p Simultaneous Intercept 3.50 0.14 33.99 24.81 0.00 Preemption 0.33 0.13 18.76 2.54 0.02 Semantics -0.05 0.13 20.38 -0.43 0.67 Table 4. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for 5-6 year olds. Est SE df t p Simultaneous Intercept 3.50 0.14 33.99 24.81 0.00 Preemption 0.33 0.13 18.76 2.54 0.02 Semantics -0.05 0.13 20.38 -0.43 0.67 Page 10 of 31 Page 10 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Est SE df t p Intentionality 0.66 0.11 30.57 5.96 0.00 SemanticsIntentionality 0.06 0.10 21.95 0.56 0.58 IntentionalityPreemption -0.06 0.10 17.86 -0.59 0.56 Single-predictor (Preemption) Intercept 3.50 0.14 36.54 25.30 0.00 Preemption 0.30 0.11 20.29 2.70 0.01 Intentionality 0.66 0.11 31.59 6.10 0.00 PreemptionIntentionality -0.03 0.09 20.10 -0.39 0.70 Single-predictor (Semantics) Intercept 3.50 0.15 31.46 22.69 0.00 Semantics 0.09 0.12 20.99 0.75 0.46 Intentionality 0.66 0.11 31.42 6.08 0.00 SemanticsIntentionality 0.03 0.09 25.93 0.34 0.74 Table 5. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for 9-10 year olds. Est SE df t p Simultaneous Intercept 3.69 0.12 45.15 31.58 0.00 Preemption 0.15 0.09 18.42 1.62 0.12 Semantics 0.03 0.09 19.10 0.36 0.73 Intentionality 0.57 0.09 34.91 6.35 0.00 SemanticsIntentionality -0.11 0.08 27.05 -1.36 0.19 IntentionalityPreemption 0.12 0.08 25.44 1.40 0.17 Single-predictor (Preemption) Intercept 3.69 0.11 48.20 32.29 0.00 Preemption 0.16 0.08 20.57 1.98 0.06 Intentionality 0.57 0.09 35.77 6.26 0.00 PreemptionIntentionality 0.08 0.08 27.57 1.00 0.33 Single-predictor (Semantics) Intercept 3.69 0.12 44.43 30.88 0.00 Semantics 0.10 0.08 20.92 1.19 0.25 Intentionality 0.57 0.09 31.87 6.23 0.00 SemanticsIntentionality -0.05 0.07 27.30 -0.69 0.50 Table 5. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for 9-10 year olds. Children aged 5;6–6;6i Page 11 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Table 6. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for Adults. Est SE df t p Simultaneous Intercept 3.84 0.10 75.11 37.59 0.00 Preemption 0.29 0.07 44.01 4.15 0.00 Semantics 0.06 0.07 43.68 0.88 0.38 Intentionality 0.65 0.10 70.12 6.39 0.00 SemanticsIntentionality 0.00 0.07 39.88 0.06 0.95 IntentionalityPreemption -0.06 0.07 41.16 -0.89 0.38 Single-predictor (Preemption) Intercept 3.84 0.10 75.56 37.62 0.00 Preemption 0.30 0.07 47.66 4.46 0.00 Intentionality 0.65 0.10 70.36 6.43 0.00 PreemptionIntentionality -0.06 0.07 42.56 -0.91 0.36 Single-predictor (Semantics) Intercept 3.84 0.11 78.66 34.57 0.00 Semantics 0.13 0.08 44.84 1.66 0.10 Intentionality 0.65 0.10 70.52 6.41 0.00 SemanticsIntentionality -0.01 0.07 41.60 -0.18 0.86 Table 7. Raw Zero Marked Forms: Simultaneous and Single-predictor Models for 5–6 year olds. Est SE df t p Simultaneous Intercept 3.31 0.11 39.21 29.68 0.00 Preemption -0.10 0.10 18.79 -1.03 0.31 Semantics 0.04 0.09 21.40 0.41 0.69 Intentionality 0.52 0.12 26.37 4.25 0.00 SemanticsIntentionality -0.02 0.12 20.43 -0.21 0.84 IntentionalityPreemption -0.07 0.12 17.43 -0.63 0.53 Single-predictor (Preemption)a Intercept 3.32 0.11 47.71 30.08 0.00 Preemption -0.08 0.09 26.12 -0.95 0.35 Intentionality 0.50 0.06 1327.96 8.05 0.00 PreemptionIntentionality -0.08 0.06 1327.96 -1.26 0.21 Table 6. Raw Ne Marked Forms: Simultaneous and Single-predictor Models for Adults. Table 6. Raw Ne Marked Forms: Simultaneous and Single predictor Models for Adults. Est SE df t p Simultaneous Intercept 3.84 0.10 75.11 37.59 0.00 Preemption 0.29 0.07 44.01 4.15 0.00 Semantics 0.06 0.07 43.68 0.88 0.38 Intentionality 0.65 0.10 70.12 6.39 0.00 SemanticsIntentionality 0.00 0.07 39.88 0.06 0.95 IntentionalityPreemption -0.06 0.07 41.16 -0.89 0.38 Single-predictor (Preemption) Intercept 3.84 0.10 75.56 37.62 0.00 Preemption 0.30 0.07 47.66 4.46 0.00 Intentionality 0.65 0.10 70.36 6.43 0.00 PreemptionIntentionality -0.06 0.07 42.56 -0.91 0.36 Single-predictor (Semantics) Intercept 3.84 0.11 78.66 34.57 0.00 Semantics 0.13 0.08 44.84 1.66 0.10 Intentionality 0.65 0.10 70.52 6.41 0.00 SemanticsIntentionality -0.01 0.07 41.60 -0.18 0.86 Table 7. Raw Zero Marked Forms: Simultaneous and Single-predictor Models for 5–6 year olds. Children aged 5;6–6;6i Est SE df t p Simultaneous Intercept 3.31 0.11 39.21 29.68 0.00 Preemption -0.10 0.10 18.79 -1.03 0.31 Semantics 0.04 0.09 21.40 0.41 0.69 Intentionality 0.52 0.12 26.37 4.25 0.00 SemanticsIntentionality -0.02 0.12 20.43 -0.21 0.84 IntentionalityPreemption -0.07 0.12 17.43 -0.63 0.53 Single-predictor (Preemption)a Intercept 3.32 0.11 47.71 30.08 0.00 Preemption -0.08 0.09 26.12 -0.95 0.35 Intentionality 0.50 0.06 1327.96 8.05 0.00 PreemptionIntentionality -0.08 0.06 1327.96 -1.26 0.21 Table 7. Raw Zero Marked Forms: Simultaneous and Single-predictor Models for 5–6 year olds. Page 12 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Est SE df t p Single-predictor (Semantics) Intercept 3.31 0.11 40.51 29.68 0.00 Semantics -0.01 0.08 23.85 -0.07 0.94 Intentionality 0.52 0.12 27.64 4.32 0.00 SemanticsIntentionality -0.06 0.10 23.00 -0.57 0.58 aThe fully maximal single-predictor model failed to converge and so, the model was run excluding random effects of intentionality. Est SE df t p Single-predictor (Semantics) Intercept 3.31 0.11 40.51 29.68 0.00 Semantics -0.01 0.08 23.85 -0.07 0.94 Intentionality 0.52 0.12 27.64 4.32 0.00 SemanticsIntentionality -0.06 0.10 23.00 -0.57 0.58 aThe fully maximal single-predictor model failed to converge and so, the model was run excluding random effects of intentionality. Table 8. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for 9–10 year olds. Est SE df t p Simultaneous Intercept 3.45 0.11 46.68 30.90 0.00 Preemption 0.01 0.08 20.69 0.09 0.93 Semantics -0.04 0.08 19.36 -0.54 0.60 Intentionality 0.64 0.09 26.92 7.16 0.00 SemanticsIntentionality -0.05 0.07 14.95 -0.64 0.53 IntentionalityPreemption -0.06 0.08 14.32 -0.84 0.42 Single-predictor (Preemption) Intercept 3.45 0.11 48.45 31.30 0.00 Preemption -0.02 0.07 24.08 -0.20 0.84 Intentionality 0.64 0.09 27.61 7.25 0.00 PreemptionIntentionality -0.08 0.07 17.14 -1.21 0.24 Single-predictor (Semantics) Intercept 3.45 0.11 47.96 31.31 0.00 Semantics -0.04 0.07 22.25 -0.59 0.56 Intentionality 0.64 0.09 26.95 7.30 0.00 SemanticsIntentionality -0.07 0.06 17.43 -1.16 0.26 Table 8. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for 9–10 year olds. Table 9. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for Adults. Est SE df t p Simultaneous Intercept 3.48 0.09 69.23 40.84 0.00 Preemption 0.04 0.06 49.10 0.77 0.45 Semantics -0.02 0.05 39.00 -0.43 0.67 Table 9. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for Adults. Table 9. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for Adults. Table 9. Raw Zero Marked Forms: Simultaneous and Single- predictor Models for Adults. Children aged 5;6–6;6i Page 13 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Est SE df t p Intentionality 0.42 0.09 61.03 4.85 0.00 SemanticsIntentionality -0.00 0.05 38.43 -0.08 0.94 IntentionalityPreemption -0.15 0.05 40.41 -2.70 0.01 Single-predictor (Preemption) Intercept 3.48 0.08 69.20 40.99 0.00 Preemption 0.04 0.06 51.69 0.69 0.49 Intentionality 0.42 0.09 60.77 4.88 0.00 PreemptionIntentionality -0.15 0.05 42.48 -2.80 0.01 Single-predictor (Semantics) Intercept 3.48 0.09 69.27 40.92 0.00 Semantics -0.01 0.05 40.02 -0.22 0.83 Intentionality 0.42 0.09 63.98 4.72 0.00 SemanticsIntentionality -0.04 0.06 39.52 -0.75 0.46 ure 1. Relationship between Preemption counts and Difference scores across clause-level-semantics conditions and age oups. Figure 1 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and Est SE df t p Intentionality 0.42 0.09 61.03 4.85 0.00 SemanticsIntentionality -0.00 0.05 38.43 -0.08 0.94 IntentionalityPreemption -0.15 0.05 40.41 -2.70 0.01 Single-predictor (Preemption) Intercept 3.48 0.08 69.20 40.99 0.00 Preemption 0.04 0.06 51.69 0.69 0.49 Intentionality 0.42 0.09 60.77 4.88 0.00 PreemptionIntentionality -0.15 0.05 42.48 -2.80 0.01 Single-predictor (Semantics) Intercept 3.48 0.09 69.27 40.92 0.00 Semantics -0.01 0.05 40.02 -0.22 0.83 Intentionality 0.42 0.09 63.98 4.72 0.00 SemanticsIntentionality -0.04 0.06 39.52 -0.75 0.46 Figure 1. Relationship between Preemption counts and Difference scores across clause-level-semantics conditions and age groups. Figure 1 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and difference scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). gure 1. Relationship between Preemption counts and Difference scores across clause-level-semantics conditions and ag roups. Figure 1 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) an Figure 1. Relationship between Preemption counts and Difference scores across clause-level-semantics conditions and age groups. Figure 1 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and difference scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Page 14 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Figure 2. Relationship between Preemption counts and Raw scores across clause-level-semantics conditions and age groups. Figure 2 shows the relationship between preemption (higher corpus relative verb frequency triggering ne form) counts and raw scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Figure 2. Relationship between Preemption counts and Raw scores across clause-level-semantics conditions and age groups. Children aged 9;6–10;6i Children aged 9;6–10;6i a narrowly significant effect of verb-level semantics (i.e., patient-affectedness) was observed (p =0.03), but only in the single-predictor model (though it would have been more com- fortably significant if we had pre-registered a one-tailed direc- tional statistical test alongside our directional prediction). The effects of Intentionality (clause-level semantics) and Preemp- tion – but not verb-level semantics – were also observed (again for both simultaneous and single-predictor models) for ne marked sentences (p values < .01). For zero-marked sen- tences, the only significant effects that were observed were unexpected and difficult-to-interpret: a main effect of Inten- tionality and an interaction of Intentionality x Preemption. In summary, the adults showed clear evidence of Preemption and Intentionality. g Although no significant main effects of predictors or interac- tions were observed for difference-scores (Table 2), the older children did show the predicted Intentionality (clause-level semantics) effect for the raw ne marked sentences, in both the single-predictor and simultaneous models. The Preemp- tion effect was nearing significance in the single-predictor model for raw ne marked sentences (p < .06). For zero-marked sentences, only a relatively-uninformative main effect of Inten- tionality was observed (again, presumably reflecting a gen- eral preference). In summary, the 5–6 year-olds showed clear evidence of Intentionality though – surprisingly – not preemption. Children aged 5;6–6;6i Figure 2 shows the relationship between preemption (higher corpus relative verb frequency triggering ne form) counts and raw scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Study 1 (grammatical acceptability judgments): summaryi Focussing again on the most-informative difference-score analyses (Table 3), main effects of Intentionality (p < .01) and Preemption (p < .01) were significant in both the simul- taneous and single-predictor models. Unlike for children, Although the fine-grained pattern of results is rather compli- cated, overall, a clear picture emerges: All age groups showed Page 15 of 31 Page 15 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Figure 3. Relationship between verb-level Semantics and Difference scores across clause-level-semantics conditions and age groups. Figure 3 shows the relationship between verb-level semantics (Patient-affectedness ratings) and difference scores across clause- level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Figure 3. Relationship between verb-level Semantics and Difference scores across clause-level-semantics conditions and age groups. Figure 3 shows the relationship between verb-level semantics (Patient-affectedness ratings) and difference scores across clause- level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Figure 3. Relationship between verb-level Semantics and Difference scores across clause-level-semantics conditions and age groups. Figure 3 shows the relationship between verb-level semantics (Patient-affectedness ratings) and difference scores across clause- level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). clear effects of Intentionality (clause-level semantics) and – apart from the middle age group – Preemption (though this likely reflects a chance finding, rather than genuine U-shaped learn- ing). That is, ergative ne marking is preferred when (a) relative to other verbs, the relevant verb is more likely to occur with ne than zero-marking in the input and (b) the event is intentional, rather than unintentional. Both of these factors therefore seem to play a key role in learning. which can be noisy with young children and – even more crucially – may not be directly reflective of the linguistic mechanisms that they use when producing speech (includ- ing errors of ergative ne marking). We therefore investigated the potential effects of preemption and verb-level semantics in a production study. As discussed earlier, this second study did not investigate intentionality since (a) we were not confi- dent that children could reliably determine intentionality from the animations and (b) we already have clear evidence of a role for intentionality – for all age-groups – from Study 1. Much more limited evidence was obtained for the effect of verb-level semantics (i.e., patient-affectedness, as deter- mined by the semantic ratings task). Study 1 (grammatical acceptability judgments): summaryi However, the fact that such an effect was observed for adults – though only for the single-predictor difference-score model – suggests that, while this factor may play little-to-no role in acquisition per se – it may have historically determined which verbs come to prefer ne versus zero subject marking. Study 2: Elicited production The sample size, methods and data-analysis plan were regis- tered prior to data collection. For the preregistration document, please see Extended Data (Ambridge et al., 2023b). Procedure The experiment ran on the Gorilla platform and children completed the task over Zoom along with a parent or guardian. The experimenter was present during the Zoom session in order to answer any questions that the parents might have when super- vising their child but did not conduct the experiment, which was entirely computerized. The task was set up as a game wherein the child watched a video depicting the action denoted by each verb and built a sentence describing the action using the clue word (the verb corresponding to the depicted action, inflected for past tense/perfective inflection) provided by a talk- ing dog. The video was presented first followed by the clue word, and the child’s responses audio-recorded. The parent or the guardian was asked to assist the child in audio-recording their responses on the computer, but not to provide any hints regarding the form of the responses themselves. The child Participants 48 children were recruited in each age group, 4;0–5;0 (M: 4.70, SD: 0.34) and 5;6–6;6 (M: 6.00, SD: 0.32) years, for the elic- ited production task. Recruitment methods (four schools were A potential concern surrounding these conclusions, though, is that they are based entirely on acceptability judgment data, Page 16 of 31 Page 16 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Figure 4. Relationship between verb-level Semantics and Raw scores across clause-level-semantics conditions and age groups. Figure 4 shows the relationship between verb-level semantics (Patient-affectedness ratings) and raw scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Figure 4. Relationship between verb-level Semantics and Raw scores across clause-level-semantics conditions and age groups. Figure 4 shows the relationship between verb-level semantics (Patient-affectedness ratings) and raw scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). Figure 4. Relationship between verb-level Semantics and Raw scores across clause-level-semantics conditions and age groups. Figure 4 shows the relationship between verb-level semantics (Patient-affectedness ratings) and raw scores across clause-level-semantics conditions (Intentional=Deliberate, Unintentional=Accidental) and age groups (5;6–6;6. 9;6–10;6, Adults). dry, and khiilaana (feed) were used for the practice trials, which depicted an action involving a boy or an inanimate object. approached and all agreed to take part in the study) and ethi- cal approval were same as Study 1. Parents who super- vised their children in completing the task received 500INR compensation for their time. We ran a pilot study with seven children aged 4–5 in order to ascertain the feasibility of running an elicited-production task with this age group. These data are not included in the analyses presented below. Materials For the elicited production task, the same list of 40 verbs and animations developed in Study 1 were used. Children watched the animations from the intentional condition only. The animations were accompanied by a “clue word”: the relevant inflected verb form in the simple past tense in both audio and written formats. The verb forms were recorded by a female near-native Hindi speaker on Audacity 2.1.2 (Audacity Team, 2016). Seven different animations, depicting the actions corre- sponding to Hindi verbs jalaana (burn), kaaTnaa (cut), nach- aana (dance), (sajaanaa) decorate, (giraanaa) drop, (sukhaana) Page 17 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 group separately (4;00–5;00; 5;6–6;6). As in Study 1, we ran both simultaneous and single-predictor models. group separately (4;00–5;00; 5;6–6;6). As in Study 1, we ran both simultaneous and single-predictor models. was instructed in Hindi on how to complete the task. For the detailed instructions for the task, translated into English, see Extended data (Ambridge et al., 2023b). Results For both age groups, main effects of preemption were significant in both the simultaneous and single-predictor models (see Table 10). As expected, the higher the (Z-score standard- ized) chi-square value operationalizing preemption, the greater the children’s production probability of using the ergative marker ne on the subject versus zero marking (see Figure 5). Signifi- cant effects of Verb Semantics (as measured using the patient affectedness measure) were observed in single-predictor models, but not in the simultaneous models, for both the age groups (see Table 10 & Figure 6). What this suggests is that effects of both preemption and verb semantics are present in the data – and, indeed show broadly similar effect sizes – but that we cannot pick them apart as they are inevitably highly corre- lated: Verbs that are high in transitivity (Verb level seman- tics) tend to occur frequently with ergative ne- marking in the input (preemption). That said, across both studies, preemption seems to be larger and more robust effect. All children saw the same list of 40 verbs. The order of pres- entation of the verbs was fully randomised for each child, using the randomisation function of the Gorilla platform. Each child completed the experiment in a single session which took approximately 30–40 minutes per child. All children completed seven practice trials prior to the main task. In the practice trials, children described the event and received feed- back on their responses. The feedback consisted of a cor- rect description of the event which the parent read out to the child. Predictors The frequency counts (chi-square measure) and patient- affectedness ratings obtained in Study 1 served as the preemp- tion and verb-level semantics predictors for Study 2. For this production study, the preemption account predicts that the higher the (scaled and centred) chi-square value (such that positive and negative values represent a bias towards the ne marked and zero marked subjects respectively), the greater the probabil- ity of children using the ergative ne versus zero marking on the subject (with all other responses discarded as missing data). The verb-level semantics account predicts that the higher the (scaled and centred) patient-affectedness score, the greater the probability of children using the ergative ne versus zero marking on the subject. Discussion (Study 2) In summary, the results of Study 2 (elicited production) echo those of Study 1 (acceptability judgments) in relation to the effects of preemption which were observed in every analysis. Unlike in Study 1, however, effects of verb level semantics were also observed; though these cannot statistically be teased apart from preemption. Taken together, then Study 1 (accept- ability judgments) and Study 2 (elicited production) suggest that statistical preemption is the main mechanism by which children learn which verbs do and do not trigger ergative ne marking. However, evidence from Study 1 suggests that clause-level semantics (in the form of intentionality) also influences judg- ments about the acceptability of ergative ne marking. The role of verb-level semantics is less clear: such effects were observed only for adults in Study 1, and only in the single-predictor models for Study 2. Thus, it seems that effects of Verb-level semantics are present in the data, but we cannot tell whether they are actu- ally utilized by children in learning, or have merely histori- cally determined which verbs tend to occur with and without ne- marking in the first place. Statistical analyses The data were analysed using R version 3.6.3 (R Studio Version 1.2.5042) (R Core Team, 2020). All analyses were conducted exactly according to our pre-registered analysis plan which is reproduced below. The preregistered analysis plan was as follows: The above mentioned hypotheses will be tested in three ways. First, we will run a maximal (as far as will converge) mixed effects model (using lme4 in R) investigating the verb semantics predictor (Hypothesis 2) only - since this is the primary pre- dictor of interest. P values will be calculated via the "drop1" method. Second, we will run a maximal (as far as will converge) mixed effects model (using lme4 in R) investigating the preemption predictor (Hypoth- esis 1) only. P values will be calculated via the "drop1" method. Third, we will run a maximal (as far as will converge) mixed effects model (using lme4 in R) investigating both the verb semantics and the preemp- tion predictors. P values will be calculated via the "drop1" method (see attached syntax). This is con- sidered a secondary analysis due to likely collinearity between the predictors. General discussion A question that lies at the very heart of language acquisition research is how children learn generalizations with exceptions (e.g., the English plural rule that yields cats, dogs, etc, with exceptions feet and men). Previous research has provided evi- dence for two accounts. Statistical preemption (e.g., Goldberg, 1995; Goldberg, 2006; Goldberg, 2011; Goldberg, 2019) holds that children are sensitive to the competition between forms with the same (or similar) meanings. For example, in the domain of verb argument structure, repeatedly hearing [A] made [B] dance (e.g., Somebody made the boy dance) probabilistically outcompetes – that is, statistically pre-empts – [A] danced [B] (e.g., Somebody danced the boy). Semantic accounts hold that learners are guided in part by meaning. For example, the reason we can say Somebody boiled the water but not Somebody Mixed effects models were run using the lme4 package (Bates et al., 2015); p values were obtained using the ‘drop1’ method (i.e., nested models were compared using the likeli- hood ratio test). Statistical analyses were conducted for each age Page 18 of 31 Page 18 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Table 10. Elicited Production Responses: Simultaneous and Single-predictor Models for Both Age Groups. 4;0-5;0 year olds 5;6-6;6 year olds Est SE z p Est SE z p Simultaneous Intercept 5.23 0.62 8.47 0.00 3.50 0.37 9.48 0.00 Preemption 1.86 0.51 3.66 0.00 1.25 0.32 3.87 0.00 Verb Semantics -0.38 0.44 -0.86 0.39 0.58 0.38 1.53 0.13 Single-predictor (Preemption) Intercept 4.79 0.51 9.39 0.00 3.48 0.37 9.37 0.00 Preemption 1.49 0.40 3.75 0.00 1.42 0.32 4.39 0.00 Single-predictor (Semantics) Intercept 4.46 0.47 9.58 0.00 3.58 0.45 7.91 0.00 Verb Semantics 0.91 0.36 2.50 0.01 0.91 0.45 2.05 0.04 gure 5. Relationship between Preemption counts and Elicited Production across Age groups. Figure 5 shows the relationship etween preemption counts (higher corpus relative verb frequency triggering ne form) and elicited production across age groups –5, 5;6–6;6). Table 10. Elicited Production Responses: Simultaneous and Single-predictor Models for Both Age Groups. General discussion 4;0-5;0 year olds 5;6-6;6 year olds Est SE z p Est SE z p Simultaneous Intercept 5.23 0.62 8.47 0.00 3.50 0.37 9.48 0.00 Preemption 1.86 0.51 3.66 0.00 1.25 0.32 3.87 0.00 Verb Semantics -0.38 0.44 -0.86 0.39 0.58 0.38 1.53 0.13 Single-predictor (Preemption) Intercept 4.79 0.51 9.39 0.00 3.48 0.37 9.37 0.00 Preemption 1.49 0.40 3.75 0.00 1.42 0.32 4.39 0.00 Single-predictor (Semantics) Intercept 4.46 0.47 9.58 0.00 3.58 0.45 7.91 0.00 Verb Semantics 0.91 0.36 2.50 0.01 0.91 0.45 2.05 0.04 Table 10. Elicited Production Responses: Simultaneous and Single-predictor Models for Both Age Groups. Table 10. Elicited Production Responses: Simultaneous and Single-predictor Models for Both Age Groups. Figure 5. Relationship between Preemption counts and Elicited Production across Age groups. Figure 5 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and elicited production across age groups (4–5, 5;6–6;6). Figure 5. Relationship between Preemption counts and Elicited Production across Age groups. Figure 5 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and elicited production across age groups (4–5, 5;6–6;6). Figure 5. Relationship between Preemption counts and Elicited Production across Age groups. Figure 5 shows the relationship between preemption counts (higher corpus relative verb frequency triggering ne form) and elicited production across age groups (4–5, 5;6–6;6). Page 19 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Figure 6. Relationship between verb-level Semantics and Elicited Production across Age groups. Figure 6 shows the relationship between verb-level semantics (Patient Affectedness) and elicited production across age groups (4–5, 5;6–6;6). Figure 6. Relationship between verb-level Semantics and Elicited Production across Age groups. Figure 6 shows the relationship between verb-level semantics (Patient Affectedness) and elicited production across age groups (4–5, 5;6–6;6). show a greater preference (Study 1) for ne over zero-marked subjects. danced the boy is that “boiling” but not “dancing” is an activ- ity that an external causer can more-or-less force another entity to undergo (Ambridge et al., 2020; Ambridge et al., 2022; Pinker, 1989; Shibatani & Pardeshi, 2002). Overall, the findings from the acceptability-judgment study (with 5–6 year-olds, 9–10 year-olds and adults) and the production study (with 4–5 and 5–6 year olds) yield a clear picture. General discussion Find- ings of statistical preemption were observed across the board, suggesting that the main way Hindi-speaking children learn which verbs do and do not trigger ne marking is probabilistic input-based learning at the verb level. At the same time, the acceptability-judgment study shows that learners also seem to be sensitive to a clause-level semantic constraint such that intentional actions require ne marking to a greater extent than unintentional ones. Effects of verb-level semantics, how- ever, were observed only sporadically, primarily in the adult acceptability judgment data and elicited production data from children. These findings indicate that to some extent, ne mark- ing is associated with higher transitivity actions, but this effect disappears when controlling for preemption. Most likely then, transitivity (verb-level semantics) determines which verbs prefer ne- versus zero marking historically, but learners primarily The present study tested the preemption and semantics hypoth- eses for Hindi ergative ne marking: another semi-regular system characterized by exceptions. The preemption account predicts that the greater the frequency with which a particu- lar verb appears with versus without ne marking on the sub- ject – relative to other verbs – the greater the extent to which child and adult participants will (Study 1) accept and (Study 2) produce ne over zero-marked subjects. The semantics hypoth- esis was tested at both the verb and the clause level: At the verb level, this account predicts that the greater the verb’s semantic transitivity (as determined in a separate semantic-rating task of patient affectedness) the greater the extent to which child and adult participants will (Study 1) accept and (Study 2) pro- duce ne over zero-marked subjects. At the clause level, this account predicts that when an action is portrayed as inten- tional rather than unintentional, child and adult participants will Page 20 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 learn these patterns statistically on a verb-by-verb basis (i.e., via statistical preemption). a. Ergative_Judgment_PracticeAnimations (contains animations and audio files that accompanied the animations in the practice trial of Study 1) These results therefore add to a growing body of work which suggests that learners acquire exception-filled generalizations by (a) learning probabilistically from the input which surface form is used by adult speakers to convey a particular meaning (i.e., via statistical preemption) and (b) forming overarch- ing generalizations based on semantics, which allow them to generalize to new scenarios. • ErgativeStudy_Data&RCode-ORE.zip • ErgativeStudy_Data&RCode-ORE.zip a. Judgment_Study_Data (contains data and R code for Study 1) Data availability Underlying data • raw-semantics-20.csv (contains the raw data obtained from 20 participants on the semantics ratings task) OSF: Ergative marking in Hindi: Stimuli, data and R code General discussion For Hindi ergative ne marking, these semantic effects seem mainly to operate at the level of the clause, specifically reflecting intentionality. That is, in terms of avoiding overgeneralizations, children learn that the excep- tions to ergative ne- marking occur in contexts with low levels of intentionality. The role of verb-level semantic effects war- rants further investigation. Future studies investigating other language systems characterized by partial productivity should therefore take seriously, and investigate empirically, the possibil- ity that effects of semantics exist. In the meantime, the present findings have demonstrated that, for a previously understudied phenomenon – ergative ne- marking – learners acquire exception- filled generalizations using both verb-by-verb learning (i.e., statistical preemption) and forming overarching semantic generalizations (here, intentionality). b. Ergative_Production_PracticeAnimations (contains animations and audio files that accompanied the animations in the practice trial of Study 2) ErgativeStudy_VerbLists_Practice&Main.zip ErgativeStudy_VerbLists_Practice&Main.zip a. Ergative_Hindi_VerbLists.xlsx (contains three sub- sheets which have details on the verbs that were used for adults and children - one adult list; two child lists - Child_List1, Child_List2) a. Ergative_Hindi_VerbLists.xlsx (contains three sub- sheets which have details on the verbs that were used for adults and children - one adult list; two child lists - Child_List1, Child_List2) b. ErgativeGJ_PracticeTrialsList.xlsx (contains list of the trials used for the grammatical judgment practice task in Study 1) b. ErgativeGJ_PracticeTrialsList.xlsx (contains list of the trials used for the grammatical judgment practice task in Study 1) c. Ergative_EP_PracticeTrialsList.xlsx (contains list of the trials used for the elicited production practice task in Study 1) • Ergative_SemanticsTaskMaterials.zip (contains materials used in the semantics ratings task) Ethics and consent The study was approved by ethics committees at the University of Liverpool, UK (RETH001041) and International Institute of Information Technology – Hyderabad, India (IIITH-IRB- PRO-2021-02). Written informed consent was obtained using online and physical consent forms, depending on whether the task was completed face-to-face or online. For children, writ- ten informed consent was obtained from parents. Children provided verbal assent. • finalrawdata-wp2-GJ-5-6-nopreemp.csv (raw data for 5–6 year olds) • finalrawdata-wp2-GJ-9-10-nopreemp.csv (raw data for 9–10 year olds) • finalrawdata-wp2-GJ-adults-nopreemp.csv (raw data for adults) • PreemptionCounts.csv (contains the frequency counts for all 40 verbs) https://doi.org/10.17605/OSF.IO/7KS63 (Ambridge et al., 2023a). • semantics-20-mean scores.xlsx (contains the mean ratings across participants for each verb on the semantics ratings task) This project contains the following underlying data: This project contains the following underlying data: • File name.pdf/xlsx/mp4 (brief description of file (in a few words)) • V2_Ergative_Judgments.R (R code for Study 1) • ErgativeStudy_Animations.zip (contains animations for the grammatical judgment and elicited production tasks in intentional (e.g., BREAK.mp4) and uninten- tional (e.g., BREAK_A.mp4) conditions; only ani- mations in the intentional condition were used for the elicited production task) b. Production_Study_Data (contains data and R code for Study 2) b. Production_Study_Data (contains data and R code for Study 2) • Production_FinalCodeSheet_4–5 (contains raw data from 4–5 year olds) • Production_FinalCodeSheet_5–6 (contains raw data from 5–6 year olds) • Ergative_Judgment_SentencesAudio.zip (contains audio files that accompanied the animations in the main trial of Study 1) • PreemptionCounts.csv (contains the frequency counts for all 40 verbs) • ErgativeStudy_PracticeAnimations.zip Page 21 of 31 Page 21 of 31 Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 • Pre-registration document for Study 1 (Ambridge, B., Maitreyee, R., Narasimhan, B., Sharma, D. M., Nair, R. B., & Samanta, S. (2019). Development of erga- tive case-marking in Hindi: Evidence from a grammaticality judgment study (preregistration). Open Science Framework. https://doi.org/10.17605/OSF.IO/ Q5RT8 • raw-semantics-20.csv (contains the raw data obtained from 20 participants on the semantics ratings task) • semantics-20-mean scores.xlsx (contains the mean ratings across participants for each verb on the semantics ratings task) • Ergative_Prod.R (R code for Study 2) • Pre-registration document for Study 2 (Maitreyee, R., Ambridge, B., Narasimhan, B., Saxena, G., Sharma, D. M., & Nair, R. B. (2019). The roles of preemp- tion and semantics in the production of ergative marking in Hindi speaking children (preregistration). Open Science Framework. https://doi.org/10.17605/ OSF.IO/H678K Extended data OSF: CLASS: Cross Linguistic Acquisition of Sentence Structure OSF: CLASS: Cross Linguistic Acquisition of Sentence Structure Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Publisher Full Text Ambridge B, Blything RP: A connectionist model of the retreat from verb argument structure overgeneralization. J Child Lang. 2016; 43(6): 1245–76. PubMed Abstract \| Publisher Full Text Ambridge B, Pine JM, Rowland CF, et al.: The retreat from overgeneralization in child language acquisition: Word learning, morphology, and verb argument structure. Wiley Interdiscip Rev Cogn Sci. 2013; 4(1): 47–62. PubMed Abstract \| Publisher Full Text Ambridge B, Pine JM, Rowland CF, et al.: The retreat from overgeneralization in child language acquisition: Word learning, morphology, and verb argument structure. Wiley Interdiscip Rev Cogn Sci. 2013; 4(1): 47–62. PubMed Abstract \| Publisher Full Text Ambridge B, Brandt S: Lisa filled water into the cup: The roles of entrenchment, preemption and verb semantics in German speakers’ L2 acquisition of English locatives. Zeitschrift für Anglistik und Amerikanistik. 2013; 61(3): 245–263. 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PubMed Abstract \| Publisher Full Text Ambridge B, Pine JM, Rowland CF: Semantics versus statistics in the retreat from locative overgeneralization errors. Cognition. 2012a; 123(2): 260–79. PubMed Abstract \| Publisher Full Text Ambridge B: How do children restrict their linguistic generalizations? An (un-)grammaticality judgment study. Cogn Sci. 2013; 37(3): 508–43. PubMed Abstract \| Publisher Full Text \| Free Full Text Ambridge B: How do children restrict their linguistic generalizations? An (un-)grammaticality judgment study. Cogn Sci. 2013; 37(3): 508–43. PubMed Abstract \| Publisher Full Text \| Free Full Text Ambridge B, Pine JM, Rowland CF, et al.: A semantics-based approach to the “no negative evidence” problem. Cogn Sci. 2009; 33(7): 1301–1316. PubMed Abstract \| Publisher Full Text Ambridge B, Ambridge C: The retreat from transitive-causative overgeneralization errors: A review and diary study. In: C. F. Rowland, A. L. Theakston, B. Ambridge, & K. E. Twomey (Eds.), Current perspectives on child language acquisition: How children use their environment to learn. John Benjamins, 2020; 113–130. Ambridge B, Pine JM, Rowland CF, et al.: Avoiding dative overgeneralisation errors: semantics, statistics or both? Lang Cogn Neurosci. 2014; 29(2): 218–243. Publisher Full Text Ambridge B, Pine JM, Rowland CF, et al.: The effect of verb semantic class and verb frequency (entrenchment) on children’s and adults’ graded judgements of argument-structure overgeneralization errors. Cognition. 2008; 106(1): 87–129. Ambridge B, Pine JM, Rowland CF, et al.: The effect of verb semantic class and verb frequency (entrenchment) on children’s and adults’ graded judgements of argument-structure overgeneralization errors. Cognition. 2008; 106(1): 87–129. Publisher Full Text Publisher Full Text Ambridge B, Bidgood A, Twomey KE, et al.: Preemption versus Entrenchment: Towards a Construction-General Solution to the Problem of the Retreat from Verb Argument Structure Overgeneralization. PLoS One. 2015; 10(4): e0123723. Ambridge B, Pine JM, Rowland CF, et al.: The roles of verb semantics, entrenchment, and morphophonology in the retreat from dative argument-structure overgeneralization errors. Language. 2012b; 88(1): 45–81. PubMed Abstract \| Publisher Full Text \| Free Full Text https://doi.org/10.17605/OSF.IO/PAVM7 (Ambridge et al., 2023b). This project contains the following extended data: Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). • File name.pdf/xlsx/mp4 (brief description of file (in a few words)) Publisher Full Text Ambridge B, Doherty L, Maitreyee R, et al.: Testing a computational model of causative overgeneralizations: Child judgment and production data from English, Hebrew, Hindi, Japanese and K’iche’ [version 2; peer review: 2 approved, 1 approved with reservations]. Open Res Eur. 2022; 1(1). Publisher Full Text Barak L, Goldberg AE, Stevenson S: Comparing computational cognitive models of generalization in a language acquisition task. Proceedings of the 2016 Conference on Empirical Methods in Natural Language Processing. Association for Computational Linguistics, 2016; 96–106. Publisher Full Text Ambridge B, Maitreyee R, Saxena G, et al.: Ergative marking in Hindi: Stimuli, data and R code. Open Science Framework. 2023a. 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The grammar of causation and interpersonal manipulation. John Benjamins, 2002; 85–126. PubMed Abstract \|� Publisher Full Text Hopper PJ, Thompson SA: Transitivity in grammar and discourse. Language. 1980; 56(2): 251–299. Aaricia Ponnet Linguistics, Ghent University, Pietersnieuwstraat, Gent, Belgium This article adds some fundamental findings to ongoing research about how children learn certain verbal constructions and how they deal with exceptions and semantic restrictions when learning verbal event constructions. Moreover, they do this by focussing on Hindi, a language which is, despite being the third most spoken language in the world, still under-researched in linguistic research and especially in research with a language acquisition and learnability perspective. Moreover, the study focuses on child speakers of Hindi in India of two different age groups (with a younger pilot study group) as well as a group of adult speakers. This means that the study not only provides interesting data from a learnability perspective (i.e. how do children deal with learning the different semantic and syntactic restrictions that are related to the acquisition of the construction under study) but also adds to the growing body of work on the Hindi language and additionally gives valuable insights on the distribution of ne marking with adult speakers of Hindi in India. It taps into to the body of work that researches the use and acquisition of differential case marking with children and adults, in this case, split ergativity (with a focus on active, transitive, two-participant verbs). One of the strong points of this study is that it not only provides a wide range of data with considerably large sample groups with more than 140 participants in total (given that this type of research in India is still rather novel, and given the restrictions during the COVID pandemic during which this research has been performed, this is quite an impressive number), but it also provides a solid hypothesis and contrasts two different linguistic theoretical approaches that the study aims to test by analysing the data of the different speaker groups. On the one hand, the study aims to predict and test ‘statistical preemption’ (based on recent work by Goldberg and collaborators) which poses that certain constructions or linguistic forms probabilistically outcompete others in the input distribution of children, which means that they will prefer certain constructions or forms over others. On the other, the study aims to elaborate on previous work on the role of semantics in the learnability of certain constructions (building on previous work by Ambridge and collaborators), and typological work on Hindi (a.o. Version 1 Reviewer Report 29 August 2023 https://doi.org/10.21956/openreseurope.16872.r33459 © 2023 Ponnet A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Publisher Full Text Wonnacott E, Newport EL, Tanenhaus MK: Acquiring and processing verb argument structure: distributional learning in a miniature language. Cogn Psychol. 2008; 56(3): 165–209. PubMed Abstract \| Publisher Full Text \| Free Full Text List JM, Cysouw M, Forkel R: Concepticon: A resource for the linking of concept lists. In: Proceedings of the Tenth International Conference on Language Resources and Evaluation. European Language Resources Association (ELRA). Page 23 of 31 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Open Peer Review Current Peer Review Status: Version 1 Reviewer Report 29 August 2023 https://doi.org/10.21956/openreseurope.16872.r33459 Open Peer Review Current Peer Review Status: Aaricia Ponnet Mohanan 1994) that highlights the effects of verb-level semantics, in the case of split ergativity: transitivity, and clause level semantics, in the case of split ergativity: volitionality or intentionality. Verbs that rate higher on the transitivity cline Page 24 of 31 Open Research Europe Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 or that are associated with a higher sense of intentionality are predicted to be associated with ne with a higher range of frequency. It is important to note that the study focuses on active, transitive, two-participant verbs – intransitive verbs that can in some cases take the ergative were not included. Data were collected using an acceptability judgment task (three groups, children of different age groups and adults) and an elicited production task (two groups, a younger group and a group aged 5-6). These are presented as two separate studies (study 1 and study 2), after which the results are compared in the discussion section. Study 1 looks at the effect of statistical preemption, intentionality and transitivity. Study 2 investigates the effect of statistical preemption and transitivity. A large online Hindi corpus was perused to account for statistical preemption with regard to the quantitative analysis. Another strong feature of the study is that it openly shares the material that was used to collect the data online, as well as the analyses in R. This means that the study is replicable, but also that reviewers and readers can have all the necessary information needed to interpret and check the results of this study. Both the studies (acceptability judgment and elicited production) find an effect of statistical preemption over the different age groups (except for the middle age group in Study 1). In Study 1, additionally, a significant effect for intentionality was attested, but not for transitivity. In Study 2, to a lesser extent, an effect was found also for transitivity. This leads the authors to the conclusion that children rely both on frequency as well as semantics to learn certain regularities as well as exceptions to these regularities. In the case of ergative ne marking then, and specifically with regard to transitive perfective verbs, children learn, according to the authors, on a verb-by-verb basis, and with those verbs where ne marking is optional in particular, are further guided by semantics, in this case, intentionality. Aaricia Ponnet As per my knowledge, this is the first study on Hindi ergative marking that has been carried out at such a large scale, which makes it a necessary, innovative contribution to the field of linguistics. The study is in my opinion a valuable and perhaps even groundbreaking contribution with regard to the several aspects that I summarized and highlighted above. I do have, however, some minor reservations/questions regarding the following: A list of the 40 verbs is given in an online repository, but for the interpretation of the results of the study, I find it essential to include at least an overview of the verbs in the article itself (preferably with the original Hindi verb as well as its English counterpart). ○ A list of the 40 verbs is given in an online repository, but for the interpretation of the results of the study, I find it essential to include at least an overview of the verbs in the article itself (preferably with the original Hindi verb as well as its English counterpart). ○ In Hindi, the subject is generally null-marked (nominative). What makes the split ergative construction in Hindi challenging is that there is an opposition between ergative ne marking and nominative null marking. Children have to learn that the opposition transitive/intransitive is morphologically marked with verbs that have the perfective aspect. The verbs that have been included in this study only concern verbs that can have ergative marking but not typical intransitive verbs such as jaanaa ‘to go’, bhaagnaa ‘to run’, baiThnaa ‘to sit’, khaaMsnaa ‘to cough’, naacnaa ‘to dance’ and so on, neither does it include such intransitive verbs that can sometimes have subject marked with the ergative such as chiikh ‘to scream’. On the one hand, this focus on two-participant transitive verbs could be made clearer in the introduction of the study (and, consequently, the exclusion of single- participant intransitive verbs), on the other hand, the inclusion of intransitive perfective verbs is in my opinion elementary to the research of the learnability of split ergativity as a whole, not only the semantics that are involved but also the morphosyntactic opposition In Hindi, the subject is generally null-marked (nominative). What makes the split ergative construction in Hindi challenging is that there is an opposition between ergative ne marking and nominative null marking. Aaricia Ponnet Children have to learn that the opposition t iti /i t iti i h l i ll k d ith b th t h th f ti t ○ Page 25 of 31 Open Research Europe Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 between ergative and nominative. This is particularly so since there are certain intransitive verbs that allow for ne marking when volitionality/intentionality is at play – and these verbs were not included in the study. Moreover, when testing statistical preemption, there will also be a role of the frequency of certain lemmas in the subject function – there can only be ne marking of the subject in the perfective aspect verb form, all other aspects have nominative subjects. Since other verb forms were not included in the study, the effect of frequency of nominative on the subject forms is therefore not accounted for in this study. Especially from a language development perspective, the question remains how children shift from mainly nominative to mainly ergative – if such a shift is present at all. This aspect is not covered by the study as most of the age groups perform similarly to the adult group, which suggests that by age 4-5 these nuances are already acquired. Another issue regards the rating of the affectedness of the patient to rate the level of transitivity, by which the authors aim to test the influence of verb-level semantics on the use of ne with active, transitive two-participant verbs. This was tested by presenting participants passivized forms of the 40 verbs that were included in the test. My concern is that the effect of transitivity cannot be entirely tested in this way. Moreover, with regard to the transitivity cline, there are several aspects that have been stated by Hopper & Thompson (1980) that influence the level of transitivity, which concerns the agent as well as the patient. Moreover, according to Hopper & Thompson (1980), transitivity is “a global property of an entire clause” (251). Maitreyee et al. distinguish between intentionality at the clause level and transitivity as verb-level semantics, while these features may very well both be positioned at the clause level. I believe these data and analyses are extremely valuable and I hope that for future Aaricia Ponnet Either the choice for this distinction at the semantic level needs to be made more clear by the authors in the text, or the focus should be more on ‘patient affectedness’ and less on transitivity, as transitivity encompasses more than affectedness alone. y y p Moreover, volitionality/agentivity (which highly overlaps with the feature ‘intentionality’ that is being investigated in this study) is one of the features stated by Hopper and Thompson that influences the rate of transitivity as well (252). Within this line of reasoning, the authors have researched the effect of two transitivity features on the use of ne (i.e. intentionality vs. patient affectedness) rather than intentionality vs. transitivity. Intentionality was tested by presenting the participants with four sentences, alternately marked with ne or null-marked in the nominative, and by adding an adverb galtii se ‘by mistake’. In this way sentences were created that were, according to the authors, more intentional than others within the meaning of that particular verb. However, 1) an adverb expressing higher intentionality was not added, and 2) the range of intentionality between the different verbs that were tested was not accounted for. The figures in the article show a clear difference between sentences that have the adverb ‘by mistake’ and verbs that have not, which confirms a successful operationalization of ‘less intentional’, but there is also a cline to be observed between the verbs at the same level of intentionality. This probably goes beyond the scope of the study but confirms that differences in intentionality are inherent to the verbs and not solely attributable to the addition of adverbs. Moreover, actions with intransitive verbs can also be highly intentional (consider, e.g., to laugh and constructions such as laughing with vs. laughing at) and intentionality thus inevitably also interacts with the discourse-pragmatic level. I would have expected the authors to mention this briefly in the discussion section of the study. I believe these data and analyses are extremely valuable and I hope that for future Page 26 of 31 Open Research Europe Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 research, the researchers are considering adding a qualitative component as well. From a language development perspective, I would have loved to see a few cases and example sentences highlighted that give us more insight into the details of the acquisition and use of ergative marking with Hindi-speaking children. Aaricia Ponnet This evidently goes beyond the scope of the study, but a qualitative analysis will give us more information on the between-verb differences in use at the same level as what the authors operationalised as intentionality and transitivity, as well as interindividual differences, between the different age groups as well as between the different participants within the same age group. Is there a learning curve or have all these children indeed acquired the semantic and syntactic constraints? Are some verbs more difficult than others? What were the highest or clearest contrasts in language use in the use of ergative marking within the adult groups, and what does that tell us about the regularity vs. optionality of ne marking, vs. the different features that interact with ne marking? I applaud the authors for creating the possibility of answering such questions with their collected data and am excited to read more of their work in the future. Please find an annotated pdf of the article with further comments here. research, the researchers are considering adding a qualitative component as well. From a language development perspective, I would have loved to see a few cases and example sentences highlighted that give us more insight into the details of the acquisition and use of ergative marking with Hindi-speaking children. This evidently goes beyond the scope of the study, but a qualitative analysis will give us more information on the between-verb differences in use at the same level as what the authors operationalised as intentionality and transitivity, as well as interindividual differences, between the different age groups as well as between the different participants within the same age group. Is there a learning curve or have all these children indeed acquired the semantic and syntactic constraints? Are some verbs more difficult than others? What were the highest or clearest contrasts in language use in the use of ergative marking within the adult groups, and what does that tell us about the regularity vs. optionality of ne marking, vs. the different features that interact with ne marking? I applaud the authors for creating the possibility of answering such questions with their collected data and am excited to read more of their work in the future. Please find an annotated pdf of the article with further comments here. If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data and materials underlying the results available? Yes Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Second language acquisition, Hindi, General Linguistics, Morphosyntax, Syntax-semantics interface, differential case marking I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 26 July 2023 tps://doi.org/10.21956/openreseurope.16872.r33451 Page 27 of 3 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 © 2023 Kapatsinski V. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Department of Linguistics, University of Oregon, Eugene, OR, USA This paper addresses the long-standing question of how speakers learn lexical restrictions on construction use in a new (to this literature) language, Hindi. It argues that the choice of the ergative particle 'ne' is constrained by both semantic transitivity of the sentence and the specific verb, with the verbs that most often occur with 'ne' being judged as less acceptable without it. This latter effect is said to provide evidence for statistical preemption. 6-year-olds, 10-year-olds and adults are tested. The effects of preemption / lexical idiosyncrasy are stronger than those of semantics. The paper makes a valuable contribution to the literature by extending this debate to a new language, and a new construction. There is also methodological innovation in developing a way to rate semantic transitivity. Another strength of the paper is that both judgments and production data are collected. One aspect of the paper that is not clear to me is whether it actually tests preemption. The tested prediction is "the greater the frequency with which a particular verb appears with versus without ne marking on the subject – relative to other verbs in the test set – the greater the extent to which ne marked forms will be preferred over zero-marked forms in the judgment task... we operationalized this measure using a scaled and centred chi-square statistic which measures how often, in past tense/perfective aspect active sentences, a particular verb triggers ergative case marking as compared with all other verbs in the test set of 40". However, this conflates frequency in the rated construction (e.g., with ne) and frequency in the other construction (without ne). The latter is an implementation of preemption but the former is not. How do we know that frequency in the other construction matters for the judgments? One way to address this issue would be to include frequency in the presented construction as a separate predictor. Less of an issue, but this also does not necessarily differentiate preemption and entrenchment, the frequency with which the verb occurs in other constructions that are not synonymous with the 'ne'. A limitation of the analyses is that no interactions between age/subject group and the other predictors are fitted. These would show whether the effects of preemption and semantics actually change significantly over the tested age span. Are the conclusions drawn adequately supported by the results? Yes https://doi.org/10.21956/openreseurope.16872.r33451 Page 27 of 31 Open Research Europe Open Research Europe Vsevolod Kapatsinski Department of Linguistics, University of Oregon, Eugene, OR, USA https://doi.org/10.21956/openreseurope.16872.r33450 © 2023 Bhatia A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Are all the source data and materials underlying the results available? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Learning theory, usage-based linguistics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Are all the source data and materials underlying the results available? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Learning theory, usage-based linguistics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 26 July 2023 https://doi.org/10.21956/openreseurope.16872.r33450 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes Page 28 of 31 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 Archna Bhatia Florida Institute for Human and Machine Cognition, Ocala, Florida, USA This article explores how children learn semi-regular systems (exceptions-filled generalizations) in languages through an example of ergative case marking in Hindi which shows irregularity in that it appears in sentences with transitive verbs in perfective aspect and in sentences with some intransitive verbs that involve volitionality but not with intransitive verbs that lack volitionality also in perfective aspect, also they may be optional in sentences with intransitive verbs. Authors are interested in testing whether such systems are learnt as probabilistic generalizations or as formal rules with memorized exceptions (semantically restricted generalizations). Authors conduct a grammaticality judgement task and an elicited production task to test the probabilistic generalization hypothesis and the semantically restricted generalization hypothesis for learning semi-regular systems. For the probabilistic generalization hypothesis, they study whether the ne-marked forms’ frequency compared to zero-marked forms’ frequency with a particular verb has an impact on its acceptability or production. To test the hypothesis about learning guided by semantic rules, e.g., only verbs high on transitivity trigger ergative ne-marking, they study the extent of ne- vs zero- marked subjects appearing with verbs rated higher on transitivity as being acceptable or produced. To test the semantics hypothesis at the verb-level, transitivity is operationalized in terms of the degree to which the patient is affected by the action denoted by the verb. To test the Page 29 of 31 Open Research Europe Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 semantics hypothesis at the clause-level, authors study the impact of an action appearing intentional rather than unintentional on the extent to which ne-marking forms compared to zero- marked forms are rated as acceptable. Authors recruited children in age groups 5-6 and 9-10 and adults in their studies. The studies’ results indicated that learners acquire exceptions-filled generalizations by learning probabilistically based on the input as well as forming overarching semantic generalizations and learning semantics based exceptions to avoid overgeneralization. The probabilistic generalization (statistical preemption) effects were observed across the board. In terms of semantics, authors observed that learners were sensitive to a clause level semantic constraint involving intentionality of the actions. Authors found limited evidence for the effect of verb-level semantics (patient affectedness), however for adults this effect was observed indicating potential historical links between patient-affectedness and whether the verb would come to prefer ne vs zero marking. Archna Bhatia The work is presented in the paper clearly. The study design is appropriate, and sufficient details are provided about the study design as well as methods and analyses used for the studies in general. The work is technically sound. Authors have made the data they prepared and used in their studies publicly available which will make the replication of the work possible as well as provide data for further analyses and for use in further studies. In my understanding, the statistical analyses and the interpretations seem appropriate, however a statistician may provide further comments. The conclusions are well-supported. Regarding the 40 selected action verbs, did authors look at the inter-annotator agreement between the two native speakers providing intuitions about the action verbs whether they appeared with ergative ne-marking zero-marking or optional ne-marking? Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Speech and Natural Language Processing, Linguistics Page 30 of 31 Open Research Europe Open Research Europe 2023, 3:49 Last updated: 29 AUG 2023 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Page 31 of 31
https://openalex.org/W2963884073	http://epub.jku.at/obvulioa/content/titleinfo/3444328/full.pdf	English	null	On the normality of p-ary bent functions	Cryptography and communications	2,017	cc-by	7,987	Cryptogr. Commun. https://doi.org/10.1007/s12095-017-0259-0 (2018) 10:1 –1049 037 On the normality of p-ary bent functions Wilfried Meidl1,2 · ´Isabel Pirsic3 Received: 22 December 2016 / Accepted: 26 September 2017 © The Author(s) 2017. This article is an open access publication / Published online: 17 October 2017 Abstract Depending on the parity of n and the regularity of a bent function f from Fn p to Fp, f can be affine on a subspace of dimension at most n/2, (n−1)/2 or n/2−1. We point out that many p-ary bent functions take on this bound, and it seems not easy to find exam- ples for which one can show a different behaviour. This resembles the situation for Boolean bent functions of which many are (weakly) n/2-normal, i.e. affine on a n/2-dimensional subspace. However applying an algorithm by Canteaut et.al., some Boolean bent functions were shown to be not n/2-normal. We develop an algorithm for testing normality for func- tions from Fn p to Fp. Applying the algorithm, for some bent functions in small dimension we show that they do not take on the bound on normality. Applying direct sum of functions this yields bent functions with this property in infinitely many dimensions. Keywords Bent function · p-ary bent function · Normal bent function · k-normal Mathematics Subject Classification (2010) 06E30 · 05B10 · 11T71 1 Introduction Let p be a prime, and let f be a function from an n-dimensional vector space Vn over Fp to Fp. The Walsh transform of f is the complex valued function f (u) = x∈Vn ϵf (x)−⟨u,x⟩ p , ϵp = e2πi/p, where ⟨u, x⟩is a (nondegenerate) inner product in Vn. The classical frameworks are Vn = Fn p, in which case we take the conventional dot product as inner product, and Vn = Fpn, for which the standard inner product is ⟨u, x⟩= Trn(ux), where Trn(z) is the absolute trace of z in Fpn. /2 where ⟨u, x⟩is a (nondegenerate) inner product in Vn. The classical frameworks are Vn = Fn p, in which case we take the conventional dot product as inner product, and Vn = Fpn, for which the standard inner product is ⟨u, x⟩= Trn(ux), where Trn(z) is the absolute trace of z in Fpn. p The function f is called a bent function if \| f (b)\| = pn/2 for all b ∈Vn. For Boolean bent functions we have f (b) = (−1)f ∗(b)2n/2 for a Boolean function f ∗, called the dual of f . When p is odd, then a bent function f satisfies (cf. [10]) f (b) = ±ϵf ∗(b) p pn/2 : pn ≡1 mod 4; ±iϵf ∗(b) p pn/2 : pn ≡3 mod 4, (1) (1) for a function f ∗from Vn to Fp. Accordingly f is called regular if p−n/2 f (b) = ϵf ∗(b) p for all b ∈Vn, which for a Boolean bent function always holds. If p−n/2 f (b) = ζ ϵf ∗(b) p for some ζ ∈{±1, ±i}, independent from b, we call f weakly regular, otherwise f is called non-weakly regular. Note that regular implies weakly regular. Weakly regular bent functions always come in pairs, since the dual is bent as well. This does in general not hold for non-weakly regular bent functions, see [6, 8]. Note that Boolean bent functions only exist for even n, which is different when p is odd, where bent functions exist in even and in odd dimension. Bent functions are interesting objects due to applications in cryptography and coding, and due to rich connections to objects in combinatorics and geometry. In particular, bent functions define relative difference sets in the elementary abelian p-group. Mathematics Subject Classification (2010) 06E30 · 05B10 · 11T71 Mathematics Subject Classification (2010) 06E30 · 05B10 · 11T71 This article is part of the Topical Collection on Special Issue on Sequences and Their Application ´Isabel Pirsic isa.pirsic@gmail.com 1 Johann Radon Institute for Computational and Applied Mathematics, Austrian Academy of Sciences, Linz, Austria 2 Otto-von-Guericke University Magdeburg, Universit¨atsplatz 2, 39106 Magdeburg, Germany 2 Otto-von-Guericke University Magdeburg, Universit¨atsplatz 2, 39106 Magdeburg, Germany 3 Institute of Financial Mathematics and Applied Number Theory, Johannes Kepler Universit¨at, Linz, Austria 3 Institute of Financial Mathematics and Applied Number Theory, Johannes Kepler Universit¨at, Linz, Austria 1038 Cryptogr. Commun. (2018) 10:1 –1049 037 1 Introduction Many construc- tions and infinite classes of bent functions are known, hence research focuses on the nature and properties of bent functions, rather than on discovering new formulas for bent functions. In this article we investigate normality for p-ary bent functions, which then also describes a feature of the corresponding relative difference set. We start by recalling the relevant definitions as given in the literature, see [2, 3, 9, 14] or [17, pp.81], [18, pp.155]. A function f : Vn →Fp is called k-normal if there exists a k-dimensional affine subspace of Vn restricted to which f is constant. If f is affine on a k- dimensional affine subspace of Vn, then f is called weakly-k-normal. When n is even and k = n/2, then f is called (weakly)-normal. We emphasize that a weakly-k-normal function can be transformed into a k-normal function by adding an affine function. As bentness is invariant under addition of affine functions, the distinction between normality and weak normality is not relevant for describing the properties of a bent function. Many classical constructions of Boolean bent functions like Maiorana-McFarland and PS+ yield normal functions. This is very different for random Boolean functions, which are not likely to be constant (affine) on an affine subspace with “large” dimension [3]. The question if there exist non-(weakly)-normal Boolean bent functions was open for several years. In [2] it was shown that the Kasami bent function in dimension 14 is non-weakly- normal. Non-weakly-normal bent functions in dimension 10 (and 12) were presented in 1039 Cryptogr. Commun. (2018) 10:1 –1049 037 [14]. By [2, Lemma 25] this guarantees the existence of non-weakly-normal Boolean bent functions in (even) dimension n ≥10. k-normality may also be of cryptographic significance. As pointed out in [3], k-normality is a quite natural complexity criterion, since any affine function is constant on an affine hyperplane. Moreover there is a relation between normality and nonlinearity for Boolean functions, see [3, Proposition 2]. As also mentioned in [3], the k-normality was not yet related to explicit attacks on ciphers, however the situation was the same for nonlinearity when it was introduced. In fact, meanwhile the attack on the stream cipher Grain-128 in [16] is based on the 5-normality of the 9-variable filter function (which can be seen as a modification of the standard quadratic bent function in 8 variables), used in the sequence generation. 1 Introduction The situation for bent functions from Vn to Fp, p odd, is somewhat different from the Boolean case. In [7] it is pointed out that a weakly regular but not regular bent function in even dimension n cannot be normal. However some results indicate that also for odd p, bent functions exhibit a typical normality behaviour. It may not be easy to find bent functions for which one can prove a different behaviour. In this paper, we first present a p-ary equivalent of a result of Carlet in [3] showing that - as one would expect - an arbitrary p-ary function is with high probability not (weakly)-k- normal for any not very small value of k. We then show the p-ary equivalent of a relation between nonlinearity and normality for Boolean functions, [3, 9]. We summarize some known results on normality for p-ary bent functions, which indicate that many have a “typ- ical” behaviour with respect to normality, similar as it was observed in the Boolean case: Many p-ary bent functions are k-normal, where k is as large as it is theoretically possible for a bent function. In Section 3 we present an algorithm for testing (weak)-k-normality for p-ary functions. Our algorithm is not a straightforward generalization of the algorithm in [2], which was used to find non-weakly-normal Boolean bent functions in dimension 14 [2], and 10 and 12 [14]. Applying this algorithm we find the first examples of p-ary bent functions (in small dimen- sions) which do not possess k-normality with maximal possible k. Generalizing Lemma 25 of [2] we then can obtain bent functions with this property in every larger dimension of the same parity. 2 Normality results One target in this paper is to pave the way for a systematic analysis of the behaviour of p-ary bent functions with respect to normality. We hence start with showing some p-ary equivalents of results on the normality behaviour of Boolean (bent) functions. Our first proposition, is the p-ary version of Theorem 3 and Proposition 1 in [3]. The proof resembles the proof in [3]. Proposition 1 Let kn be a sequence of integers such that limn→∞ pkn nkn = ∞. The density of the functions which are weakly-kn-normal in the set of functions from Vn to Fp, tends to 0 if n tends to infinity. Let ln be a sequence of positive integers such that ln/√n tends to infinity if n tends to infinity. The density of the set of weakly ln-normal functions from Vn to Fp of degree at most 3 in the set of all functions of degree at most 3, tends to 0 if n tends to infinity. Cryptogr. Commun. (2018) 10:1 –1049 037 1040 Proof For the proof we may identify Vn with Fn p. The number of linear subspaces of Fn p of dimension kn is Proof For the proof we may identify Vn with Fn p. The number of linear subspaces of Fn p of dimension kn is n kn = (pn −1)(pn −p)(pn −p2) · · · (pn −pkn−1) (pkn −1)(pkn −p)(pkn −p2) · · · (pkn −pkn−1), hence the number of kn-dimensional affine subspaces of Fn p is hence the number of kn-dimensional affine subspaces of Fn p is λn = pn−kn n kn . Let μn be the number of functions from Fn p to Fp which are affine on a fixed kn-dimensional affine subspace A (which does not depend on the choice of A). To determine μn, we choose A = Fkn 2 × {(0, . . . , 0)}. Observe that the restriction of a p-ary function to A is affine if and only if its ANF contains no monomial of degree at least 2 which only contains variables in {x1, x2, . . . , xkn}. The number of such functions is ppn−pkn+kn+1, hence the number ωkn of weakly-kn-normal functions is at most λnppn−pkn+kn+1 = pn−kn n kn ppn−pkn+kn+1. 2 Normality results With n kn < pnkn−k2n+kn (p −1)kn ≤pnkn−k2n+kn−kn logp 2, we obtain that ωkn ≤λnppn−pkn+kn+1 < pn−knpnkn−k2n+kn−knlogp2ppn−pkn+kn+1 = ppnpn(kn+1)−k2n−knlogp2+kn+1−pkn < ppnpn(kn+1)−pkn . Since pkn nkn tends to infinity when n tends to ∞, the exponent n(kn + 1) −pkn tends to −∞. As a consequence, limn→∞ ωkn ppn = 0. pp Let νn be the number of functions from Fn p to Fp of degree at most 3 which are affine on A = Fln 2 × {(0, . . . , 0)}. Similarly as above we see that νn = p1+n+(n 2)+(n 3)−(ln 2)−(ln 3), and the number of weakly-ln-normal functions of degree at most 3 is at most pn(ln+1)−l2n+1+n+(n 2)+(n 3)−(ln 2)−(ln 3). The density of this set in the set of p-ary functions of degree at most 3 is therefore upper bounded by pn(ln+1)−l2n−(ln 2)−(ln 3), – V be a k-dimensional subspace of Vn, and let W be a complement of V in Vn, which tends to 0 if n tends to infinity. Then u∈W ⊥ f (u) f (u) = pn−k a∈V fa(0) fa(0). Vn. Then u∈W ⊥ f (u) f (u) = pn−k a∈V fa(0) fa(0). Proof Applying (b), (a), (c) (in this order) we get Proof Applying (b), (a), (c) (in this order) we get u∈W ⊥ f (u) f (u) = pn−k b∈W Dbf (0) = pn−k b∈W a∈V Dbfa(0) = pn−k a∈V b∈W Dbfa(0) = pn−k a∈V fa(0) fa(0). The next lemma is a p-ary version of [1, Corollary V3]. The next lemma is a p-ary version of [1, Corollary V3]. The next lemma is a p-ary version of [1, Corollary V3]. Lemma 2 With the above notations we have Lemma 2 With the above notations we have Lemma 2 With the above notations we have Lemma 2 With the above notations we have a∈V \| fa(0)\|2 ≤max u∈Vn \| f (u)\|2. Moreover, max v∈Vn \| fa(v)\| ≤max u∈Vn \| f (u)\|. a∈V \| fa(0)\|2 ≤max u∈Vn \| f (u)\|2. max v∈Vn \| fa(v)\| ≤max u∈Vn \| f (u)\|. Moreover, Moreover, which tends to 0 if n tends to infinity. which tends to 0 if n tends to infinity. which tends to 0 if n tends to infinity. We remark that the proof of Proposition 1 also shows that the existence of a not weakly k- normal function from Vn to Fp is guaranteed whenever pn(k+1)−k2+k+1−pk (p−1)k < 1. For instance, there are not (weakly) normal functions for p = 3 and n = 6, and for p = 5 and n = 4. For Boolean functions, in [3, 9] relations between normality and Walsh coefficients have been explored. We next generalize these results to p-ary functions. Some identities for Boolean functions which play a role in the analysis can straightforwardly be generalized to odd p, hence we omit the proof. Let – V be a k-dimensional subspace of Vn, and let W be a complement of V in Vn, 1041 Cryptogr. Commun. (2018) 10:1 –1049 037 – fa be defined on W by fa(x) = f (a + x), x ∈W, for a function f : Vn →Fp and a ∈Vn, n, – Dbf (x) = f (x) −f (x + b) the derivative of f in direction b. n, – Dbf (x) = f (x) −f (x + b) the derivative of f in direction b. Then Then Then Then (a) Dbf (0) = a∈V Dbfa(0) for any b ∈W, (a) Dbf (0) = a∈V Dbfa(0) for any b ∈W, (a) Dbf (0) = a∈V Dbfa(0) for any b ∈W, (b) u∈V f (u + a) f (u + a) = pk b∈V ⊥ϵ⟨a,b⟩ p Dbf (0) (Lemma V2 in [1]), (c) a∈V b∈W Dbfa(0) = a∈V fa(0) fa(0). (b) u∈V f (u + a) f (u + a) = pk b∈V ⊥ϵ⟨a,b⟩ p Dbf (0) (Lemma V2 in [1]), The following lemma is the p-ary analog of Theorem V1 in [1] (Equation (4) in [9]). Lemma 1 Let W be a k-dimensional subspace of Vn and let V be a complement of W in Vn. Then u∈W ⊥ f (u) f (u) = pn−k a∈V fa(0) fa(0). Lemma 1 Let W be a k-dimensional subspace of Vn and let V be a complement of W in Vn. Moreover, max v∈Vn \| fa(v)\| ≤max u∈Vn \| f (u)\|. Proof By Lemma 1, with \|W ⊥\| = pn−k, we have pn−k a∈V \| fa(0)\|2 = u∈W ⊥ \| f (u)\|2 ≤pn−k max u∈Vn \| f (u)\|2. pn−k a∈V \| fa(0)\|2 = u∈W ⊥ \| f (u)\|2 ≤pn−k max u∈Vn \| f (u)\|2. This in particular implies This in particular implies \| fa(0)\| = \| x∈W ϵf (x+a) p \| ≤max u∈Vn \| f (u)\| (2) (2) for all a ∈V . We may apply the same arguments to the function ˜f (x) = f (x) + ⟨v, x⟩ for some v ∈Vn (which has the same Walsh spectrum as f , hence maxu∈Vn \|˜f (u)\| = maxu∈Vn \| f (u)\|). Then (2) converts to for all a ∈V . We may apply the same arguments to the function ˜f (x) = f (x) + ⟨v, x⟩ for some v ∈Vn (which has the same Walsh spectrum as f , hence maxu∈Vn \|˜f (u)\| = maxu∈Vn \| f (u)\|). Then (2) converts to \|˜fa(0)\| = \| x∈W ϵf (x+a)+⟨v,x⟩+⟨v,a⟩ p \| ≤max u∈Vn \| f (u)\| (3) (3) \| ˜fa(0)\| = \| x∈W ϵf (x+a)+⟨v,x⟩+⟨v,a⟩ p \| ≤max u∈Vn \| f (u)\| (3) for all a ∈V , and the claim of the lemma follows. x∈W u∈Vn for all a ∈V , and the claim of the lemma follows. for all a ∈V , and the claim of the lemma follows. for all a ∈V , and the claim of the lemma follows. for all a ∈V , and the claim of the lemma follows. for all a ∈V , and the claim of the lemma follows. 1042 Cryptogr. Commun. (2018) 10:1 –1049 037 With Lemma 2 we get the relation between normality and Walsh coefficient more general for functions from Vn to Fp for arbitrary primes p. Corollary 1 Let f be a function from Vn to Fp. If f is (weakly) k-normal, then pk ≤ maxu∈Vn \| f (u)\|. Proof Suppose that f is weakly k-normal, i.e. f (x) = ⟨v, x⟩+c, for some v ∈Vn, c ∈Fp, and all x ∈a + W for some k-dimensional subspace W of Vn and some a in a complement V of W. Moreover, Then, using Lemma 2 we have \| x∈W ϵf (x+a)+⟨v,x⟩+⟨v,a⟩ p \| = pk ≤max u∈Vn \| f (u)\|. For a bent function f : Vn →Fp, Corollary 1 implies that f can be at most ⌊n/2⌋- normal. Moreover, for bent functions in even dimension which are weakly regular but not regular the following result has been shown in [7, Theorem 6(i)]: Proposition 2 Let n be even, p an odd prime, and f : Fn p →Fp be a bent function. If f is weakly regular but not regular, then f is not (weakly) normal. Hence, a weakly regular but not regular bent function in even dimension can be at most (n/2 −1)-normal. However, whereas an arbitrary p-ary function is with high probabil- ity “highly non-normal” (see Proposition 1), many bent functions in odd characteristic are (weakly) k-normal with k as large as the theory allows. That is, many p-ary bent functions in even dimension are weakly normal, except from those which are weakly regular but not regular, of which many are n/2 −1-normal, many p-ary bent functions in odd dimension are weakly-(n −1)/2-normal. The following results on normality of p-ary bent functions support this observation. Note that the large classes of completed Maiorana-McFarland and PS+ bent functions (all of which members are regular bent functions in even dimension) are normal by their definition. – A quadratic bent function Q : Vn →Fp, p odd, is normal if n is even and Q is regular, (n/2 −1)-normal if n is even and Q is weakly regular but not regular, and (n −1)/2-normal if n is odd, see [7]. – A quadratic bent function Q : Vn →Fp, p odd, is normal if n is even and Q is regular, (n/2 −1)-normal if n is even and Q is weakly regular but not regular, and (n −1)/2-normal if n is odd, see [7]. – [13, Proposition 5] A regular bent function of the form – [13, Proposition 5] A regular bent function of the form f (x) = Trn αxl(pn/2−1) + ϵx(pn−1)/2 is normal. (For the bentness conditions see [13, Theorem 1].) is normal. (For the bentness conditions see [13, Theorem 1].) – [7, Theorem 7] The regular Coulter-Matthews bent functions are normal. [7, Theorem 7] The regular Coulter-Matthews bent functions are normal. [7, Theorem 7] The regular Coulter Matthews bent functions are normal. Moreover, – The secondary construction of non-weakly regular bent functions f : Vn →Fp in [4, 5], yields (weakly) normal bent functions when n is even and (weakly) (n−1)/2-normal bent functions when n is odd. – The secondary construction of non-weakly regular bent functions f : Vn →Fp in [4, 5], yields (weakly) normal bent functions when n is even and (weakly) (n−1)/2-normal bent functions when n is odd. – [7, Example 1] f : F34 →F3, f (x) = Tr4(ω10x22 + x4), ω primitive element of F34, is normal. – [7, Example 1] f : F34 →F3, f (x) = Tr4(ω10x22 + x4), ω primitive element of F34, is normal. The last example presented in [11], was one of the first known examples for a non-weakly regular bent function. As pointed out in [7], the function does not have a bent dual. One may 1043 Cryptogr. Commun. (2018) 10:1 –1049 037 expect that this in some sense not smooth bent function exhibits a more chaotic behaviour, which however does not apply with regard to normality in this case. We here remark that differently to Boolean functions in dimension 4 (see [3]), functions from F34 to F3 which are not weakly normal do exist. Examples are the quadratic bent functions from F34 to F3 which are weakly regular but not regular, and then by Proposition 2 not weakly normal. expect that this in some sense not smooth bent function exhibits a more chaotic behaviour, which however does not apply with regard to normality in this case. We here remark that differently to Boolean functions in dimension 4 (see [3]), functions from F34 to F3 which are not weakly normal do exist. Examples are the quadratic bent functions from F34 to F3 which are weakly regular but not regular, and then by Proposition 2 not weakly normal. In general it seems not to be easy to find p-ary bent functions which do not exhibit this “typical” behaviour with regard to normality as described above. This resembles the situation for Boolean bent functions of which most standard examples are (weakly) normal. However, it has been shown that there are not (weakly) normal Boolean bent functions in every (even) dimension n ≥10, see [2, 14], which shows that normality is not a feature of Boolean bent functions. Moreover, We attempt to prove a different than the described behaviour with respect to normality for some p-ary bent functions. Candidates for non-weakly normal bent functions may be sporadic examples of non-weakly regular bent functions (other than the last example in the list above): g1 : F36 →F3 with g1(x) = Tr6(ξ7x98), where ξ is a primitive element of F36, [10], 7 14 35 70 1. g1 : F36 →F3 with g1(x) = Tr6(ξ7x98), where ξ is a primitive element of F36, [10], 2. g2 : F36 →F3 with g2(x) = Tr6(ξ7x14 + ξ35x70), where ξ is a primitive element of F36, [12]. 1. g1 : F36 →F3 with g1(x) = Tr6(ξ7x98), where ξ is a primitive element of F36, [10], 2. g2 : F36 →F3 with g2(x) = Tr6(ξ7x14 + ξ35x70), where ξ is a primitive element of F36, [12]. Recently, the first construction of non-weakly regular bent functions for which the dual is not bent was presented, see [8]. This construction may also provide candidates for non- weakly normal bent functions: Let 1, α, β ∈Fpn be linearly independent over Fp, and let f (x) = Trn(x2), h1(x) = Trn(αx2), h2(x) = Trn(βx2). Then the bent function F : Fpn×F2 p →Fp F(x, y1, y2) = f (x) + (y1 + h1(x))(y2 + h2(x)) is in general non-weakly regular. As pointed out in the next section, the sporadic examples g1, g2 given as above are in fact not weakly-normal, and the construction in [8] potentially yields not weakly-normal bent functions. is in general non-weakly regular. As pointed out in the next section, the sporadic examples g1, g2 given as above are in fact not weakly-normal, and the construction in [8] potentially yields not weakly-normal bent functions. 3 Testing normality In particular, for p = 3, p i=1(ai +U) is an affine subspace if and only if a1 +a2 +a3 = 0. In particular, for p = 3, p i=1(ai +U) is an affine subspace if and only if a1 +a2 +a3 = 0. Proof First assume that {a1, a2, . . . , ap} is an affine subspace which w.l.o.g. we can write as a1 + ⟨a0⟩with a0 = a2 −a1. Then p i=1 (ai + U) = p−1 t=0 (a1 + t(a2 −a1) + U) = a1 + p−1 i=0 (t(a2 −a1) + U) = a1 + ⟨a0⟩+ U. Since 0 ̸= a0 = a2 −a1 ∈Uc implies a0 ̸∈U, the dimension of U′ := ⟨a0⟩+ U is s + 1. p Since 0 ̸= a0 = a2 −a1 ∈Uc implies a0 ̸∈U, the dimension of U′ := ⟨a0⟩+ U is s + 1. Conversely, let the union p i=1(ai + U) = a1 + U′ be an affine subspace for some pairwise distinct a1, . . . , ap ∈Uc. Again we have a0 = a2 −a1 ̸∈U, but a2 −a1 ∈U′. Hence we can write U′ as ⟨a0⟩+ U. For 1 < s ≤p we can write the element as −a1 of U′ as as −a1 = u + ta0 for some t ∈Fp and u ∈U. Hence γ = as −a1 −ta0 = u ∈U. Since 0 ̸= a0 = a2 −a1 ∈Uc implies a0 ̸∈U, the dimension of U′ := ⟨a0⟩+ U is s + 1. Conversely let the union p 1(ai + U) = a1 + U′ be an affine subspace for some Conversely, let the union p i=1(ai + U) = a1 + U′ be an affine subspace for some pairwise distinct a1, . . . , ap ∈Uc. Again we have a0 = a2 −a1 ̸∈U, but a2 −a1 ∈U′. Hence we can write U′ as ⟨a0⟩+ U. For 1 < s ≤p we can write the element as −a1 of U′ as as −a1 = u + ta0 for some t ∈Fp and u ∈U. Hence γ = as −a1 −ta0 = u ∈U. Since γ ∈Uc we must have γ = u = 0, and hence as = a1 + ta0. 3 Testing normality It is not easy to show (weak) normality for a given function, and it is even harder to disprove (weak) normality. There is no approach known, how to prove non-weak-normality by hand. In [2, 14], to show the non-weak-normality of some Boolean bent function in dimension 10,12,14, a computer algorithm is used, see [2]. In this section, based on the principles of the algorithm for Boolean functions in [2], we develop an algorithm for p-ary functions. Similarly as in [2] for Boolean functions, the strategy is to combine cosets of a subspace U of dimension s on which f is a fixed constant c to an affine subspace of dimension s + 1 on which f is constant c. Differently to the Boolean case, where the union of two cosets of a linear subspace U is always an affine subspace, the union of p cosets of a subspace U of Fn p is in general not an affine subspace. Hence the algorithm in [2] does not transfer straightforward to p-ary functions. To generate a complete list of the cosets of a subspace U (without repetitions) we fix a complement Uc of U. We then get a partition of Fn p into cosets of U as {a + U : a ∈Uc}. We will use the following two simple lemmas for which we include the proof for the convenience of the reader. Cryptogr. Commun. (2018) 10:1 –1049 037 1044 Lemma 3 Let U be a linear subspace of Vn = Fn p of dimension s < n, let Uc be a complement of U and let a1, a2, . . . , ap be distinct elements of Uc. Then the union Lemma 3 Let U be a linear subspace of Vn = Fn p of dimension s < n, let Uc be a complement of U and let a1, a2, . . . , ap be distinct elements of Uc. Then the union p i=1 (ai + U) p i=1 (ai + U) p i=1 (ai + U) is an affine subspace a1 + U′ of dimension s + 1, if and only if {a1, a2, . . . , ap} is an affine subspace {a1 + (a2 −a1)t : 0 ≤t ≤p −1} of Uc. Then a1 + U′ = a1 + ⟨a2 −a1⟩+ U. a1 + U′ = a1 + ⟨a2 −a1⟩+ U. 3 Testing normality Lemma 4 Let f be a function from Vn to Fp and A = a1 + U′ be an affine subspace of dimension s + 1 ≤n of Vn. Then the restriction of f to A is affine but nonconstant if and only if U′ = ⟨a0⟩+ U such that f is constant on each coset (a1 + ta0) + U of U, and affine (but nonconstant) on a1 + ⟨a0⟩. Remark 1 The function f is then constant on the cosets a1 + ta0 + U of U, 0 ≤t ≤p −1, with pairwise distinct constants for pairwise distinct 0 ≤t1, t2 ≤p −1. For the special case that p = 3, the condition in Lemma 4 simplifies: The function f is affine (but not constant) on a+U if and only if a+U is the union of three affine subspaces a1 +U′, a2 +U′, a3 +U′ for a subspace U′ of Fm 3 of dimension s −1, such that f\|(a1+U′) = c, f\|(a2+U′) = c + 1 and f\|(a3+U′) = c + 2. Remark 1 The function f is then constant on the cosets a1 + ta0 + U of U, 0 ≤t ≤p −1, with pairwise distinct constants for pairwise distinct 0 ≤t1, t2 ≤p −1. For the special case that p = 3, the condition in Lemma 4 simplifies: The function f is affine (but not constant) on a+U if and only if a+U is the union of three affine subspaces a1 +U′, a2 +U′, a3 +U′ for a subspace U′ of Fm 3 of dimension s −1, such that f\|(a1+U′) = c, f\|(a2+U′) = c + 1 and f\|(a3+U′) = c + 2. Proof of the Lemma Let f be affine on A, i.e. there exists a linear function L such that f (a1 + u′) = L(u′) + f (a1) for u′ ∈U′. Since we suppose that f is not constant on A, the linear function L is not the zero-function on U′, hence has an s-dimensional kernel U in U′. We can write U′ as U′ = ⟨a0⟩+ U for some a0 ∈U′ \ U, and observe that for all t ∈Fp and u ∈U, f (a1 + ta0 + u) = L(ta0 + u) + f (a1) = tL(a0) + L(u) + f (a1) = tL(a0) + f (a1). 3 Testing normality In particular, f is affine on a1 + ⟨a0⟩, and constant tL(a0) + f (a1) on a1 + ta0 + U for every fixed t. f (a1 + ta0 + u) = L(ta0 + u) + f (a1) = tL(a0) + L(u) + f (a1) = tL(a0) + f (a1). In particular, f is affine on a1 + ⟨a0⟩, and constant tL(a0) + f (a1) on a1 + ta0 + U for every fixed t. y Conversely let A = a1 + ⟨a0⟩+ U, and suppose that f is constant on (a1 + ta0) + U for every fixed 0 ≤t ≤p−1, and affine on a1 +⟨a0⟩. Then for some linear function L we have f (a1 + ta0 + u) = f (a1 + ta0) = tL(a0) + f (a1), Hence f is affine on a1 + U′. (Note that U is in the kernel of L). Hence f is affine on a1 + U′. (Note that U is in the kernel of L). 1045 Cryptogr. Commun. (2018) 10:1 –1049 037 Lemma 3 and Lemma 4 suggest the following procedure to construct an affine subspace of dimension s+1 on which f : Fn p →Fp is constant, from such affine subspaces of dimen- sion s. For a linear subspace U of dimension s fix a complement Uc and find a1, . . . , ap ∈ Uc such that f is constant with the same c on all affine subspaces a1 + U, . . . , ap + U. If {a1, . . . ap} form a one-dimensional affine subspace, then take the union of those cosets. Note that this union then equals a1 + U′ with U′ = ⟨U, a2 −a1⟩. (In the following we use the term 1-flat for a one-dimensional affine subspace.) We applied our algorithm to several known bent functions, and observed that many of them are in fact weakly k-normal with k as large as the theory allows. But we also found Cryptogr. Commun. (2018) 10:1 –1049 037 1046 examples with a different behaviour. We collect some of the experimental results, which we find interesting in the following. For the first two examples we choose bent functions which have maximal possible normality. The other functions we present below do not meet the upper bound on k-normality. examples with a different behaviour. We collect some of the experimental results, which we find interesting in the following. For the first two examples we choose bent functions which have maximal possible normality. The other functions we present below do not meet the upper bound on k-normality. I The weakly regular and not regular Coulter-Matthews bent function Tr6(ξ3x(37+1)/2)) from F36 to F3, where ξ is a primitive element of F36, is 2-norm I The weakly regular and not regular Coulter-Matthews bent function I The weakly regular and not regular Coulter-Matthews bent function Tr6(ξ3x(37+1)/2)) from F36 to F3, where ξ is a primitive element of F36, is 2-normal. 138 24 184 336 Tr6(ξ3x(37+1)/2)) from F36 to F3, where ξ is a primitive element of F36, is 2-norm 138 24 184 336 Tr6(ξ3x(37+1)/2)) from F36 to F3, where ξ is a primitive element of F36, is 2-normal. II The regular bent function in dimension 4, Tr4(ξ138x24 + ξ184x336), from F54 to F5,where ξ is a primitive element of F54, is 2-normal (Ex.7.1 in [15]). II The regular bent function in dimension 4, Tr4(ξ138x24 + ξ184x336), from F54 to F5,where ξ is a primitive element of F54, is 2-normal (Ex.7.1 in [15]). III The weakly regular Coulter-Matthews bent function in odd dimension 7, Tr7(ξ6x(39+1)/2)), where ξ is a primitive element of F37, is 2-normal but not (weakly) 3-normal. IV The weakly regular Coulter-Matthews bent function in odd dimension 9, Tr9(ξ5x(311+1)/2)), where ξ is a primitive element of F39, is 3-normal but not (weakly) 4-normal. Tr9(ξ5x(311+1)/2)), where ξ is a primitive element of F39, is 3-normal but not (weakly) 4-normal. V The non-weakly regular bent function g1 : F36 →F3 with g1(x) = Tr6(ξ7x98) where ξ is a primitive element of F36, is not (weakly) normal. 7 14 V The non-weakly regular bent function g1 : F36 →F3 with g1(x) = Tr6(ξ7x98) where ξ is a primitive element of F36, is not (weakly) normal. 3 VI The non-weakly regular bent function g2 : F36 →F3 with g2(x) = Tr6(ξ7x14 + ξ35x70), where ξ is a primitive element of F36, is not (weakly) normal. VII The non-weakly regular bent function F : F34×F2 3 →F3 with F(x, y1, y2) = Tr4(x2) + (y1 + Tr4(ξ73x2))(y2 + Tr4(ξ76x2)), where ξ is a primitive element of F34, is not (weakly) normal. Examples III and IV are both bent functions in odd dimension, which are not (weakly) (n−1)/2-normal. As our experimental results indicate, being solely ((n−1)/2−1)-normal seems to be the typical behaviour of Coulter-Matthews bent functions in odd dimension. To the best of our knowledge, the last three examples are the first (non-binary) examples of bent functions in even dimension (not in the class of weakly regular but not regular bent functions) which are shown to be not (weakly) normal. Though we do not see a causal relationship between bent functions without a bent dual and non-normality, we note that all functions in V,VI,VII are non-weakly regular bent functions for which the dual is not bent, see [6, 8]. Once a bent function in dimension n is known which is not (weakly) k-normal for some k, we can construct bent functions in any dimension N = n+2s, s ≥1, that is not (weakly) (k + s)-normal, applying the following lemma which is a generalization of Lemma 25 in [2] for Boolean functions in dimension n and k = n/2. In particular we can construct not weakly normal (not weakly (N −1)/2, N/2 −1-normal) bent functions in dimension N, starting from such bent functions in dimension n. Lemma 5 For a p-ary function f : Fn p →Fp the following properties are equivalent. (1) f is (weakly) k-normal, (2) g : Fn p×F2 p →Fp given by g(x, y, z) = f (x) + yz is (weakly) (k + 1)-normal. In particular, f is (weakly) normal if and only if g is weakly normal (n even). Lemma 5 For a p-ary function f : Fn p →Fp the following properties are equivalent. (2) g : Fn p×F2 p →Fp given by g(x, y, z) = f (x) + yz is (weakly) (k + 1)-normal. In particular, f is (weakly) normal if and only if g is weakly normal (n even). In particular, f is (weakly) normal if and only if g is weakly normal (n even). Proof First suppose that f is (weakly) k-normal, and E is a k-dimensional affine sub- space restricted to which f is constant (affine). Then g is constant (affine) on the (k + 1)-dimensional affine subspace E′ = {(x, y, 0) : x ∈E, y ∈Fp} of Fn p×F2 p. 1047 Cryptogr. Commun. (2018) 10:1 –1049 037 Conversely suppose that g is weakly (k + 1)-normal, and let E′ = w + U′, w = (w1, w2, w3), be a (k + 1)-dimensional affine subspace of Fn p×F2 p restricted to which g is constant or affine. Then for (x, y, z) ∈E′ we have g(x, y, z) = ⟨γ, x⟩+ αy + βz + c (4) F F b F d fi g(x, y, z) = ⟨γ, x⟩+ αy + βz + c (4) ∈Fp. For a, b ∈Fp define g(x, y, z) = ⟨γ, x⟩+ αy + βz + c (4) for some γ ∈Fn p, α, β, c ∈Fp. For a, b ∈Fp define Ea,b = {x∈Fn p : (x, a, b) ∈E′}. (5) g(x, y, z) = ⟨γ, x⟩+ αy + βz + c (4) g(x, y, z) = ⟨γ, x⟩+ αy + βz + c (4) Ea,b = {x∈Fn p : (x, a, b) ∈E′}. (5) (5) If ¯x ∈Ea,b, then Ea,b = ¯x + U, where U is the subspace of Fn p given by U = {x∈Fn p : (x, 0, 0) ∈U′} (straightforward). Observe that restricted to Ea,b, the function If ¯x ∈Ea,b, then Ea,b = ¯x + U, where U is the subspace of Fn p given by U = {x∈Fn p : (x, 0, 0) ∈U′} (straightforward). Observe that restricted to Ea,b, the function f (x) −⟨γ, x⟩= αa + βb + c −ab (6) f (x) −⟨γ, x⟩= αa + βb + c −ab (6) is constant. If U has dimension k we are done. Suppose that dim(U) < k. Since E′ is the union a,b{(x, a, b) : x ∈Ea,b} (some Ea,b may be the same, some the empty set), we have pk+1 = \|E′\| ≤ a,b \|Ea,b\|. As we assume that dim(U) < k, this implies that dim(U) = k −1, i.e. \|Ea,b\| = pk−1 for all (a, b)∈F2 p and all Ea,b are distinct. We then define E as the disjoint union E = a∈Fp Ea,α = ¯x + ¯U E = a∈Fp Ea,α = ¯x + ¯U for an element ¯x ∈E, where ¯U = {x∈Fn p : (x, a, 0) ∈E′ for some a ∈Fp}, and observe that f (x) −⟨γ, x⟩= βα + c is constant on this k-dimensional affine subspace. for an element ¯x ∈E, where ¯U = {x∈Fn p : (x, a, 0) ∈E′ for some a ∈Fp}, and observe that f (x) −⟨γ, x⟩= βα + c is constant on this k-dimensional affine subspace. Combining our sporadic examples in low dimension with Lemma 5 we get the following result. Theorem 1 There are not (weakly) normal ternary bent functions, which do not belong to the class of bent functions which are weakly regular but not regular, in every even dimension n ≥6. We remark that since our sporadic examples, Examples V, VI, VII, are non-weakly reg- ular bent functions for which the dual is not bent, by [8, Theorem 2] all bent functions obtained from these functions with Lemma 5 also have this property. To the best of our knowledge, no example for a regular p-ary bent function which is not (weakly) normal is known. 4 Perspectives In this article we contribute to the analysis of k-normality for p-ary bent functions. Depend- ing on the regularity of a bent function f from Vn to Fp and the parity of n, many bent functions seem to be (weakly) normal, (n/2−1)-normal or (n−1)/2-normal, which is dras- tically different from the average behaviour of a p-ary function. It seems not easy to find bent functions for which one can show a different behaviour. This resembles the situation for Boolean bent functions. We develop an algorithm for testing normality for p-ary functions. Applying this algorithm we verify that some ternary non-weakly regular bent functions in Cryptogr. Commun. (2018) 10:1 –1049 037 1048 even dimension n are not weakly normal. For odd dimension n we found examples in the class of Coulter-Matthews bent functions which are not weakly (n −1)/2-normal. With Lemma 5 we then can construct from such functions in dimension n, bent functions with the same property in any dimension n + 2s, s ≥1. even dimension n are not weakly normal. For odd dimension n we found examples in the class of Coulter-Matthews bent functions which are not weakly (n −1)/2-normal. With Lemma 5 we then can construct from such functions in dimension n, bent functions with the same property in any dimension n + 2s, s ≥1. There are many interesting open questions on normality for p-ary bent functions. We close with a collection of some of them, which can now be attacked using our presented algorithm. – Find regular p-ary bent functions in even dimension which are not normal. – Find regular p-ary bent functions in even dimension which are not normal. – Find weakly regular but not regular p-ary bent functions in even dimension which are not (n/2 −1)-normal. – Find weakly regular but not regular p-ary bent functions in even dimension which are not (n/2 −1)-normal. To the best of our knowledge there are no such examples known. – Show that the weakly regular but not regular Coulter-Matthews bent functions in even dimension are (n/2 −1)-normal or find counter-examples. – Show that the weakly regular but not regular Coulter-Matthews bent functions in even dimension are (n/2 −1)-normal or find counter-examples. The question on the average behaviour of Boolean and p-ary bent functions with respect to normality seems not easy to be answered. 4 Perspectives Are (most) bent functions affine on affine subspaces of large dimension, or do they behave like arbitrary Boolean and p-ary functions, normal, (n/2 −1)-normal, ((n −1)/2)-normal bent functions are only easier to find? Acknowledgements Open access funding provided by Johannes Kepler University Linz. Wilfried Me s supported by the Austrian Science Fund (FWF) Project no. M1767-N26. ´ pp y j ´Isabel Pirsic is supported by the Austrian Science Fund (FWF) Project no. 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https://openalex.org/W2254511209	https://www.matec-conferences.org/articles/matecconf/pdf/2015/13/matecconf_isot2015_01003.pdf	English	null	MEMS acceleration sensor with remote optical readout for continuous power generator monitoring	MATEC web of conferences	2,015	cc-by	3,220	Maurizio Tormen1,a, Branislav Timotijevic1, Yves Pétremand1, Markus Lützelschwab1, Dara Zaman Bayat1, Yvan Jacqu Laurent Aebi2 ,a, Branislav Timotijevic1, Yves Pétremand1, Markus Lützelschwab1, Dara Zaman Bayat1, Yvan Jacquat2 , Maurizio Tormen1,a, Branislav Timotijevic1, Yves Pétremand1, Markus Lützelschwab1, Dara Zaman Bayat1, Laurent Aebi2 1CSEM SA, Rue J. Droz 1, 2002 Neuchatel, Switzerland 2MC-Monitoring SA, Route André Piller 19, 1762 Givisiez, Switzerland 1CSEM SA, Rue J. Droz 1, 2002 Neuchatel, Switzerland 2MC-Monitoring SA, Route André Piller 19, 1762 Givisiez, Switzerland Abstract. Miniaturized accelerometers with remote optical readout are required devices for the continuous monitoring of vibrations inside power generators. In turbo and hydro generators, end-winding vibrations are present during operation causing in the long term undesirable out-of-service repairs. Continuous monitoring of these vibrations is therefore mandatory. The high electromagnetic fields in the generators impose the use of devices immune to electromagnetic interferences. In this paper a MEMS based accelerometer with remote optical readout is presented. Advantages of the proposed device are the use of a differential optical signal to reject the common mode signal and noise, the reduced number of steps for the MEMS chip fabrication and for the system assembly, and the reduced package volume. FAS sensors are used as well in numerous other applications including rotating machinery, high voltage transformers, circuit breakers, high voltage electrical transmission lines, explosive atmospheres, etc. System monitoring leads to operating costs advantages, such as optimal planning of maintenance tasks, reduction of unplanned downtime, increase of the machine availability. DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2015 / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 0 3 , 3 2 0 00 2 1 1 3 3 DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2015 / 0 0 ( 2015) 201 conf Web of Conferences 5 MATEC atec m 0 3 , 3 2 0 00 2 1 1 3 3 a Corresponding author: maurizio.tormen@csem.ch 1 Introduction Miniaturized accelerometers with remote optical readout are required devices for the continuous monitoring of vibrations inside power generators. In turbo and hydro generators, end-winding vibrations are present during operation at twice the electrical synchronous frequency of the generator. High vibrations can lead to loosening of entire end-winding support system, wear of insulation material, rupture of coil and fatigue cracking of conductor, all of which require out-of- service repairs. Continuous monitoring of these vibrations is therefore mandatory and the high electromagnetic fields in the generators impose the use of devices by design non-conductive and immune to electromagnetic interferences. A number of different approaches to FAS have been explored: a general overview can be found in [1]. Recent works have mainly explored solutions based on Grating filters ([2, 3], Fabry-Perot filters [4], Michelson interferometer [5]. In this paper a MEMS based accelerometer with remote optical readout is presented. Advantages compared to state of the art devices for such application are a simplified electronics when compared to wavelength or phase based measurement systems, high rejection of the common mode signal and high measurement linearity thanks to a differential intensity signal approach, low cost MEMS chip given the reduced number of fabrication steps and the number of chips per wafer (more than 400 on a 6” wafer), low cost and high yield system assembly thanks to a simple and robust assembly process, low barriers to market-entry since the reduced package volume is compatible with the already allocated space in turbo generators. For electromagnetic immunity, no metal is present in the sensor head. Existing solutions in the market are based on manual assembly of a relevant number of micro-machined and micro-optical elements, often implying limited frequency range available for monitoring due to relatively high masses and, in terms of production, high manufacturing costs and low yields. A Fiber-optic Acceleration Sensor (FAS) is by design non-conductive and immune to electromagnetic interferences. Its passive principle makes it ideal for shock and vibration measurements in areas where conventional accelerometers (e.g. piezoelectric or MEMS with metal wiring) may create electrical discharges, cause changes in magnetic field distribution, prove hazardous to personnel, and impair reliable operation. FAS sensors are therefore an optimal solution to monitor end-winding vibration during generator operation. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.matec-conferences.org or http://dx.doi.org/10.1051/matecconf/20153201003 2 Opto-mechanical design MEMS optical accelerometer layout. Figure 3. (Left) Simulation of the relative signals at the output fiber ends as a function of MEMS mirror lateral displacement. (Right) Differential signal at the two output fiber ends (gray line) and its linear fit (black line). The seismic mass and the springs were designed in order to provide sufficiently high resonance frequencies for the specific application: one family has the first resonance at 1 kHz, the second at 3 kHz (Table 1). The lateral displacement of the mirror was design to be 10 m and 1m, respectively, at an acceleration of 40g. 2 Opto-mechanical design The MEMS chip is composed of a seismic mass, The MEMS chip is composed of a seismic mass, a Corresponding author: maurizio.tormen@csem.ch This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MATEC Web of Conferences suspended through springs, with opened regions to introduce a damping gel, of a 2-facet mirror, moving with the seismic mass, redirecting the optical signal from an input fiber into two output fibers. In order to reduce the system dimensions the two output fibers were not placed at 90 deg in respect to the input fiber as in [1], but at a shallower angle, 70 deg or 45 deg. Figure 1 reports the layout of the basic chip, with the input fiber and the two output fibers. separately in remote (in Figure 3 left, the simulated power expected at the two output fibers ends, as a function of the lateral displacement of the mirror). The acceleration measurement is derived from the difference of the two signals (in Figure 3 right). Deviation from linearity is expected to be less than 1% for displacements up to 10 μm for accelerations up to 40g. Analytical simulations and Zemax ray-tracing were performed in order to optimize the optical signal as a function of geometrical parameters of the system. Figure 1. MEMS optical accelerometer layout. g p y 0 10 20 30 40 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Lateral displacement of the mass (um) Relative intensity (Power in the input fiber = 1) 0 10 20 30 40 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Lateral displacement of the mass (um) Differential signal and linear fit Figure 3. (Left) Simulation of the relative signals at the output fiber ends as a function of MEMS mirror lateral displacement. (Right) Differential signal at the two output fiber ends (gray line) and its linear fit (black line). 0 10 20 30 40 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Lateral displacement of the mass (um) Relative intensity (Power in the input fiber = 1) 0 10 20 30 40 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Lateral displacement of the mass (um) Differential signal and linear fit 0 10 20 30 40 Lateral displacement of the mass (um) Figure 1. 3 Microfabrication process The microfabrication process (Figure 4) starts with 6” SOI (Silicon-On-Insulator) wafers with the following thicknesses: Handle layer 500 μm, buried oxide layer 3 μm and device layer 120 μm. The process has been designed to have a single mask and DRIE step in order to reduce the process time and cost. The structures are patterned via photolithography with AZ 9260 photoresist. The obtained mask is transferred into silicon by DRIE etching through the entire device layer down to the buried oxide. Table 1 List of the investigated FAS family designs. First resonance frequency [Hz] Mass displacement [m] at 40g Design 1 1’000 10 Design 2 3’000 1 Table 1 List of the investigated FAS family designs. Table 1 List of the investigated FAS family designs. In absence of acceleration the signals in the output fibers are nominally identical (Figure 2, on the left); when the seismic mass is subjected to acceleration, the lateral displacement of the mirror induces an increase of optical power in one fiber and a reduction in the second one (Figure 2, on the right). Figure 4. Schematic process flow of the MEMS optical accelerometer chip (cross-section view). Figure 2. Ray-tracing simulation of the optical signal arriving from the input fiber (central one) and redirected by the moving mirror into the 2 output fibers (external ones) in absence (left) and presence (right) of acceleration. Figure 2. Ray-tracing simulation of the optical signal arriving from the input fiber (central one) and redirected by the moving mirror into the 2 output fibers (external ones) in absence (left) and presence (right) of acceleration. Figure 4. Schematic process flow of the MEMS optical accelerometer chip (cross-section view). After stripping the remaining photoresist and cleaning the wafer, the buried oxide is partially etched to release the The signals from the two output fibers are collected 01003-p.2 01003-p.2 ISOT 2015 operations. The output fibers are placed at an angle of α (70 deg or 45 deg) in respect to the central input fiber (Figure 7, right). The fiber alignment is performed using a six axis alignment stage (F-206 from PI) with an arbitrary pivot point. To facilitate the fiber insertion the MEMS chip hosts some fiber alignment features; on top of that a pre-tilting of the fiber during insertion was introduced to slide the fibers into their respective channels on the MEMS chip. 3 Microfabrication process The PEEK base features as well channels for the guidance of the three fibers. The outer two channels are curved with radii, r, so that the minimal bending radius of the fiber can be maintained during alignment and operation. The radius was chosen as small as possible to reduce the overall package size and at the same time reduce the bending losses to an acceptable level (0.3-0.4 dB). movable structures (Figure 5). movable structures (Figure 5). Figure 5. SEM picture of the fabricated device. Figure 6. SEM image of the 2-facet movable mirror redirecting the optical signal and the U-grooves for optical fiber alignment. Figure 5. SEM picture of the fabricated device. Figure 5. SEM picture of the fabricated device. For mechanical protection, a glass lid is added to the package at a final step. Figure 8 shows the fully assembled device with connectorized fibers for ready for the characterization. Figure 7. Left. Assembled device. Right: geometrical features of the PEEK package. Figure 6. SEM image of the 2-facet movable mirror redirecting the optical signal and the U-grooves for optical fiber alignment. The technique used is a timed quasi-dry etching by vapour HF. During this process step, it is necessary to sufficiently etch the oxide in order to release the movable structures, but not to overetch it, to maintain intact the oxide supporting the not-moving parts of the device layer. Surface treatments are introduced to polish the lateral surfaces in order to optimize the mirror reflectivity (Figure 6). A layer of photoresist is then poured on the surface of the wafer in order to protect the structures during dicing for singulation of the chips. The process continues at chip level with a final cleaning procedure to remove the protective photoresist: at this point the chip is ready for the assembly with the optical fibers in the package. Figure 7. Left. Assembled device. Right: geometrical features of the PEEK package. Figure 8. Fully assembled and connectorized optical accelerometer sensor. 4 Packaging The package provides a solid base for the interface between the MEMS device and the optical fibers. In terms of possible materials for the package, metal parts are excluded because of the strong electromagnetic fields present in the generators; moreover temperatures of up to 155°C and a hydrogen rich atmosphere (5bars) represent further constraints in the choice of the package material. Finally, hermetic package is not required for the applications. Based on this input, PEEK was chosen because of its physical and chemical properties as well as its adequate machinability. Figure 8. Fully assembled and connectorized optical accelerometer sensor. References In order to flatten the frequency response in the frequency range of interest (10Hz-400Hz for the first family, and 50Hz-1500Hz for the second family), the second generation of devices will include damping structures. 4 . Zh on g-zh u Yan g ; Ho n g Lu o an d Sh u i- d on g Xion g " High sen sitivity fib er op tic acceler o m eter b ased on fo ld in g F-P cavity ", Pr oc. SPIE 8 9 1 4 , In ter n ation a l Sym p o siu m on Ph oto electr on ic Detection an d Im agin g 2 0 1 3 : Fib er Op tic Sen sor s an d Op tical Coh er en ce Tom ogr ap h y, 8 9 1 4 1 1 (Au gu st 2 9 , 2 0 1 3 ); 4 . Zh on g-zh u Yan g ; Ho n g Lu o an d Sh u i- d on g Xion g " High sen sitivity fib er op tic acceler o m eter b ased on fo ld in g F-P cavity ", Pr oc. SPIE 8 9 1 4 , In ter n ation a l Sym p o siu m on Ph oto electr on ic Detection an d Im agin g 2 0 1 3 : Fib er Op tic Sen sor s an d Op tical Coh er en ce Tom ogr ap h y, 8 9 1 4 1 1 (Au gu st 2 9 , 2 0 1 3 ); The dynamic range is reported in Figure 10 for the two families (continuous line for family 1, dashed line for family 2). Family 1 has a better dynamic range at lower frequency down to 0.2g. At 0.2g the deviation from 7g is still below 5% while family 2 presents more than 30% deviation. Below 0.2g the signal to noise ratio comes close 1. Figure 10. Dynamic range analysis of two chips from the two families presented in Table 1. ) 5 . References 1 . B. Gu ld im an n , ‘‘Micr om ach in ed fib er op tic acceler om eter b ased o n in ten sity m od u lation ’’, Ph D th esis, Un iver sity of Neu ch atel, 2 0 0 1 . The frequency response for two chips from the two different families is reported in Figure 9. p g Figure 9. Frequency response of two chips from the two families presented in Table 1. 2 . Ch on gyu Lin ; Hon g Lu o ; Sh u id on g Xion g an d Haitao Li; "In vestigation on a fib er op tic acceler o m eter b ased on FBG- FP in ter fer om eter ", Pr oc. SPIE 9 2 9 7 , In ter n ation al Sy m p osiu m on Op toelectr o n ic Tech n ology an d Ap p lication 2 0 1 4 : La ser an d Op tical Mea su r em en t Tech n o logy; an d Fib er Op tic Sen sor s, 9 2 9 7 3 5 (Decem b er 3 , 2 0 1 4 ); 2 . Ch on gyu Lin ; Hon g Lu o ; Sh u id on g Xion g an d Haitao Li; "In vestigation on a fib er op tic acceler o m eter b ased on FBG- FP in ter fer om eter ", Pr oc. SPIE 9 2 9 7 , In ter n ation al Sy m p osiu m on Op toelectr o n ic Tech n ology an d Ap p lication 2 0 1 4 : La ser an d Op tical Mea su r em en t Tech n o logy; an d Fib er Op tic Sen sor s, 9 2 9 7 3 5 (Decem b er 3 , 2 0 1 4 ); Figure 9. Frequency response of two chips from the two families presented in Table 1. 3 . Yeon -Gwan Lee et al, "Per for m an ce of a sin gle r eflective gr a tin g-b a sed fib er op tic acceler om eter ’’ 2 0 1 2 M eas. Sci. T ech n ol. 2 3 0 4 5 1 0 1 . 5 Characterization Table 2 reports the comparison between the simulated resonance frequencies and the ones experimentally obtained for the two designs presented in Table 1. Two different techniques were used to measure the resonance frequency. The first technique uses a shaker, a reference piezo sensor with its charge amplifier and a function generator to have a complete frequency response; the second technique makes the device vibrate at increasing frequencies with a piezo actuator and the resonance frequency is in correspondence with the maximum Figure 7 (left) shows an assembled device. The optical fibers are completely molded in UV curable adhesive, thereby providing an effective strain relief. Without it, the fibers would exert a detrimental force on the MEMS chip during both the handling as well as 01003-p.3 MATEC Web of Conferences displacement of the seismic mass. displacement of the seismic mass. 6 Conclusions Table 2. Comparison of the resonant frequency obtained, numerically and experimentally. Table 2. Comparison of the resonant frequency obtained, numerically and experimentally. In the present paper a MEMS based accelerometer with remote optical readout is presented. First results showed high rejection of the common mode signal thanks to a differential intensity signal approach, low cost MEMS chip given the reduced number of fabrication steps and the number of chips per wafer, low cost and high yield system assembly thanks to a simple and robust assembly process. Simulated resonance [Hz] First setup Second setup Design 1 989 849 837 Design 2 3132 2674 2645 Future work foresees the introduction of damping structures to improve the flatness of the frequency response in the frequency range of interest for the measurement. A difference of ~1% in measured resonance frequency is obtained between the two experimental methods. A more significant variation (~15%) is present between the numerical simulations and the experimental results. From an application point of view, despites the mentioned discrepancy, the obtained results for the resonance frequency are in line with the requirements. References Fen g Pen g, Ju n Yan g, Bin g Wu , Yon ggu i Yu an , Xin glian g Li, Ai Zh ou , an d Lib o Yu an , "Com p act fib er op tic acceler o m eter ," Ch in . Op t. Lett. 1 0 , 0 1 1 2 0 1 - (2 0 1 2 ) Figure 10. Dynamic range analysis of two chips from the two families presented in Table 1. 01003-p.4
https://openalex.org/W2801124857	https://escholarship.org/content/qt2xp8x53c/qt2xp8x53c.pdf?t=rh0h3r	English	null	Nutritional Management of Chronic Kidney Disease	New England journal of medicine/The New England journal of medicine	2,018	cc-by	2,854	UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine UC Irvine Previously Published Works Title Nutritional Management of Chronic Kidney Disease Permalink https://escholarship.org/uc/item/2xp8x53c Journal New England Journal of Medicine, 378(6) ISSN 0028-4793 Authors Kalantar-Zadeh, Kamyar Fouque, Denis Publication Date 2018-02-08 DOI 10.1056/nejmc1715765 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Nutritional Management of Chronic Kidney Disease Permalink https://escholarship.org/uc/item/2xp8x53c Journal New England Journal of Medicine, 378(6) ISSN 0028-4793 Authors Kalantar-Zadeh, Kamyar Fouque, Denis Publication Date 2018-02-08 DOI 10.1056/nejmc1715765 Copyright Information This work is made available under the terms of a Creative Commons License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Peer reviewed Peer reviewed Correspondence tension: a mimic of the Chiari I malformation. AJNR Am J Neuro- radiol 2012;33:1901-6. 2. Friedman DI, Liu GT, Digre KB. Revised diagnostic criteria for the pseudotumor cerebri syndrome in adults and children. Neurology 2013;81:1159-65. 2. Friedman DI, Liu GT, Digre KB. Revised diagnostic criteria for the pseudotumor cerebri syndrome in adults and children. Neurology 2013;81:1159-65. 5. Mader TH, Gibson CR, Pass AF, et al. Optic disc edema, globe flattening, choroidal folds, and hyperopic shifts observed in astronauts after long-duration space flight. Ophthalmology 2011;118:2058-69. 3. Alperin N, Ranganathan S, Bagci AM, et al. MRI evidence of impaired CSF homeostasis in obesity-associated idiopathic intra- cranial hypertension. AJNR Am J Neuroradiol 2013;34:29-34. 4. Aiken AH, Hoots JA, Saindane AM, Hudgins PA. Incidence of cerebellar tonsillar ectopia in idiopathic intracranial hyper- DOI: 10.1056/NEJMc1716067 1. Kalantar-Zadeh K, Fouque D. Nutritional management of chronic kidney disease. N Engl J Med 2017;377:1765-76. 2. Kopple JD, Shinaberger JH, Coburn JW, Sorensen MK, Rubini Powered by the California Digital Library University of California eScholarship.org n engl j med 378;6 nejm.org February 8, 2018 The New England Journal of Medicine Downloaded from nejm.org at Irvine (UCD) on July 25, 2022. For personal use only. No other uses without permission. Copyright © 2018 Massachusetts Medical Society. All rights reserved. Nutritional Management of Chronic Kidney Disease ME. Optimal dietary protein treatment during chronic hemo- dialysis. Trans Am Soc Artif Intern Organs 1969;15:302-8. ME. Optimal dietary protein treatment during chronic hemo- dialysis. Trans Am Soc Artif Intern Organs 1969;15:302-8. ME. Optimal dietary protein treatment during chronic hemo- dialysis. Trans Am Soc Artif Intern Organs 1969;15:302-8. To the Editor: In their review article on the nu- tritional management of chronic kidney disease, Kalantar-Zadeh and Fouque (Nov. 2 issue)1 make some statements about daily acid production re- sulting from bicarbonate losses in the gut that may contain errors, in my view. Normally, there are no losses of bicarbonate in the gut unless a person has diarrhea. Furthermore, although the metabolism of carbohydrates and fats generates organic acids, these are eventually converted back to bicarbonate and do not add net acid to the body unless the organic anions are excreted in urine. 3. Kalantar-Zadeh K, Tortorici AR, Chen JL, et al. Dietary re- strictions in dialysis patients: is there anything left to eat? Semin Dial 2015;28:159-68. 4. Gutierrez A, Alvestrand A, Wahren J, Bergström J. Effect of in vivo contact between blood and dialysis membranes on pro- tein catabolism in humans. Kidney Int 1990;38:487-94. 5. Uribarri J. DOQI guidelines for nutrition in long-term peri- toneal dialysis patients: a dissenting view. Am J Kidney Dis 2001; 37:1313-8. DOI: 10.1056/NEJMc1715765 DOI: 10.1056/NEJMc1715765 To the Editor: Kalantar-Zadeh and Fouque dis- cuss an epidemiologic study in which higher urinary potassium excretion (as a proxy for in- take) was associated with a higher risk of pro- gression of chronic kidney disease.1 However, there is accumulating evidence from several studies that indicates otherwise (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org). In the stud- ies listed in the table, higher urinary potassium excretion was associated with better renal out- comes or lower all-cause mortality but not with hyperkalemia. Experimental data also suggest that potassium is renoprotective by reducing blood pressure, vascular calcification, and in- flammation.2-4 An ongoing multicenter, double- blind, placebo-controlled study, in which the effect of potassium supplementation on renal outcomes is being evaluated in patients with stage 3 or 4 chronic kidney disease (Clinical Trials.gov number, NCT03253172), is investi- gating whether potassium repletion is renopro- tective in patients with chronic kidney disease and whether repletion outweighs the risk of hyperkalemia. Jaime Uribarri, M.D. Nutritional Management of Chronic Kidney Disease Because patients with chronic kidney disease generally consume a low-potas- sium diet (approximately 2 g per day), supple- mentation in the study (1.5 g per day) is provided To the Editor: Kalantar-Zadeh and Fouque dis- cuss an epidemiologic study in which higher urinary potassium excretion (as a proxy for in- take) was associated with a higher risk of pro- gression of chronic kidney disease.1 However, there is accumulating evidence from several studies that indicates otherwise (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org). In the stud- ies listed in the table, higher urinary potassium excretion was associated with better renal out- comes or lower all-cause mortality but not with hyperkalemia. Experimental data also suggest that potassium is renoprotective by reducing blood pressure, vascular calcification, and in- flammation.2-4 An ongoing multicenter, double- blind, placebo-controlled study, in which the effect of potassium supplementation on renal outcomes is being evaluated in patients with stage 3 or 4 chronic kidney disease (Clinical Trials.gov number, NCT03253172), is investi- gating whether potassium repletion is renopro- tective in patients with chronic kidney disease and whether repletion outweighs the risk of hyperkalemia. Because patients with chronic kidney disease generally consume a low-potas- sium diet (approximately 2 g per day), supple- mentation in the study (1.5 g per day) is provided The authors advise a daily dietary protein in- take of 1.2 to 1.4 g per kilogram of body weight in patients undergoing dialysis. In my opinion, this would provide excessive protein consump- tion that is unsupported by data and, therefore, not warranted. A protein intake of approximate- ly 1 g per kilogram per day has been reported as being sufficient to maintain a positive nitrogen balance in stable patients undergoing dialysis.2 The idea that patients undergoing dialysis should have a higher protein intake derives from obser- vational data that have shown an association be- tween higher protein intake and better outcome and that have not been tested in interventional trials3 and from the belief that dialysis is a cata- bolic event beyond dialytic losses of amino acids and proteins.4,5 Jaime Uribarri, M.D. Icahn School of Medicine at Mount Sinai New York, NY jaime.uribarri@mssm.edu No potential conflict of interest relevant to this letter was re- ported. n engl j med 378;6 nejm.org February 8, 2018 The New England Journal of Medicine Downloaded from nejm.org at Irvine (UCD) on July 25, 2022. For personal use only. No other uses without permission. Copyright © 2018 Massachusetts Medical Society. All rights reserved. Nutritional Management of Chronic Kidney Disease 583 n engl j med 378;6 nejm.org February 8, 2018 The New England Journal of Medicine Downloaded from nejm.org at Irvine (UCD) on July 25, 2022. For personal use only. No other uses without permissio Copyright © 2018 Massachusetts Medical Society. All rights reserved. The new engl and jour nal of medicine to achieve the recommended daily potassium intake (3.5 g per day).5 to achieve the recommended daily potassium intake (3.5 g per day).5 medications that interfere with the renin–angio- tensin–aldosterone system is unknown. medications that interfere with the renin–angio- tensin–aldosterone system is unknown. Jeffrey S. Berns, M.D. Jeffrey S. Berns, M.D. Erasmus Medical Center Rotterdam, the Netherlands e.j.hoorn@erasmusmc.nl Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA bernsj@uphs.upenn.edu bernsj@uphs.upenn.edu No potential conflict of interest relevant to this letter was re- ported. Liffert Vogt, M.D., Ph.D. Academic Medical Center Amsterdam, the Netherlands 1. Gansevoort RT, de Zeeuw D, de Jong PE. Additive antipro- teinuric effect of ACE inhibition and a low-protein diet in human renal disease. Nephrol Dial Transplant 1995;10:497-504. 2. Ruilope LM, Casal MC, Praga M, et al. Additive antiprotein- uric effect of converting enzyme inhibition and a low protein intake. J Am Soc Nephrol 1992;3:1307-11. 1. Gansevoort RT, de Zeeuw D, de Jong PE. Additive antipro- teinuric effect of ACE inhibition and a low-protein diet in human renal disease. Nephrol Dial Transplant 1995;10:497-504. 1. Gansevoort RT, de Zeeuw D, de Jong PE. Additive antipro- teinuric effect of ACE inhibition and a low-protein diet in human renal disease. Nephrol Dial Transplant 1995;10:497-504. Joris I. Rotmans, M.D., Ph.D. 2. Ruilope LM, Casal MC, Praga M, et al. Additive antiprotein- uric effect of converting enzyme inhibition and a low protein intake. J Am Soc Nephrol 1992;3:1307-11. No potential conflict of interest relevant to this letter was re- ported. DOI: 10.1056/NEJMc1715765 1. He J, Mills KT, Appel LJ, et al. Urinary sodium and potassium excretion and CKD progression. J Am Soc Nephrol 2016;27:1202- 12. The authors reply: Uribarri writes that there is normally no fecal bicarbonate excretion. How- ever, in one stool-measurement study,1 the mean (±SD) fecal bicarbonate excretion in healthy adults was 34.6±12.3 mmol per liter, and another study reported a value of 30 mmol per liter.2 Hence, the minimum daily loss of gastrointesti- nal bicarbonate is 10 to 15 mmol per day. The incomplete oxidation of fats and carbohydrates leads to the generation of ketoacids and lactic acid. The bulk of these acids is recycled, but the resultant net hydrogen ion residue is added to the daily acid production. 2. Terker AS, Zhang C, McCormick JA, et al. Potassium modu- lates electrolyte balance and blood pressure through effects on distal cell voltage and chloride. Cell Metab 2015;21:39-50. 3. Sun Y, Byon CH, Yang Y, et al. Dietary potassium regulates vascular calcification and arterial stiffness. JCI Insight 2017 Octo- ber 5 (Epub ahead of print). 4. medications that interfere with the renin–angio- tensin–aldosterone system is unknown. Wang W, Soltero L, Zhang P, Huang XR, Lan HY, Adrogue HJ. Renal inflammation is modulated by potassium in chronic 4. Wang W, Soltero L, Zhang P, Huang XR, Lan HY, Adrogue HJ. Renal inflammation is modulated by potassium in chronic kidney disease: possible role of Smad7. Am J Physiol Renal Physiol 2007;293:F1123-F1130. 5. Palmer BF, Clegg DJ. Achieving the benefits of a high-potas- sium, paleolithic diet, without the toxicity. Mayo Clin Proc 2016; 91:496-508. 5. Palmer BF, Clegg DJ. Achieving the benefits of a high-potas- sium, paleolithic diet, without the toxicity. Mayo Clin Proc 2016; 91:496-508. DOI: 10.1056/NEJMc1715765 DOI: 10.1056/NEJMc1715765 We recommended a daily dietary protein in- take of 1.0 g or less per kilogram in persons at high risk for chronic kidney disease and 0.6 to 0.8 g per kilogram in those with more advanced stages of chronic kidney disease or proteinuria, including patients who are transitioning to dialy- sis therapy. For patients undergoing established maintenance dialysis who have minimal residual kidney function and undergo hemodialysis three times or more per week, we recommend a higher daily dietary protein intake of more than 1.0 g per kilogram, which is a range that ap- pears to be associated with the lowest mortal- ity.3 This amount is consistent with the average protein intake in the general population. To the Editor: Kalantar-Zadeh and Fouque ad- dress the possible, although still debated, role of restricting dietary protein intake in reducing pro- teinuria and slowing the progression of chron- ic kidney disease. Not discussed, however, is whether there is a role for low-protein diets in the modern era of treatment with angiotensin- converting–enzyme (ACE) inhibitors and angio- tensin-receptor blockers (ARBs). Data regarding the additive effect of a low-protein diet and these two drug classes on proteinuria are limited.1,2 It is unclear how low-protein diets compare with appropriate use of ACE inhibitor or ARB treat- ment or whether the combination of a low-pro- tein diet and drugs that block the renin–angio- tensin–aldosterone system confers any added benefit in slowing the progression of chronic kidney disease. Low-protein diets may alleviate metabolic disturbances in some patients with chronic kidney disease, but whether such diets are effective in patients who are already receiving In response to Hoorn et al.: in our review article, we discuss that overzealous restrictions in dietary potassium intake should be avoided, since many potassium-rich foods, such as fresh fruits and vegetables with high fiber and vita- min content and low acidogenicity, are heart- healthy and less atherogenic than most low- 584 n engl j med 378;6 nejm.org February 8, 2018 The New England Journal of Medicine Downloaded from nejm.org at Irvine (UCD) on July 25, 2022. For personal use only. No other uses without permissio Copyright © 2018 Massachusetts Medical Society. All rights reserved. Correspondence et al.5 (in which higher protein intake, reflected by higher urinary level of urea nitrogen, was associated with a reduced antiproteinuric effect of olmesartan). potassium foods. n engl j med 378;6 nejm.org February 8, 2018 The New England Journal of Medicine Downloaded from nejm.org at Irvine (UCD) on July 25, 2022. For personal use only. No other uses without permission. Copyright © 2018 Massachusetts Medical Society. All rights reserved. DOI: 10.1056/NEJMc1715765 Effect of urine urea nitro- gen and protein intake adjusted by using the estimated urine creatinine excretion rate on the antiproteinuric effect of angio- tensin II type I receptor blockers. Nutrition 2015;31:1333-8. DOI: 10.1056/NEJMc1715765 1. Caprilli R, Frieri G, Latella G, Vernia P, Santoro ML. Faecal excretion of bicarbonate in ulcerative colitis. Digestion 1986;35: 136-42. DOI: 10.1056/NEJMc1715765 As shown in Table 2 of our article (available at NEJM.org), we reiterate the recommended dietary allowance of potassium of 4.7 g per day both in persons at high risk for chronic kidney disease and in patients with mild-to-moderate chronic kidney disease (stage 1, 2, or 3). However, in patients with advanced chronic kidney disease (stage 4 or 5) or frequent hyperkalemic episodes, we recommend a lower dietary potassium intake of less than 3 g per day. At this juncture, we do not recommend higher dietary potassium goals or potassium supple- ments, pending the results of clinical trials. Kamyar Kalantar‑Zadeh, M.D., Ph.D. University of California, Irvine Orange, CA kkz@uci.edu Denis Fouque, M.D., Ph.D. Université Claude Bernard Lyon Lyon, France Université Claude Bernard Lyon Lyon, France Since publication of their article, the authors report no fur- ther potential conflict of interest. In response to Berns: in most studies of low- protein diets, participating patients with chronic kidney disease have received concurrently, be- yond the diets studied, state-of-the-art therapies for chronic kidney disease, including angioten- sin-pathway–modulating medications. Such trials have confirmed the beneficial effects of restrict- ed protein intake (Table S2 in the Supplementary Appendix of our article), as have several meta- analyses, including a recent meta-analysis of the trials listed in that supplementary table.4 In ad- dition, we are aware of at least three focused studies in humans that have confirmed an addi- tive effective of a low-protein diet, including the two studies cited by Berns — by Gansevoort et al. and Ruilope et al. (studies in which additive anti- proteinuric effects of a low-protein diet and enalapril are reported) — and a study by Chin 1. Caprilli R, Frieri G, Latella G, Vernia P, Santoro ML. Faecal excretion of bicarbonate in ulcerative colitis. Digestion 1986;35: 136-42. 2. Emmett M, Palmer BF. Acid-base and electrolyte abnormali- ties with diarrhea. UpToDate, 2017 (https://www.uptodate.com/ contents/acid-base-and-electrolyte-abnormalities-with-diarrhea? search=Acid-base+and+electrolyte+abnormalities+with+diarrhea .&source=search_result&selectedTitle=1%7E150). 3. Shinaberger CS, Greenland S, Kopple JD, et al. Is controlling phosphorus by decreasing dietary protein intake beneficial or harmful in persons with chronic kidney disease? Am J Clin Nutr 2008;88:1511-8. 4. Rhee CM, Ahmadi SF, Kovesdy CP, Kalantar-Zadeh K. Low- protein diet for conservative management of chronic kidney dis- ease: a systematic review and meta-analysis of controlled trials. J Cachexia Sarcopenia Muscle 2017 November 2 (Epub ahead of print). 5. Chin HJ, Kim DK, Park JH, et al. Acute Graft-versus-Host Disease To the Editor: In their review of acute graft- versus-host disease (GVHD), Zeiser and Blazar (Nov. 30 issue)1 identify the use of peripheral- blood stem-cell grafts as a risk factor for acute GVHD. Retrospective studies have shown that the effects of stem-cell source on acute GVHD are either inconsistent or null,2,3 and two large, prospective, randomized, clinical trials in which bone marrow was compared with mobilized peripheral-blood allografts both showed that stem-cell source had no significant effect on the incidence of acute GVHD.4,5 Although stem-cell source does affect the incidence of chronic GVHD after allogeneic transplantation from un- related donors,3 the best available evidence sug- gests that stem-cell source does not alter the risk of acute GVHD. The article also describes positive results when antithymocyte globulin (ATG) is used for the prevention of acute GVHD on the basis of open-label, randomized, clinical trials. It is im- portant to note that a recent large, multicenter, prospective, placebo-controlled, double-blind, randomized clinical trial showed that prophylac- tic use of ATG significantly increased the risk of death; at 2 years, overall survival was 74% in the placebo group but only 59% in the group receiv- ing ATG. Prophylactic use of ATG for the preven- 585
https://openalex.org/W2761201902	https://eap-iea.org/index.php/eap/article/download/328/315	Bosnian	null	Twentieth Century Spectacle. Music and Power	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by-sa	13,150	DOI: 10.21301/ൾൺඉ.ඏ11ං2.12 Milan Subotić Institut za evropske studije, Beograd milsub@gmail.com Lenjinov mozak: o jednom pokušaju „materijalističkog zasnivanja genijalnosti“* Apstrakt: U ovom radu je prikazan i interpretiran pokušaj da se pomoću neurološ- kih istraživanja Lenjinovog mozga potvrdi vera u njegovu genijalnost. Smatrajući da je „kult ličnosti“ trajna i važna karakteristika političkih sistema sovjetskog tipa, autor je rekonstruisao jedan aspekt posthumne (zlo)upotrebe Lenjinovog tela preduzete u cilju konsolidacije režima i jačanja legitimnosti boljševičke vlasti. Često tematizovano sim- boličko i političko značenje procesa balsamovanja i izlaganja Lenjinovog tela u Mauzo- leju, dopunjeno je u ovom radu razmatranjem delatnosti Instituta za proučavanje mozga koji je formiran kako bi se dokazala genijalnost vođe Oktobarske revolucije. Bez obzira na ograničene rezultate, ovo korišćenje nauke u stvaranju i jačanju kulta vođe predstav- ljalo je, po mišljenju autora, paradoksalni spoj prosvetiteljske genealogije marksističke teorije i sovjetske političke prakse. Ključne reči: Lenjin, mozak, kult ličnosti, materijalizam, neurologija, nauka, politika UDK: 316.46:929 Lൾඇංඇ V. I. 616.8 UDK: 316.46:929 Lൾඇංඇ V. I. 616.8 Oඋං඀ංඇൺඅඇං ඇൺඎඹඇං උൺൽ Oඋං඀ංඇൺඅඇං ඇൺඎඹඇං උൺൽ ∗ Rad je nastao u okviru projekta Instituta za evropske studije koji finansira Ministarstvo prosvete i nauke Republike Srbije. 1 Kako je to u članku povodom pedesetog rođendana Lenjina 1920. godine istakao Maksim Gorki: „Pobornik teorije koja tvrdi da je uloga ličnosti u procesu razvoja ∗ Rad je nastao u okviru projekta Instituta za evropske studije koji finansira Ministarstvo prosvete i nauke Republike Srbije. 1 K k j t čl k d d t đ d L ji 1920 di i t k Ministarstvo prosvete i nauke Republike Srbije. 1 Kako je to u članku povodom pedesetog rođendana Lenjina 1920. godine istakao Maksim Gorki: „Pobornik teorije koja tvrdi da je uloga ličnosti u procesu razvoja Uvod: „Čovek iz budućnosti“ Jedna od brojnih paradoksalnih karakteristika režima sovjetskog tipa je činje- nica da je, s jedne strane, u brojnim teorijskim radovima marksista „o ulozi poje- dinca u istoriji“ naglašavan presudni značaj velikih društvenih grupa (klasa) u isto- rijskom razvoju, dok je, s druge strane, u stvarnosti „princip liderstva“ (вождизм) funkcionisao kao trajni konstitutivni element političke kulture tih društava. Isticanje značaja društvenih klasa, proizvodnih odnosa i „objektivnih“ istorijskih zakona bilo je istovremeno praćeno afirmacijom harizme vođa komunističkih partija kao „avangarde proletarijata“ u borbi za opšteljudsku emancipaciju.1 Stoga je boljševički „scenario vlasti“ sistematski, nizom simboličkih sredstava ∗ Rad je nastao u okviru projekta Instituta za evropske studije koji finansira Ministarstvo prosvete i nauke Republike Srbije. 1 Kako je to u članku povodom pedesetog rođendana Lenjina 1920. godine istakao Maksim Gorki: „Pobornik teorije koja tvrdi da je uloga ličnosti u procesu razvoja Етноантрополошки проблеми, н. с. год. 11 св. 1 (2016) 560 Mංඅൺඇ Sඎൻඈඍංම (ceremonija, ikonografije, ritualnih praksi, itd), „uzdizao“ vođu kao oličenje kolektivne klasne svesti, „glavu“ komunističkog pokreta i „izvršioca“ istorijskih zakona. Kada je, kao u slučaju osude Staljinove vladavine, ovaj „kult ličnosti“ naknadno kritikovan, on je obično tumačen kao slučajna „deformacija“, a ne kao princip založen u samim temeljima sistema.2 Tako je, pozivajući se na „povratak izvorima“, istorijska osuda „kulta ličnosti“ svoj legitimitet u velikoj meri zasnivala na isticanju uzornog lika Staljinovog prethodnika te je, na taj način, i sama dodatno reprodukovala „kult“ koji je kritikovala. Taj pokušaj povrataka „dobrom vođi“ bio je znatno olakšan činjenicom da je Lenjin upravo komunističku partiju (ceremonija, ikonografije, ritualnih praksi, itd), „uzdizao“ vođu kao oličenje kolektivne klasne svesti, „glavu“ komunističkog pokreta i „izvršioca“ istorijskih zakona. Kada je, kao u slučaju osude Staljinove vladavine, ovaj „kult ličnosti“ naknadno kritikovan, on je obično tumačen kao slučajna „deformacija“, a ne kao princip založen u samim temeljima sistema.2 Tako je, pozivajući se na „povratak izvorima“, istorijska osuda „kulta ličnosti“ svoj legitimitet u velikoj meri zasnivala na isticanju uzornog lika Staljinovog prethodnika te je, na taj način, i sama dodatno reprodukovala „kult“ koji je kritikovala. Taj pokušaj povrataka „dobrom vođi“ bio je znatno olakšan činjenicom da je Lenjin upravo komunističku partiju – centralizovanu organizaciju, a ne njenе vođе – smatrao ključnim istorijskim akterom i nosiocem istinske klasne svesti. „Ceo život smo se idejno borili protiv veličanja ličnosti, pojedinca – odavno smo razrešili pitanje heroja“, žalio se on svom saradniku povodom hvalospeva koji su se o njemu pojavljivali u sovjetskoj štampi (vid. Uvod: „Čovek iz budućnosti“ Бонч-Бруевич 1969, 365). Prema brojnim drugim svedočanstvima, Lenjin nije bio sklon manifestacijama javnog obožavanja njegove ličnosti.3 Ipak, s obzirom na to da su „partija“ i „klasa“ apstraktni i bezlični koncepti, oni nisu mogli poslužiti kao efikasno sredstvo ideološke i političke mobilizacije masa, pa je 1918. godine on formulisao projekat obimne „spomeničke propagande“ kojim je planirana zamena postojećih spomenika dignutih u slavu ruskih careva i „njihovih sluga“ novim statuama heroja međunarodnog i ruskog radničkog pokreta: – centralizovanu organizaciju, a ne njenе vođе – smatrao ključnim istorijskim akterom i nosiocem istinske klasne svesti. „Ceo život smo se idejno borili protiv veličanja ličnosti, pojedinca – odavno smo razrešili pitanje heroja“, žalio se on svom saradniku povodom hvalospeva koji su se o njemu pojavljivali u sovjetskoj štampi (vid. Бонч-Бруевич 1969, 365). Prema brojnim drugim svedočanstvima, Lenjin nije bio sklon manifestacijama javnog obožavanja njegove ličnosti.3 Ipak, s obzirom na to da su „partija“ i „klasa“ apstraktni i bezlični koncepti, oni nisu mogli poslužiti kao efikasno sredstvo ideološke i političke mobilizacije masa, pa je 1918. godine on formulisao projekat obimne „spomeničke propagande“ kojim je planirana zamena postojećih spomenika dignutih u slavu ruskih careva i „njihovih sluga“ novim statuama heroja međunarodnog i ruskog radničkog pokreta: „Dok su umetnici-simpatizeri boljševičkog režima stvarali slike ’radnika’ i ’seljaka’ koje je trebalo da simbolišu čitave društvene kolektivitete, Lenjinov projekat je bio zamišljen tako da pre slavi pojedince nego društvene klase. On je pozivao na podizanje ’bista ili figura čitavog tela, pa i bareljefa’ posvećenih ’pretečama socijalizma, njegovim teoretičarima i borcima, kao i onim prosveće- nim ličnostima filozofije, nauke i umetnosti koji, iako nisu neposredno vezani za socijalizam, predstavljaju istinske heroje kulture’“ (Bonnell 1997, 138). kulture zanemarljiva, V. I. Lenjin je, po mom mišljenju, onaj izvor energije bez koje Ruska revolucija ne bi mogla steći oblik koji je dobila“ (Горький 1920). 2 Osuda Staljina koju je formulisao Hruščov na XX Kongresu KPSS je primer takvog pristupa. Nasuprot njegovoj kritici „kulta ličnosti“, davno je uočeno: „Kultovi vođa jako retko su bivali rezultat jednostavne želje vođe za ličnom glorifikacijom ili javnim obožavanjem – mada u pojedinačnim slučajevima i taj faktor može biti značajan – već su u velikoj meri bili rezultat strukturnih karakteristika političkog sistema društva o kome je reč. U tom pogledu, sovjetski politički sistem je veoma relevantan primer jer ga karakteriše postojanje naglašenog kulta vođe tokom njegove dosadašnje istorije“ (Gill 1980, 167). Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 3 Na primer, Lenjin je pomenuti članak Gorkog smatrao „neumesnim“, pa je Politbirou predložio da osudi takvo pisanje o njegovoj ličnosti: „U takvim člancima ne samo da nema ništa komunističkog, već ima mnogo antikomunističkog“ (Ленин ПСС, 45, 429). Između ostalog, Gorki je pisao: „Lenjinov lični život je takav da bi on, u epohi dominacije religijskih osećanja, bio smatran svecem“ (Горький 1920). Uvod: „Čovek iz budućnosti“ p j j g g j g j j ( ) 3 Na primer, Lenjin je pomenuti članak Gorkog smatrao „neumesnim“, pa je Politbirou predložio da osudi takvo pisanje o njegovoj ličnosti: „U takvim člancima ne samo da nema ništa komunističkog, već ima mnogo antikomunističkog“ (Ленин ПСС, 45, 429). Između ostalog, Gorki je pisao: „Lenjinov lični život je takav da bi on, u epohi dominacije religijskih osećanja, bio smatran svecem“ (Горький 1920). 3 Na primer, Lenjin je pomenuti članak Gorkog smatrao „neumesnim“, pa je Politbirou predložio da osudi takvo pisanje o njegovoj ličnosti: „U takvim člancima ne samo da nema ništa komunističkog, već ima mnogo antikomunističkog“ (Ленин ПСС, 45, 429). Između ostalog, Gorki je pisao: „Lenjinov lični život je takav da bi on, u epohi dominacije religijskih osećanja, bio smatran svecem“ (Горький 1920). Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 561 Lൾඇඃංඇඈඏ ආඈඓൺ඄ Svestan važnosti uspešne propagande Lenjin je, uprkos upornom ispoljava- nju lične skromnosti, već 1919. godine pristao da se njegove biste i portreti distribuiraju širom zemlje kako bi se „nepismeni i nepoverljivi seljaci“ uverili u njegovo postojanje (vid. Эннкер 2011, 54). Uživajući nesporan autoritet u par- tijskim redovima, on je vremenom u masovnoj svesti identifikovan s partijom i klasom, postajući njihovo individualizovano „telesno ovaploćenje“.4 U svoje- vrsnom teološki koncipiranom obrtu, u Lenjinu su se tako sjedinile uloge Oca i Sina – on je istovremeno bio i tvorac (otac) ruskog radničkog pokreta i njegov istorijski nastao izdanak (sin).5 Stoga je u tekstu povodom petogodišnjice aten- tata na Lenjina, sovjetski književnik i publicista Mihail Koljcov u formi niza pi- tanja, mogao da do kraja izvede tezu o potpunom izjednačavanju kolektiva (kla- se) i pojedinca (vođe): „Zar nije krv samog proletarijata – Lenjinova krv? Nisu li radnički mišići – njegovi mišići. Nije li centralna, čvorišna razvodna stanica i glavni strateški štab borbe proletarijata – mozak Lenjina?“ (Кольцов 1923, 22). Retorički karakter navedenih pitanja bio je očigledan – u njihovoj pozadini bilo je uverenje prema kome se „proletarijat“, kao suvereni subjekt Istorije, konstituiše posredstvom svoja dva lica („hipostaze“): Partije kao avangardne organizacije, te samog Lenjina koji je, kako je to istakao Karl Radek, „kvintesencija ruske radničke revolucije, oličenje njenog kolektivnog uma i njenog smisla“ (cit. prema: Вайскопф 2001, 337). Ostavljajući po strani razmatranje boljševičkog shvatanja koncepata „klase“ i „partije“, tezom o Lenjinu kao „oličenju“ klase („Lenjin – to smo svi mi“) otvaraju se dva međusobno povezana problema. 4 „Razvijeni Lenjinov kult bio je organizovani sistem rituala i simbola čija je zajednička funkcija bila da kod učesnika kulta i posmatrača izazovu osećanje poštovanja neophodnog za stvaranje emocionalne veze između stanovništva i partije koju je Lenjin oličavao“ (Tumarkin 1997, 2–3). 5 Opširnije o tome u: Вайскопф 2001, 336–366. Status očinske figure najjasnije је formulisao Trocki u nekrologu Lenjinu: „I eto, nema više Iljiča. Partija je ostala siroče, radnička klasa je ostala siroče. Upravo se to osećanje rađa nakon vesti o smrti učitelja, vođe“ (Троцкий 1924). Uvod: „Čovek iz budućnosti“ Prvi se sastojao u tome što su boljševici, nasuprot pomenutoj marksističko- doktrinarnoj tradiciji redukovanja značaja uloge individue u istoriji, istorijskim okolnostima bili podstaknuti da „šire veru“ u posebni status svoga vođe. U industrijski nerazvijenoj Rusiji njegova ličnost nije mogla biti svedena na puki „izraz“ empirijski postojeće radničke klase već je naglašavana njegova uloga „demijurga“ Oktobarske revolucije koja je, kako je to još Gramši isticao, izvedena protivno učenju izloženom u Marksovom Kapitalu. Rečju, upravo je društvena slabost klase morala biti kompenzovana snagom organizacije (partije) i genijalnošću Lenjina kao njenog tvorca i vođe: „Snaga genija proporcionalna je veličini istorijskih zadataka koji stoje pred njegovom klasom“ (Преобра- женский 1924, 145). Stoga, nezavisno od skromnosti kao lične osobine čoveka 4 „Razvijeni Lenjinov kult bio je organizovani sistem rituala i simbola čija je zajednička funkcija bila da kod učesnika kulta i posmatrača izazovu osećanje poštovanja neophodnog za stvaranje emocionalne veze između stanovništva i partije koju je Lenjin oličavao“ (Tumarkin 1997, 2–3). 5 Opširnije o tome u: Вайскопф 2001, 336–366. Status očinske figure najjasnije је formulisao Trocki u nekrologu Lenjinu: „I eto, nema više Iljiča. Partija je ostala siroče, radnička klasa je ostala siroče. Upravo se to osećanje rađa nakon vesti o smrti učitelja, vođe“ (Троцкий 1924). Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 562 Mංඅൺඇ Sඎൻඈඍංම koji se zvao „Vladimir Iljič“, politička i socijalna konsolidacija novog post- revolucionarnog poretka zahtevala je pretvaranje „Lenjina“ u genija i proroka.6 Ako je „Vladimir Iljič Uljanov“ u individualno-biografskom smislu bio izdanak konkretne porodice, društveno-istorijskih okolnosti, intelektualnog sazrevanja, emocionalnog razvoja, itd, onda je on kao „Lenjin“ bio shvatljiv samo kao „gost iz budućnosti“: „Besprekorni borac za svetsku pravičnost, čovek iz budućnosti, predstavnik budućeg sveta koji je poslat u našu mučeničku epohu ugnjetavanja i ropstva – to je zvanje koje Lenjinu, u njegovoj pedeset i četvrtoj godini života, kategorički priznaje celo čovečanstvo“ (Кольцов 1923, 31). Već početkom iduće godine, Lenjinova smrt je sovjetsku vlast suočila s im- perativom ponovnog uspostavljanja narušenog jedinstva pomenutog „trojstva“ (klasa–partija–vođa). U rešavanju tog problema pretendenti na ulogu novog vođe poslužili su se originalnom strategijom – izgradnjom mauzoleja u kome je, pogledu stanovništva, izloženo mumificirano Lenjinovo telo kao svedočan- stvo njegove besmrtnosti. 6 „Po prirodi stvari i uvek, očigledno je da kada se na razmeđi dve epohe rodi čovek čiji mozak i srce ovaploćuju sve najbolje i najprogresivnije u čitavom čovečanstvu, u njegovoj progresivnoj klasi (u ovom slučaju – radničkoj klasi), onda takav čovek i kada sam to ne primećuje, prirodno i jednostavno formuliše proročanstva, a iznenađeni ljudi posmatraju i uviđaju kako se, malo po malo, ispunjava sve što je on predvideo“ (Зиновьев, 1924). 7 Govoreći o Lenjinovoj besmrtnosti jedan od delegata na Lenjinovoj komemoraciji, seljak iz unutrašnjosti, tvrdio je da će vođa iz groba nastaviti da rukovodi zemljom (vid. Tumarkin 1997, 156) Lenjinova bolest i smrt Bar za najbliži krug partijskih saradnika, Lenjinova smrt 21. januara 1924. godine nije bila iznenadna – od pojave prvih simptoma (snažnih glavobolja i vrtoglavica praćenih kraćim gubitkom svesti) sredinom 1921. godine, njegova bolest je, s kratkim periodima remisije, bivala sve teža.8 U poslednjem javnom nastupu na plenarnom zasedanju Moskovskog saveta 20. novembra 1922. godi- ne, Lenjin je priznao da je „počevši od decembra (prošle godine – M. S.) na dugi period izgubio radnu sposobnost“ (Ленин 1922, 300), ali je sovjetska javnost o stanju njegovog zdravlja zvanično obaveštena tek nakon teškog moždanog uda- ra koji ga je 9. maja 1923. godine lišio sposobnosti govora i paralisao mu desnu stranu tela. Šturi zdravstveni bilteni u ondašnjoj štampi bili su praćeni optimi- stičkim prognozama o brzom povratku Lenjina političkim i državničkim dužno- stima, a njegova duga odsutnost iz Kremlja objašnjavana je željom rukovodstva Partije da se on u potpunosti oporavi.9 Lekari, članovi konzilijuma sastavljenog od vodećih nemačkih i ruskih stručnjaka, podnosili su Politbirou izveštaje o stanju pacijenta koji nisu davali povoda za takav optimizam – njihove prognoze bile su veoma uzdržane, a ni sama dijagnoza bolesti nije bila jasno formulisana: „Uprkos tome, tokom leta i jeseni 1923. godine na stranicama ’Pravde’ po- vremeno su objavljivana lažna saopštenja o Lenjinovom ozdravljenju i njego- vom brzom povratku za kormilo vlasti. Ta saopštenja su predstavljala reakciju vodećih partijskih rukovodilaca na pitanja u kojima je izražavana zabrinutost zbog zdravlja vođe. Llažni optimizam oni su potkrepljivali različitim svedočan- stvima o stanju njegovog zdravlja: jednom su tvrdili da su mu lekari dozvolili da čita novine, drugi put da mu je čak dozvoljeno da piše, te da on već sasvim tečno govori“ (Эннкер 2010, 73). 8 Prema prof. Osipovu koji je lečio Lenjina: „Tok njegove bolesti može biti podeljen na tri perioda. Početak prvog je mart 1922. godine; drugog – decembra iste godine, a trećeg – marta 1923. Ovo deljenje bolesti na tri perioda ukazuje da ona nije tekla kontinuiranim tokom, već da se njen razvoj odvijao neravnomerno, sa periodima tokom kojih se bolesnik oporavljao i relativno se bolje osećao, da bi se zatim bolest pogoršavala, razvijala i napredovala“ (Осипов 1925). 9 Ministar (Narodni komesar) zdravlja Semaško je 20. oktobra 1923. 8 Prema prof. Osipovu koji je lečio Lenjina: „Tok njegove bolesti može biti podeljen na tri perioda. Početak prvog je mart 1922. godine; drugog – decembra iste godine, a trećeg – marta 1923. Ovo deljenje bolesti na tri perioda ukazuje da ona nije tekla kontinuiranim tokom, već da se njen razvoj odvijao neravnomerno, sa periodima tokom kojih se bolesnik oporavljao i relativno se bolje osećao, da bi se zatim bolest pogoršavala, razvijala i napredovala“ (Осипов 1925). 9 Ministar (Narodni komesar) zdravlja Semaško je 20. oktobra 1923. godine uveravao javnost da je Lenjinovo zdravstveno stanje sve bolje: „Zdravlje druga Lenjina se sistematski, svakim danom poboljšava… Iljič se šali, interesuje se za javne poslove osećajući da će uskoro uzeti neposredno učešće u njima… On je željan posla, ali mi ga od toga moramo odgovarati jer osećamo odgovornost za njegovo zdravlje ne samo pred ruskim, već i pred svetskim proletarijatom“ (cit. prema: Петренко 1991, 149). Uvod: „Čovek iz budućnosti“ Odbijanjem priznanja da je središnje „mesto vlasti“ sada prazno,7 borba sukobljenih frakcija za Lenjinovo nasleđe pretvorena je u interpretativni spor o pravom značenju vođinog učenja: „Tokom celokupne isto- rije Sovjetskog Saveza, Lenjin je bio glavni legitimizujući označitelj sovjetske ideologije koji je zauzimao spoljašnju poziciju u odnosu na ideološki diskurs. Drugim rečima, postulat o istinitosti i nespornosti njegovih ideja bio je polazna tačka svih ideoloških iskaza i u njima se on nije mogao dovoditi u sumnju“ (Юрчак 2007). U ovom radu se neću baviti pomenutim procesom kodifikacije „lenjinizma“ i ulogom koju je kult Lenjina imao u staljinističkoj konsolidaciji sovjetskog reži- ma. Takođe, ovde neće biti reči o problemu nastanka, značenja i smisla izlaganja Lenjinovog „netruležnog tela“ o kome se, po mom mišljenju, produktivno može pristupiti polazeći od Kantorovičeve analize srednjovekovnog učenja o „dva kraljeva tela“. Iako je po svom tematskom obimu predmet ovog rada znatno uži, on se ipak neposredno odnosi na tumačenje fenomena (zlo)upotrebe Lenjinovog tela u nastanku kulta „vođe svetskog proletarijata“. Ograničen na interpretaciju pokušaja da se naučnom analizom Lenjinovog mozga osigura „materijalistički dokaz njegove genijalnosti“, ovaj rad predstavlja prilog razumevanju nastanka 7 Govoreći o Lenjinovoj besmrtnosti jedan od delegata na Lenjinovoj komemoraciji, seljak iz unutrašnjosti, tvrdio je da će vođa iz groba nastaviti da rukovodi zemljom (vid. Tumarkin 1997, 156) Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 563 Lൾඇඃංඇඈඏ ආඈඓൺ඄ „političke teologije“ jednog političkog sistema koji je svoju genealogiju zasni- vao na tradiciji prosvećenosti, tj. na sopstvenoj naučnosti. „političke teologije“ jednog političkog sistema koji je svoju genealogiju zasni- vao na tradiciji prosvećenosti, tj. na sopstvenoj naučnosti. 10 Prema Nini Tumarkin, termin „lenjinizam“ uveden je u opticaj 3. januara 1923. godine u članku Vladimira Sorina koji je rukovodio odeljenjem agitacije u Moskovskom komitetu partije. U članku objavljenom u „Pravdi“, ovaj autor je istakao da „marksizam pre Lenjina nije bio pravi marksizam“, te da se Lenjinova dela moraju ozbiljno („s olovkom u ruci“) proučavati kako bi se „propagirao lenjinizam“, kao praktična primena marksističkog učenja u savremenim društveno-istorijskim uslovima. Takođe, on je predložio da se, nezavisno od već postojećeg Instituta Marksa i Engelsa, osnuje poseban Institut Lenjina koji bi se bavio izdavanjem njegovih dela i sakupljanjem svih materijala o njemu. Taj predlog je ozvaničen aprila 1923. godine, a tokom leta je početo prikupljanje materijala, iako je Institut zvanično otvoren posle Lenjinove smrti, 31. maja 1924. godine (vid. Tumarkin 1997, 120–126) Lenjinova bolest i smrt godine uveravao javnost da je Lenjinovo zdravstveno stanje sve bolje: „Zdravlje druga Lenjina se sistematski, svakim danom poboljšava… Iljič se šali, interesuje se za javne poslove osećajući da će uskoro uzeti neposredno učešće u njima… On je željan posla, ali mi ga od toga moramo odgovarati jer osećamo odgovornost za njegovo zdravlje ne samo pred ruskim, već i pred svetskim proletarijatom“ (cit. prema: Петренко 1991, 149). Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 564 Mංඅൺඇ Sඎൻඈඍංම Samo malobrojni Lenjinovi poznanici, kojima je bilo dozvoljeno da ga po- sete na imanju van Moskve gde je u izolaciji proveo poslednje mesece života, mogli su videti nepokretnog „vođu svetske revolucije“ u invalidskim kolicima, lišenog sposobnosti čitanja i pisanja, kao i mogućnosti verbalne komunikacije s okruženjem. Da su svesni težine Lenjinovog zdravstvenog stanja i da ni sami ne veruju u mogućnost njegovog oporavka, članovi Politbiroa posredno su po- tvrđivali uvođenjem u javni diskurs koncepta „lenjinizma“, kao i oficijelnim početkom Lenjinove „muzeifikacije“. Izveštaje o aktivnostima „vođe svetskog proletarijata“ zamenilo je isticanje značaja njegovog učenja kao „stvaralačke razrade i primene marksističke doktrine“, kao i odluka da se pristupi sistema- tičnom sakupljanju njegovih tekstova koji će ubuduće predstavljati obavezujući kanon političkog mišljenja i osnovno rukovodstvo za delovanje: „Počevši od 1923. godine, vodeći partijski propagandisti počeli su naglašavati nužnost par- tijske zakletve na vernost ’lenjinizmu’... U vreme kada je Lenjin postao objekt obožavanja i slavljenja, on više fizički nije bio u mogućnosti da ostvaruje svoju vlast“ (Эннкер 2011, 75; 31). Zvanični predlog o osnivanju Instituta Lenjina formulisan je već tokom njegovog života (aprila 1923. godine), a proces pri- kupljanja dokumentacije, rukopisa, fotografija, pisama i svih materijala vezanih za njegov život i rad počeo je paralelno s upornim objavljivanjem najava o Le- njinovom uspešnom zdravstvenom oporavku.10 U tom pogledu, odluka o for- miranju muzeja u okviru Instituta svedočila je da vrh Partije još živog Lenjina već tretira kao prošlost oko čije „ispravne interpretacije“ će sukobljene frakcije voditi žestoke sporove u borbi za „lenjinsko nasleđe“. Čini se da je i sam Lenjin, u trenucima kratkoročnih poboljšanja zdravstvenog stanja (posle drugog udara u decembru 1922, do trećeg u martu 1923. godine), bio svestan da ga najbliži partijski saradnici polako pretvaraju u muzejski eksponat – diktirajući Nadež- di Krupskoj svoj „testament“ (Pismo kongresu), nije kritikovao samo Staljina, već i sve ostale potencijalne „naslednike“. Lenjinova bolest i smrt Uprkos teškom zdravstvenom stanju, on je u ovom Pismu „manifestovao neukrotivu volju ka političkoj dominaciji i vlasti, volju koja je bila glavna osobina njegove ličnosti“ (Khlevniuk 2015, Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 565 Lൾඇඃංඇඈඏ ආඈඓൺ඄ 74). Ipak, ta „Lenjinova poslednja bitka“ (Lewin 2007) okončana je njegovim porazom – konačnim trijumfom fizičke smrti otvoren je put simboličkoj („me- tafizičkoj“) instrumentalizaciji njegovog tela. 74). Ipak, ta „Lenjinova poslednja bitka“ (Lewin 2007) okončana je njegovim porazom – konačnim trijumfom fizičke smrti otvoren je put simboličkoj („me- tafizičkoj“) instrumentalizaciji njegovog tela. U noći 21/22. januara vajar Sergej Merkurov doveden je u daču pokojnika da izradi njegovu posmrtnu masku – uzbuđen, videvši o kome je reč, vajar je za puls u svojim prstima pomislio da su otkucaji Lenjinovih karotida (Меркуров 2012, 160–162). Bogatog prethodnog iskustva u izradi posmrtnih maski, Merkurov je uspešno završio svoj posao – otisak Lenjinovog lica koji je napravio poslužiće kasnije za masovnu reprodukciju i kao model za bezbrojne biste i spomenike dignute u čast Lenjina širom Sovjetskog Saveza.11 Posle vajara, Lenjinovo telo preuzeli su patolozi – tim od osam Lenjinovih lekara, kojima je pridružen tadašnji najugledniji ruski patolog Abrikosov (Абрикосов), uz učešće nemačkog neurologa Ferstera (Fёrster) i prisustvo ministra zdravlja Semaška (Семаш- ко, 1874–1949), obavio je 22. januara autopsiju pokojnikovog tela. Zvanično „Saopštenje o bolesti i smrti V. I. Lenjina“ objavljeno je u „Pravdi“ 24. januara – na osnovu nalaza patološko-anatomske autopsije, zaključeno je da je Lenjin bolovao od teškog oblika arterioskleroze koja je izazvala trajne promene u moždanom tkivu, te da je neposredni uzrok njegove smrti bio masivan izliv krvi u mozak (vid. u: Пелевин 1924, 133–134).12 S obzirom na to da su identifikovane i opisane promene krvnih sudova bile netipične za dob pokojnika,13 lekari su bili svesni da se u domaćoj i svetskoj javnosti opravdano može postaviti pitanje o tome „zašto i kako se kod čoveka od 53 godine koji je živeo umerenim životom, nije pio ni pušio, razvio takav degenerativni proces?“ (Осипов 1925). Sumnje u dijagnozu i nalaze autopsije dodatno su bile podsticane činjenicom da je tok U noći 21/22. januara vajar Sergej Merkurov doveden je u daču pokojnika da izradi njegovu posmrtnu masku – uzbuđen, videvši o kome je reč, vajar je za puls u svojim prstima pomislio da su otkucaji Lenjinovih karotida (Меркуров 2012, 160–162). 11 Merkurov je 1910. godine uradio Tolstojevu posmrtnu masku, a do kraja života (1952) napravio je otiske lica preko stotinu slavnih pokojnika. Prvih dvadeset kopija Lenjinove maske je 1924. godine uradio za vodeće partijske rukovodioce, a Staljin je svoj primerak u staklenoj kutiji držao izložen u svom kabinetu. Opširnije o tradiciji izrade posmrtnih maski u Rusiji videti u: Neumeyer 2015. Lenjinova bolest i smrt Bogatog prethodnog iskustva u izradi posmrtnih maski, Merkurov je uspešno završio svoj posao – otisak Lenjinovog lica koji je napravio poslužiće kasnije za masovnu reprodukciju i kao model za bezbrojne biste i spomenike dignute u čast Lenjina širom Sovjetskog Saveza.11 Posle vajara, Lenjinovo telo preuzeli su patolozi – tim od osam Lenjinovih lekara, kojima je pridružen tadašnji najugledniji ruski patolog Abrikosov (Абрикосов), uz učešće nemačkog neurologa Ferstera (Fёrster) i prisustvo ministra zdravlja Semaška (Семаш- ко, 1874–1949), obavio je 22. januara autopsiju pokojnikovog tela. Zvanično „Saopštenje o bolesti i smrti V. I. Lenjina“ objavljeno je u „Pravdi“ 24. januara – na osnovu nalaza patološko-anatomske autopsije, zaključeno je da je Lenjin bolovao od teškog oblika arterioskleroze koja je izazvala trajne promene u moždanom tkivu, te da je neposredni uzrok njegove smrti bio masivan izliv krvi u mozak (vid. u: Пелевин 1924, 133–134).12 S obzirom na to da su identifikovane i opisane promene krvnih sudova bile netipične za dob pokojnika,13 lekari su bili svesni da se u domaćoj i svetskoj javnosti opravdano može postaviti pitanje o tome „zašto i kako se kod čoveka od 53 godine koji je živeo umerenim životom, nije pio ni pušio, razvio takav degenerativni proces?“ (Осипов 1925). Sumnje u dijagnozu i nalaze autopsije dodatno su bile podsticane činjenicom da je tok „Saopštenje o bolesti i smrti V. I. Lenjina“ objavljeno je u „Pravdi“ 24. januara – na osnovu nalaza patološko-anatomske autopsije, zaključeno je da je Lenjin bolovao od teškog oblika arterioskleroze koja je izazvala trajne promene u moždanom tkivu, te da je neposredni uzrok njegove smrti bio masivan izliv krvi u mozak (vid. u: Пелевин 1924, 133–134).12 S obzirom na to da su identifikovane i opisane promene krvnih sudova bile netipične za dob pokojnika,13 lekari su bili svesni da se u domaćoj i svetskoj javnosti opravdano može postaviti pitanje o tome „zašto i kako se kod čoveka od 53 godine koji je živeo umerenim životom, nije pio ni pušio, razvio takav degenerativni proces?“ (Осипов 1925). Sumnje u dijagnozu i nalaze autopsije dodatno su bile podsticane činjenicom da je tok 11 Merkurov je 1910. godine uradio Tolstojevu posmrtnu masku, a do kraja života (1952) napravio je otiske lica preko stotinu slavnih pokojnika. Prvih dvadeset kopija Lenjinove maske je 1924. godine uradio za vodeće partijske rukovodioce, a Staljin je svoj primerak u staklenoj kutiji držao izložen u svom kabinetu. Opširnije o tradiciji izrade posmrtnih maski u Rusiji videti u: Neumeyer 2015. 12 Osim arterioskleroze koja je snažno deformisala arterije velikog mozga i silaznu aortu, hipertrofije desne srčane komore i višestrukih promena tkiva leve hemisfere mozga (encephalomalacia), patološki nalaz je sadržavao i opis promena na kosti i mekim tkivima (callus) levog ramena – posledica atentata koji je 1918. izvršila F. Kaplan. Popularne verzije o uticaju atentata („otrovanih metaka“) na Lenjinovu smrt nemaju nikakvu drugu osnovu osim ovog uzgrednog pominjanja tragova ranjavanja u patološko-anatomskom nalazu. Lenjinova bolest i smrt 12 Osim arterioskleroze koja je snažno deformisala arterije velikog mozga i silaznu aortu, hipertrofije desne srčane komore i višestrukih promena tkiva leve hemisfere mozga (encephalomalacia), patološki nalaz je sadržavao i opis promena na kosti i mekim tkivima (callus) levog ramena – posledica atentata koji je 1918. izvršila F. Kaplan. Popularne verzije o uticaju atentata („otrovanih metaka“) na Lenjinovu smrt nemaju nikakvu drugu osnovu osim ovog uzgrednog pominjanja tragova ranjavanja u patološko-anatomskom nalazu. p 13 Prema opisu Semaška: „Glavna arterija koja hrani krvlju skoro ¾ mozga – ’unutrašnja karortidna arterija’ (art. carotis interna) – bila je u samom ulasku u lobanju tako otvrdla da su njeni zidovi u značajnoj meri zatvarali protok krvi, a na nekim mestima je bila toliko kalcifikovana da je pinceta udarala po njima kao po kosti“ (Семашко 1924). 13 Prema opisu Semaška: „Glavna arterija koja hrani krvlju skoro ¾ mozga – ’unutrašnja karortidna arterija’ (art. carotis interna) – bila je u samom ulasku u lobanju tako otvrdla da su njeni zidovi u značajnoj meri zatvarali protok krvi, a na nekim mestima je bila toliko kalcifikovana da je pinceta udarala po njima kao po kosti“ (Семашко 1924). Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 566 Mංඅൺඇ Sඎൻඈඍංම Lenjinove bolesti ličio na „progresivnu paralizu“ koja, po brojnim simptomima, karakteriše zapušteni (tercijalni) stadijum sifilisa (neurosyphilis ili endarteritis luetica). Mogućnost širenja glasina o takvoj prirodi bolesti bila je zasnovana na činjenici da su tokom lečenja bar dvojica nemačkih članova lekarskog konzilijuma (prof. Strümpell i prof. Henschen)14 bili skloni da etiologiju Lenjinovog stanja traže u veneričnom oboljenju stečenom u emigrantskoj mladosti pacijenta, te su savetovali primenu terapije koja je tada široko korišćena u lečenju sifilisa. Istina, standardni test (Wassermann test) Lenjinove cerebrospinalne tečnosti i krvi bio je negativan, ali današnji zagovornici ove dijagnoze ističu visok procenat njegove nepouzdanosti.15 Nasuprot njima, drugi lekari su skloniji da, na osnovu dostupnih podataka, okarakterišu Lenjinovu bolest kao oblik demencije uslovljene višestrukim moždanim udarima – multi-infarct dementia (Kreutzberg et al. 1992, 363). ) Nezavisno od (ne)mogućnosti naknadnog utvrđivanja tačne dijagnoze, jasno je da bi već samo pominjanje sifilisa vodilo socijalnoj i političkoj stigmatizaciji ne samo Lenjina, već i komunističkog pokreta koji je on predvodio, te je sasvim razumljivo uporno nastojanje sovjetskog političkog vrha da takvu dijagnozu od- baci kao zlonamernu i politički motivisanu „glasini“. 14 Prema dnevničkoj belešci prof. Henšena: “Pacijent boluje od višestrukog arteriosklerotičnog smekšavanja mozga (encephalomalacia) za koga su nemački lekari, više ili manje ubeđeni, da je u pitanju cerebralni sifilis, te preporučuju anti-luetičku terapiju“. Drugi lekar, prof. Štrimpel je postavio dijagnozu sifilis aorte (endarteritis luetica), te insistirao na odgovarajućoj terapiji (cit. prema: Klatzo 2002, 29). ), g j j p j ( p , ) 15 „Cerebralno-spinalna tečnost (CSF) je bila normalna i Vasermnanov test je bio negativan, ali u slučajevima tercijalnog sifilisa taj test na uzorku CSF je lažno negativan u 34–90% slučajeva, dok je test krvi takav u 5% slučajeva“ (Lerner et al. 2004, 374). 16 Da su takve glasine bile raširene indirektno je potvrdio Zinovjev u svom govoru na zasedanju Lenjingradskog saveta: „Drugovi, vama su svakako poznate sve one glupe izmišljotine koje su naši protivnici pustili u opticaj kako bi ’objasnili’ uzrok bolesti Iljiča. Sada su najistaknutiji predstavnici nauke potpuno razrušili takve glasine – od njih nije ostao ni kamen na kamenu!“ (Зиновьев 1924). Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) Lenjinova bolest i smrt Stoga je Komesar narod- nog zdravlja isticao da je upravo objavljivanjem protokola autopsije sa dijagno- zom arterioskleroze „stavljena tačka na sve pretpostavke (pa, i glasine) koje su, još za života Vladimira Iljiča, u pogledu karaktera njegove bolesti kolale u nas i u inostranstvu“ (Семашко 1924).16 Ipak, preuranjeni teški oblik skleroze zah- tevao je dodatna objašnjenja koja su tražena u dva međusobno komplementarna smera. Prvi, ideološki manje važan, ticao se isticanja uticaja naslednog faktora – Lenjinov otac je takođe umro od moždanog udara u pedeset petoj godini života, kao i njegova majka koja je, istina, doživela sedamdeset godina. Uz nasleđe lo- ših krvnih sudova mozga, dodato je naglašavano njihovo prekomerno „habanje“ (изнашивание) izazvano Lenjinovim napornim radom, kao i preživljenim stre- sovima tokom emigracije, revolucije i rukovođenja sovjetskom državom. Upra- 14 Prema dnevničkoj belešci prof. Henšena: “Pacijent boluje od višestrukog arteriosklerotičnog smekšavanja mozga (encephalomalacia) za koga su nemački lekari, više ili manje ubeđeni, da je u pitanju cerebralni sifilis, te preporučuju anti-luetičku terapiju“. Drugi lekar, prof. Štrimpel je postavio dijagnozu sifilis aorte (endarteritis luetica), te insistirao na odgovarajućoj terapiji (cit. prema: Klatzo 2002, 29). ssues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 567 Lൾඇඃංඇඈඏ ආඈඓൺ඄ vo je ovaj, pored nasleđa, drugi identifikovani uzročnik Lenjinovog stanja bio ideološki važan činilac u stvaranju predstave o neumornom, genijalnom vođi proleterijata koji je sopstveno zdravlje žrtvovao za „stvar revolucije“. Njegova skleroza je, kako se tvrdilo, bila Abnutzungssklerose – posledica prekomernog „trošenja“ organizma: „Najvažniju ulogu nisu igrali nasledni, već ’stečeni’ uzroci – skleroza je više od svega pogodila mozak, tj. onaj organ koji je tokom života Vladimir Iljič na- jintenzivnije koristio. Bolest obično pogađa ’najranjivije mesto’ (locus minoris resistentiae), a takvo ’ranjivo’ mesto je u Vladimira Iljiča bio veliki mozak – on je bio u stalnom napornom radu, sistematski je bio izložen preteranom ’trošenju’ i sva uzbuđenja ’udarala’ su, pre svega, na njega“ (Семашко 1924). Tačnije, kako je objašnjavajući mehanizam nastanka anatomskih promena Lenjinovog mozga to istakao jedan drugi ruski neurolog, istinsko „ranjivo me- sto“ bili su krvni sudovi kao „pomoćni organi“ misaone aktivnosti, a ne sam centralni organ: „Iako je mozak titanski radio, nije se umorio, izdržao je – oslabili su krvni su- dovi, njihovo vezivno tkivo, njihovo obnavljanje... Moždano tkivo nije se istro- šilo, već su se pre vremena istrošili krvni sudovi ... Nesavršena telesna školjka nije izdržala duhovno naprezanje. 17 Sačuvana je potvrda: „Ja, niže potpisani Arosev, za Institut V. I. Lenjina preuzeo sam od druga Beljenkovog 24. januara u 18h i 25 minuta uveče staklenu posudu sa mozgom, srcem Iljiča, kao i metkom izvađenim iz njegovog tela. Obavezujem se da ću primljeno čuvati u Institutu V. I. Lenjina i lično odgovarati za njihovu celovitost i bezbednost“ (Спивак 2010, 14). Аросев (Александр Аросев, 1890–1938) bio je Lenjinova bolest i smrt Mozak je bio pobednik, ali pomoćno, spored- no vezivno tkivo u njemu pokazalo se nepostojanim – otuda lipidne promene, skleroza, kalcifikacija, krtost, suženje krvnih sudova, te smekšavanje moždanog tkiva i izliv krvi u mozak“ (Мельников-Разведенков 1924). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti 18 „Od druge polovine XIX veka kada su istraživanja anatomije i patologije mozga imala zapažen razvoj i uticaj na neurologiju, proučavanje strukture mozga pojedinaca različitih umeća i intelektualnih sposobnosti smatrano je sredstvom koje bi moglo pružiti putokaz u razumevanju mentalnih fenomena… U vreme istraživanja Lenjinovog mozga proučavanje mozgova intelektualno obdarenih pojedinaca nije bio novi poduhvat. Taj pravac istraživanja počeo je … u Nemačkoj, gde je Rudolf Vagner (1805–1864) proučavao mozak fizičara i matematičara Karla Fridriha Gausa (1777– 1855)“ (Bentivoglio 1998, 291; 294). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti Ostavljajući po strani debate o dijagnozi i gotovo sholastičku raspravu o defini- sanju „ranjivog mesta“, za razumevanje posthumnog statusa Lenjina u sovjetskoj „političkoj teologiji“ – pored pogrebnog rituala, odluke o balsamovanju, izgradnji Mauzoleja i izlaganju tela u njemu – zanimljivo je praćenje dalje sudbine njego- vog mozga. Naime, neposredno posle izvršene autopsije u Institut Lenjina dopre- mljena je posuda koja je sadržavala mozak, srce i metak izvađen iz pokojnikovog tela.17 Tako je Institut – osnovan prevashodno radi izdavanja Lenjinovih Sabranih dela i sakupljanja različite dokumentacije o njegovom životu i radu – dobio dva Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 568 Mංඅൺඇ Sඎൻඈඍංම organa koja su, oličavajući voljne i intelektualne sposobnosti bivšeg „vlasnika“, imali nesumnjivu simboličku vrednost. Takav običaj čuvanja organa „velikih lju- di“ bio je relativno raširen, ali nije praktikovano njihovo javno izlaganje u muzej- skim postavkama. Iako je u okviru Instituta bio osnovan muzej, Lenjinov mozak nije bio dopremljen kao budući eksponat. On je trebalo da posluži drugoj, važnijoj svrsi – naučnom istraživanju od koga je očekivano ne samo da potvrdi Lenjinovu genijalnost, već i da jasno identifikuje njenu „materijalističku osnovu“. Rečju, polazeći od opšteg filozofskog uverenja da „biće određuje svest“, boljševici su bili uvereni da se Lenjinove teorijske i političko-praktične sposobnosti mogu dovesti u korelaciju s materijalnim (strukturnim i fizičkim) karakteristikama njegovog mozga, te da je neurološko-anatomskim istraživanjima moguće odgonetnuti „taj- nu“ njegove svetsko-istorijske veličine i izuzetnosti. Sličan pristup u objašnjenju mentalnih sposobnosti nisu delili samo zagovornici „dijalektičkog materijalizma“ u Sovjetskom Savezu, već i mnogi naučnici u Evropi koji su se bavili istraživanji- ma anatomije mozga intelektualno obdarenih ljudi.18 Istina, u pitanju su najčešće bili mozgovi naučnika (među poslednjim, Ajnštajnov), kao i velikih umetnika, a ne istaknutih političkih ličnosti. Ali, za boljševike Lenjin je bio mnogo više od pu- kog „političara“: „Interes za izučavanje njegovog mozga raste ako se ima na umu da je to mozak genijalnog čoveka... čiji su se voljni impulsi (čelična volja) i geni- jalne misli rađale, razvijale u velikom mozgu; ... [čovek] čija je konstitucija bila ’celebralna’ i čiji je mozak bio izvanredno razvijen“ (Мельников-Разведенков 1924). Genijalnost Lenjina nije dovođena u sumnju, a naučne analize strukture i karakteristika njegovog mozga trebalo je samo da potvrde i „materijalistički obja- sne“ ovo aksiomatsko polazište. Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) ) ( g ) 19 Pol Broka (Paul Pierre Broca, 1824 –1880), osnivač francuskog Antropološkog društva, 1861. godine je pisao: „Generalno, mozak je kod odraslih osoba veći nego kod dece, kod muškaraca nego kod žena, kod istaknutih ljudi nego kod onih osrednjeg talenta, kod viših rasa nego kod nižih… Kad su osobe u drugim stvarima jednake, postoji rukovodilac odeljenja rukopisa Instituta Lenjina, kasnije sovjetski diplomata i žrtva čistki 1938. godine. Institut za istraživanje mozga: ćelijska arhitektura genijalnosti Opis fizičkih karakteristika mozga je na samom početku čitavog poduhvata stvarao ozbiljan problem – deo moždane mase bio je oštećen bolešću, a veličina i težina mozga nije potvrđivala u XIX veku rašireno uverenje o pozitivnoj ko- relaciji između obima moždane mase i intelektualnih sposobnosti.19 Već tokom rukovodilac odeljenja rukopisa Instituta Lenjina, kasnije sovjetski diplomata i žrtva čistki 1938. godine. ) ( g ) 19 Pol Broka (Paul Pierre Broca, 1824 –1880), osnivač francuskog Antropološkog društva, 1861. godine je pisao: „Generalno, mozak je kod odraslih osoba veći nego kod dece, kod muškaraca nego kod žena, kod istaknutih ljudi nego kod onih osrednjeg talenta, kod viših rasa nego kod nižih… Kad su osobe u drugim stvarima jednake, postoji 19 Pol Broka (Paul Pierre Broca, 1824 –1880), osnivač francuskog Antropološkog društva, 1861. godine je pisao: „Generalno, mozak je kod odraslih osoba veći nego kod dece, kod muškaraca nego kod žena, kod istaknutih ljudi nego kod onih osrednjeg talenta, kod viših rasa nego kod nižih… Kad su osobe u drugim stvarima jednake, postoji Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 569 Lൾඇඃංඇඈඏ ආඈඓൺ඄ autopsije ustanovljeno je da težina Lenjinovog mozga iznosi svega 1340 grama, što je bilo malo ispod prosečne težine od 1400 g., pa je rešenje nađeno u sniža- vanju proseka i isticanju uticaja bolesti na gubitak moždane mase: „Prosečna težina ljudskog mozga je 1300 –1400 grama, a ako zamislimo zdravi mozak Vladimira Iljiča onda, imajući u vidu njegov sastav, možemo za- ključiti da je on bio težak oko 1400 grama, tj. nešto više od prosečnog. Zdravi delovi mozga su bili veoma dobro razvijeni, što ukazuje na moćan mozak...“ (Осипов 1925).20 Uverenje o postojanju jasne pozitivne korelacije između inteligencije (kao i stvaralačkog talenta) i veličine mozga u naučnim krugovima je već krajem XIX veka bilo poljuljano nizom istraživanja u kojima je pokazano da teški krimi- nalci u tom pogledu često nadmašuju ugledne univerzitetske profesore,21 a da su u slučaju umetnika razlike u težini mozga – na primer, između Turgenjeva (2012 gr.) i Anatola Fransa (1017 gr.) – veoma velike. Uprkos isticanju važnosti gustine i strukture moždanih vijuga, kao i razvijenosti različitih regija velikog mozga, uverenje o primarnom značaju veličine ostalo je široko rasprostranjeno, pa ga ni argumentacija zagovornika Lenjinove genijalnosti nije mogla zanema- riti. 20 Zinovjev je još više preterivao u opisu fizičkih karakteristika Lenjinovog mozga: „Najbolji nemački profesori su rekli - ‘Od mozga Vladimira Iljiča koji nije sagoreo u radu ostala je samo četvrtina’. I moramo se diviti moćnom mozgu druga Lenjina koji je i u takvim uslovima uspeo da sačuva tako mnogo intelektualne snage i da stanje stvari razume mnogo dublje nego čovek zdravog mozga“ (Зиновьев 1924). izvanredan odnos između razvoja inteligencije i zapremine mozga“ (cit. prema: Gould 1996, 115). Broka je bio i jedan od osnivača Društva za uzajamnu autopsiju (Société d’autopsie mutuelle) koje je okupljalo lekare-donore sopstvenih posmrtnih ostataka za potrebe naučnih istraživanja. 21 Nemački anatom i biolog Teodor fon Bišof (Theodor von Bischoff, 1807–1882) 1880. godine je objavio studiju o istraživanju mozgova 119 kriminalaca koji su u velikoj većini bili znatno iznad prosečne veličine, kao i veći od mozgova brojnih profesora Univerziteta u Getingenu koje je, pored Gausovog, sistematski proučavao već pomenuti Rudolf Vagner (Opširnije u: Gould 1996, 124–127). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti Ipak, u razvoju projekta istraživanja Lenjinovog mozga prioritet su imali savremeniji naučni pristupi, pa je odlukom Vlade postojeća laboratorija koja je bila odeljenje „Instituta V. I. Lenjina“ ubrzo (1927. godine) prerasla u zaseban Institut za proučavanje mozga (Института мозга). Osnivanje posebne naučne institucije zadužene za istraživanje Lenjinovog mozga zahtevalo je prethodnu obuku istraživača i nabavku odgovarajuće tehničke opreme. Obe ove pretpo- stavke ostvarene su pomoću „nemačke veze“, tj. uspostavljanjem saradnje sa berlinskim Institutom za istraživanje mozga (Kaiser Wilhelm Institut für Hirn- forschung) i njegovim direktorom Oskarom Fogtom (Oscar Vogt, 1870–1959) koji je pozvan da rukovodi radom laboratorije i budućeg Instituta u Moskvi. izvanredan odnos između razvoja inteligencije i zapremine mozga“ (cit. prema: Gould 1996, 115). Broka je bio i jedan od osnivača Društva za uzajamnu autopsiju (Société d’autopsie mutuelle) koje je okupljalo lekare-donore sopstvenih posmrtnih ostataka za potrebe naučnih istraživanja. 20 Zinovjev je još više preterivao u opisu fizičkih karakteristika Lenjinovog mozga: „Najbolji nemački profesori su rekli - ‘Od mozga Vladimira Iljiča koji nije sagoreo u radu ostala je samo četvrtina’. I moramo se diviti moćnom mozgu druga Lenjina koji je i u takvim uslovima uspeo da sačuva tako mnogo intelektualne snage i da stanje stvari razume mnogo dublje nego čovek zdravog mozga“ (Зиновьев 1924). 21 Nemački anatom i biolog Teodor fon Bišof (Theodor von Bischoff, 1807–1882) 1880. godine je objavio studiju o istraživanju mozgova 119 kriminalaca koji su u velikoj većini bili znatno iznad prosečne veličine, kao i veći od mozgova brojnih profesora Univerziteta u Getingenu koje je, pored Gausovog, sistematski proučavao već pomenuti Rudolf Vagner (Opširnije u: Gould 1996, 124–127). Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 570 Mංඅൺඇ Sඎൻඈඍංම Donoseći odluku o angažovanju Fogta i njegove saradnice i supruge Sesil (Cécile Vogt-Mugnier, 1875–1962), Politbiro je bio rukovođen višestrukim ra- zlozima. Jedan od najvažnijih bio je svakako ugled koji je Fogt stekao u ondaš- njim naučnim krugovima svojim inovatorskim radovima o ćelijskoj arhitekturi (cytoarchitectonic) ljudskog mozga. Predavanjem koje je u januaru 1923. go- dine održao na prvom Kongresu sovjetskih neurologa on je ostavio izvanredan utisak na svoje moskovske kolege (vid. Richter 2007, 138). Od brojnih Fogtovih istraživačkih interesovanja, posebnu pažnju šire javnosti privlačili su njegovi radovi o „ćelijskoj arhitektonici elitnih mozgova“: „Njegova pretpostavka o direktnoj vezi između funkcije i strukture moz- ga implicirala je da se visoki intelektualni kvaliteti ljudi moraju odraziti u strukturnim karakteristikama posebnih oblasti korteksa pojedinaca. 22 „Prema konačnom sporazumu koji je potpisan u Moskvi 22. maja 1925. godine, predviđeno je da će Rusi biti zaduženi za administrativna pitanja, da će preuzeti finansijsku odgovornost za nalaženje zgrade, osiguranje potrebnih kapaciteta i osoblja. Predviđeno je da nekolicini ruskih doktora koje će Fogt izabrati bude omogućeno da provedu izvesno vreme u njegovom institutu u Berlinu kako bi se obučili tehnikama koje se tamo koriste u obradi mozga, kao i da steknu iskustva u istraživanju citoarhitektonike. U redovnim putovanjima između Berlina i Moskve, Fogt je dobijao 1.000$ za svaki boravak u Moskvi“ (Klatzo 2002, 31). 23 Govor je objavio kao Izveštaj o radu moskovskog Instituta mozga: „Bericht iiber die Arbeiten des Moskauer Staatsinstituts fiir Hirnforschung“, Journal for Psychologie und Neurologie, 1929, 40:108–118. Delove referata ovde navodim prema sažetom prevodu referata objavljenom u: Klatzo 2002, 33). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti To ga je navodilo da traga za korelacijama između izuzetnih intelektualnih kvalite- ta pojedinaca i citoarhitektonskih karakteristika pojedinih oblasti njihovog mozga. Ove korelacije su takođe mogle biti proučavane potragom za opštim citoarhitektonskim razlikama koje bi se identifikovale poređenjem mozgova intelektualne elite sa mozgovima mentalno ograničenih ili psihotičnih ljudi“ (Klatzo 2002, 23). Zainteresovani za prvi navedeni pravac Fogtovih istraživanja, članovi Poli- tbiroa su 19. februara 1925. godine nemačkom naučniku i njegovim ruskim ko- legama otvoreno postavili pitanje: „Može li istraživanje citoarhitektonike moz- ga ukazati na materijalnu zasnovanost genijalnosti V. I. Lenjina?“ (Источник 1994, 73). Fogtov pozitivan odgovor rezultovao je njegovim imenovanjem za direktora moskovskog Instituta mozga, kao i odlaskom na obuku u Berlin neko- licine njegovih ruskih saradnika.22 Naravno, osim naučne reputacije Fogt je po- sedovao „političku podobnost“ za obavljanje odgovornog i politički osetljivog posla u Moskvi – bio je simpatizer nemačke socijaldemokratije i jedan od osni- vača berlinskog ogranka „Društva prijatelja SSSR“, blizak prijatelj diplomate i budućeg sovjetskog ministra spoljnih poslova Maksima Litvinova, a njegovo angažovanje preporučila je i Klara Cetkin (Zetkin). Posle obuke nekoliko sovjetskih neurologa u Berlinu i uvoza potrebne teh- ničke opreme (mikrotona za rezanje uzoraka mozga debljine 20 mikrona), Fogt i saradnici su, nakon sečenja Lenjinovog mozga na nekoliko „blokova“ i njiho- Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 571 Lൾඇඃංඇඈඏ ආඈඓൺ඄ vog zalivanja u parafin, do kraja 1927. godine napravili preko 30.000 preparata spremnih za mikroskopsko istraživanje. Prve rezultate dvogodišnjeg rada Fogt je saopštio pred vodećim sovjetskim lekarima i političkim zvaničnicima u In- stitutu za proučavanje mozga 10. novembra 1929. godine.23 Ukratko, njegov najvažniji nalaz ticao se veličine piramidalnih ćelija u trećem sloju24 Lenjinovog korteksa: „U mnogim oblastima trećeg sloja kore velikog mozga, a naročito u njegovim dubljim delovima, našao sam piramidalne ćelije takve veličine i brojnosti kakve nikada ranije nisam video“. Pored toga, kao i u slučaju drugih „elitnih mozgova“ koje je Fogt analizirao, treći sloj Lenjinovog korteksa bio je znatno širi od četvrtog.25 Pošto je smatrao da je veličina piramidalnih neurona u trećem sloju ključna za više intelektualne funkcije, Fogt je zaključio da se Lenjin može smatrati „atletom asocijativnog mišljenja“ (Assoziationsathleten) jer upravo „opisani veliki neuroni čine razumljivim Lenjinovu izuzetnu brzinu u shvatanju i razmišljanju, kao i njegov osećaj za realnost“. 24 Fogt je razlikovao sedam slojeva korteksa koji se po svojoj ćelijskoj strukturi i funkciji međusobno razlikuju. 25 „Činilo se da su Fogtova citoarhitektonska istraživanja otkrivala izvesnu oppštu razliku između mozgova veoma inteligentnih pojedinaca, pripadnika ‘elite’ i mozgova kriminalaca i retardiranih osoba. Razlika se zasnivala na opozitnom razvoju trećeg i četvrtog sloja kore korteksa. Jer, dok je treći sloj kod ‘intelektualaca’ upadljiv po gustini i veličini njegovih neurona, četvrti je znatno uzaniji i nerazvijeniji – obrnut odnos je identifikovan u mozgu kriminalaca i mentalno retardiranih pojedinaca“( Klatzo 2002, 42). 27 Videti Dopis komesara Vojno-medicinske akademije Lamkina (11. 01. 1928) o stavovima ruskih neurologa koji smatraju da „Fogtova škola danas više ne daje sve ono što se u toj oblasti moglo i trebalo uraditi korišćenjem drugih istraživačkih pristupa“(Источник 1994, 77–78). Staljin je bio upoznat s ovim dopisom. 26 „Tadašnja zajednica neurologa smatrala je te tvrdnje u naučnom pogledu sumnjivim interpretacijama, a postoje indicije da je kasnije i sam Fogt kajao što se suviše eksponirao u čitavoj toj aferi“ (Kreutzberg et al. 1992, 363). 28 Pol Gregori ključni razlog ovog oštrog napada na Fogta vidi u činjenici da je on bio izvan mogućnosti kontrole sovjetskog vrha: „Fogt je delovao u međunarodnim naučnim okvirima u kojima su debate i kontra-hipoteze dobrodošle, a ne u kontrolisanom okruženju sovjetske nauke. Njegovi nalazi o Lenjinovoj genijalnosti mogli su biti javno osporeni, pa čak i potpuno preokrenuti kontra-argumentom da Lenjinove ‘gigantske piramidalne ćelije’ mogu biti indikator mentalne retardacije. U ‘sovjetskoj nauci’ nije bilo protiv-argumenata, naročito kada je zvanični partijski stav bio da je Lenjin ‘genijalan’“ (Gregory 2008, 33). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti da se praktično odustane od daljih istraživanja – M. S.); (2) „da se prekinu odnosi s prof. Fogtom i da se u Berlin pošalju drugovi koji će od njega preuzeti isečke i dijapozitive Lenjinovog mozga kako bi se okončale mahinacije buržoaskih profesora“ (Источник 1994, 78–9).28 Uprkos iskazanoj „ideološkoj budnosti“ Steckog, njegovi predlozi nisu prihvaćeni – Odlukom Politbiroa od 13. 03. 1932. godine Institut je stavljen pod neposrednu kontrolu Naučnog komiteta Vlade (ЦИК СССР), a Fogt je, iako od 1930. godine nije više dolazio u Moskvu, ipak ostao (nominalni) direktor Instituta, dok su istra- živanja nastavljena pod rukovodstvom njegovog zamenika, ruskog neurologa Sarkisova (Саркисов). Dolaskom Hitlera na vlast Fogtov direktorski položaj u berlinskom Institutu bio je ugrožen – 1935. godine on je penzionisan, da bi ubrzo, uz obilatu donatorsku podršku porodice Krup (Krupp), u unutrašnjosti Nemačke (Neustadt) osnovao sopstveni, privatni institut (Institut für Hirnfors- chung und allgemeine Biologie). U Fogtovom životu „moskovski intermeco“ retardiranih pojedinaca.26 Odjeci ovih debata u nemačkoj štampi zabrinuli su sovjetske zvaničnike, pa su oni počeli da dovode u pitanje produžetak saradnje sa Fogtom i njegov ostanak na funkciji direktora moskovskog Instituta.27 Po- sebnu zabrinutost izazivale su vesti da je Fogt u Berlin odneo jedan preparat Lenjinovog mozga koji je pokazivao tokom svojih predavanja (poredeći ga s isečkom mozga „prostitutke“), kao i to što on u štampi nije odgovarao na „zlob- ne komentare“ o Lenjinu. U pismu Staljinu od 10. januara 1932. godine Aleksej Stecki (Стецкий, 1896–1938), rukovodilac kulturno-propagandnog odeljenja CK, kritički je referisao o stanju u Institutu i Fogtovom radu: „Profesor Fogt je na osnovu anatomske analize Lenjinovog mozga formulisao mehanističku teo- riju genijalnosti zasnovanu na postojanju velikog broja i svojevrsnog raspore- da piramidalnih ćelija“. Pismo je završio predlozima: (1) da se Lenjinov mozak preda Mauzoleju (tj. da se praktično odustane od daljih istraživanja – M. S.); (2) „da se prekinu odnosi s prof. Fogtom i da se u Berlin pošalju drugovi koji će od njega preuzeti isečke i dijapozitive Lenjinovog mozga kako bi se okončale mahinacije buržoaskih profesora“ (Источник 1994, 78–9).28 Uprkos iskazanoj „ideološkoj budnosti“ Steckog, njegovi predlozi nisu prihvaćeni – Odlukom Politbiroa od 13. 03. 1932. godine Institut je stavljen pod neposrednu kontrolu Naučnog komiteta Vlade (ЦИК СССР), a Fogt je, iako od 1930. godine nije više dolazio u Moskvu, ipak ostao (nominalni) direktor Instituta, dok su istra- živanja nastavljena pod rukovodstvom njegovog zamenika, ruskog neurologa Sarkisova (Саркисов). Institut za istraživanje mozga: ćelijska arhitektura genijalnosti U zaključku svog referata Fogt je istakao neophodnost daljeg komparativnog istraživanja „arhi- tektonike“ Lenjinovog mozga i mozgova drugih istaknutih pojedinaca, kao i prosečnih pripadnika različitih etničkih grupa Sovjetskog Saveza. Mnogo godi- na kasnije, pred svoju smrt, on je u razgovoru s jednim svojim saradnikom (prof. Heinz Schulze) priznao: „Prirodno, snažno sam naglasio moguću vezu velikih neurona trećeg sloja kore velikog mozga sa Lenjinovim asocijativnim sposob- nostima da bih zadovoljio očekivanja prisutnog Komesara zdravlja Semaška – od njegovog izveštaja Politbirou zavisila je podrška našem (moskovskom) Institutu“ (Klatzo 2002, 118). Skromni rezultati istraživanja bili su popularisani u sovjetskoj štampi i pu- blicistici kao naučno zasnovana potvrda da je Lenjinov mozak „nesumnjivo prototip mozga budućeg natčoveka“ (vid. Спивак 2010, 18). Ali, pojedini na- učnici u Nemačkoj su Fogtovu teza o direktnoj korelaciji između veličine pira- midalnih ćelija trećeg sloja korteksa i izuzetne intelektualne obdarenosti kriti- kovali ističući slučajeve postojanja istih takvih ćelija u moždanoj kori mentalno 24 Fogt je razlikovao sedam slojeva korteksa koji se po svojoj ćelijskoj strukturi i funkciji međusobno razlikuju. Č 25 „Činilo se da su Fogtova citoarhitektonska istraživanja otkrivala izvesnu oppštu razliku između mozgova veoma inteligentnih pojedinaca, pripadnika ‘elite’ i mozgova kriminalaca i retardiranih osoba. Razlika se zasnivala na opozitnom razvoju trećeg i četvrtog sloja kore korteksa. Jer, dok je treći sloj kod ‘intelektualaca’ upadljiv po gustini i veličini njegovih neurona, četvrti je znatno uzaniji i nerazvijeniji – obrnut odnos je identifikovan u mozgu kriminalaca i mentalno retardiranih pojedinaca“( Klatzo 2002, 42). Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 572 Mංඅൺඇ Sඎൻඈඍංම retardiranih pojedinaca.26 Odjeci ovih debata u nemačkoj štampi zabrinuli su sovjetske zvaničnike, pa su oni počeli da dovode u pitanje produžetak saradnje sa Fogtom i njegov ostanak na funkciji direktora moskovskog Instituta.27 Po- sebnu zabrinutost izazivale su vesti da je Fogt u Berlin odneo jedan preparat Lenjinovog mozga koji je pokazivao tokom svojih predavanja (poredeći ga s isečkom mozga „prostitutke“), kao i to što on u štampi nije odgovarao na „zlob- ne komentare“ o Lenjinu. U pismu Staljinu od 10. januara 1932. godine Aleksej Stecki (Стецкий, 1896–1938), rukovodilac kulturno-propagandnog odeljenja CK, kritički je referisao o stanju u Institutu i Fogtovom radu: „Profesor Fogt je na osnovu anatomske analize Lenjinovog mozga formulisao mehanističku teo- riju genijalnosti zasnovanu na postojanju velikog broja i svojevrsnog raspore- da piramidalnih ćelija“. Pismo je završio predlozima: (1) da se Lenjinov mozak preda Mauzoleju (tj. 29 Fogtov naučni rad je bio veoma obiman, a u mnogim oblastima neurologije po- stigao je zapažene rezultate čija procena zahteva specijalističko znanje. Nezavisno od istraživanja Lenjinovog mozga, on danas slovi kao jedan od utemeljivača moderne ne- urologije. 30 Na primer, mozak tvorca periodičnog sistema elemenata D. I. Mendeljejeva (1834–1907), lekara A. Koževnikova (1836-1902), S. Korsakova (1854–1900), matematičara P. Behmetjeva (1860-1913), kao i kompozitora A. Rubinštajna (1829- 1894), pisca Saljtikova-Šćedrina (1826–1889). Vid. u: Капустин (1925); Vein, (2008) Institut za istraživanje mozga: ćelijska arhitektura genijalnosti Dolaskom Hitlera na vlast Fogtov direktorski položaj u berlinskom Institutu bio je ugrožen – 1935. godine on je penzionisan, da bi ubrzo, uz obilatu donatorsku podršku porodice Krup (Krupp), u unutrašnjosti Nemačke (Neustadt) osnovao sopstveni, privatni institut (Institut für Hirnfors- chung und allgemeine Biologie). U Fogtovom životu „moskovski intermeco“ 28 Pol Gregori ključni razlog ovog oštrog napada na Fogta vidi u činjenici da je on bio izvan mogućnosti kontrole sovjetskog vrha: „Fogt je delovao u međunarodnim naučnim okvirima u kojima su debate i kontra-hipoteze dobrodošle, a ne u kontrolisanom okruženju sovjetske nauke. Njegovi nalazi o Lenjinovoj genijalnosti mogli su biti javno osporeni, pa čak i potpuno preokrenuti kontra-argumentom da Lenjinove ‘gigantske piramidalne ćelije’ mogu biti indikator mentalne retardacije. U ‘sovjetskoj nauci’ nije bilo protiv-argumenata, naročito kada je zvanični partijski stav bio da je Lenjin ‘genijalan’“ (Gregory 2008, 33). Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 573 Lൾඇඃංඇඈඏ ආඈඓൺ඄ (Klatzo) bio je završen, a o svom istraživanju Lenjinovog mozga on više nije pisao i govorio.29 ), p j ( ) у ( ); , ( ) 31 Бехтерев В.М. „О создании Пантеона в СССР: В порядке обсуждения“, Известия,1927, 19 июня. 29 Fogtov naučni rad je bio veoma obiman, a u mnogim oblastima neurologije po- stigao je zapažene rezultate čija procena zahteva specijalističko znanje. Nezavisno od istraživanja Lenjinovog mozga, on danas slovi kao jedan od utemeljivača moderne ne- urologije. 30 Na primer, mozak tvorca periodičnog sistema elemenata D. I. Mendeljejeva (1834–1907), lekara A. Koževnikova (1836-1902), S. Korsakova (1854–1900), matematičara P. Behmetjeva (1860-1913), kao i kompozitora A. Rubinštajna (1829- 1894), pisca Saljtikova-Šćedrina (1826–1889). Vid. u: Капустин (1925); Vein, (2008) 31 Бехтерев В.М. „О создании Пантеона в СССР: В порядке обсуждения“, Известия,1927, 19 июня. 34 „Teza o korelaciji specifičnih citoarhitektonskih karakteristika korteksa sa nekim Lenjinovim izuzetnim mentalnim sposobnostima zahtevala bi analizu psihološkog portreta njegovog intelekta, te je Fogt u svom referatu sugerisao da bi to zahtevalo korišćenje specijalnog upitnika sastavljenog u tu svrhu. (Klatzo 2002, 35). Sovjetski „Panteon mozgova“ Sovjetski „Panteon mozgova“ Jedan od pravaca budućeg rada Instituta, na koji je Fogt ukazao u svom re- feratu iz 1928. godine, odnosio se na neophodnost obimnijih komparativnih istraživanja u kojima bi se Lenjinov mozak uporedio s primercima mozgova drugih istaknutih pojedinaca. U prošlosti – kako u Evropi, tako i u carskoj Rusiji – porodice preminulih „velikana“ teško su davale dozvolu da se organi njihovih bližnjih pretvore u objekte naučnih istraživanja. Ipak, mozgovi izvesnog broja istaknutih pojedinaca – pretežno prirodnjaka i lekara, kao i pojedinih umetnika – porodice preminulih „velikana“ teško su davale dozvolu da se organi njihovih bližnjih pretvore u objekte naučnih istraživanja. Ipak, mozgovi izvesnog broja istaknutih pojedinaca – pretežno prirodnjaka i lekara, kao i pojedinih umetnika – bili su i pre Oktobarske revolucije analizirani i opisivani u ruskoj naučnoj lite- raturi.30 Neurolog i psihijatar, akademik Vladimir Behterjev (Бехтерев, 1857– 1927) koji je istraživao mozak Mendeljejeva, bio je u Rusiji jedan od najuporni- jih zagovornika ideje o potrebi sistematskog sakupljanja, čuvanja i proučavanja mozgova istaknutih pojedinaca. Ugledni osnivač i direktor Psiho-neurološkog instituta u Sankt Petersburgu (1907), predložio je 1927. godine osnivanje so- vjetskog „Panteona mozgova“31 koji bi se razlikovao od pariskog jer bi, pored memorijalne, imao veliku naučnu vrednost: „Panteon koji bi mogla sagraditi Sovjetska Rusija bi morao biti veoma ko- risna naučna institucija, a istovremeno i javna institucija dostupna pogledu svih onih koji to žele. On bi bio kolekcija konzervisanih mozgova koji su pripada- li talentovanim ljudima bez obzira na oblasti delatnosti kojima su se bavili, a istovremeno bi propagirao materijalistički pogled na razvoj ljudske stvaralačke aktivnosti“ (cit. prema: Спивак 2010, 23). Pozivajući se na dobrobit nauke (napredak u odgonetanju „tajanstvene sfin- ge koju nazivamo genijem“), Behterjev je sasvim realistično procenio da se u Sovjetskom Savezu osnovna teškoća u ostvarenju čitavog poduhvata – otpor srodnika – može jednostavno otkloniti dekretom vlasti. Zato je on predložio da 29 Fogtov naučni rad je bio veoma obiman, a u mnogim oblastima neurologije po- stigao je zapažene rezultate čija procena zahteva specijalističko znanje. Nezavisno od istraživanja Lenjinovog mozga, on danas slovi kao jedan od utemeljivača moderne ne- urologije. Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 574 Mංඅൺඇ Sඎൻඈඍංම se formira jedan poseban državni komiteti koji bi imao ovlašćenja da formuliše kriterijume izbora u Panteon, kao i da naredi preuzimanje organa nakon autopsije istaknutih pokojnika. 32 Glavni argument ministra zdravlja bio je Lenjinov mozak: „U Institutu za proučavanje mozga nalazi se mozak V.- I. Lenjina. Samim tim, suštinski je predodređeno da Panteon bude osnovan u Institutu. Ako bi se on nalazio u Lenjingradu, nužno bi bilo tamo preneti Lenjinov mozak. Transport 30 hiljada preparata bi, samo po sebi, bilo veoma težak posao, povezan sa velikim opasnostima. U Institutu mozak V. I. nalazi se pod stalnom zaštitom OGPU“ (cit. prema Спивак 2010, 43). p ( p ) 33 Videti dokumentarni tv film «Комната №19»: https://www.youtube.com/ watch?v=H-zUAICzBmE Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) Sovjetski „Panteon mozgova“ Prema zamisli Behterjeva, najpogodnije mesto za osniva- nje Panteona bi bio njegov Institut, ali formiranje moskovskog Instituta za prou- čavanje (Lenjinovog) mozga uticalo je da od „dve prestonice“, za sedište „Pan- teona“, ipak bude određena ona starija.32 Pošto je 24. decembra 1927. godine usled trovanja hranom iznenadno umro u Moskvi, mozak Behterjeva pridružen je Lenjinovom – njegova ideja o „Svesaveznom Panteonu“ ostvarena je u okviru moskovskog Instituta u kome je formirana kolekcija „elitnih mozgova“. Umesto anonimnih beskućnika i kriminalaca, sada su komparativni okvir za istraživanje Lenjinovog mozga činili egzemplari sakupljeni u Panteonu. Vremenom, kolek- cija je postajala sve bogatija – mozgovi političara (Kirov, Lunačarski, Kujbišev, Kalinjin, Menžinski, K. Cetkin, N. Krupska, Staljin...), književnika (Majakovski, Bagricki, Bjeli, Gorki, Stanislavski...) i naučnika (Pavlov, Vigotski, Bogdanov, Cijalkovski, Mičurin, Landau, Saharov) sačuvani su u moskovskom Institutu. Istina, oni nisu bili dostupni pogledu posetilaca – u sobi br. 19. oronule zgrade u kojoj je smešten nekadašnji Institut (sada Odeljenje istraživanja mozga ’Nauč- nog centra neurologije’ Ruske akademije medicinskih nauka), njihovi parafinom konzervirani mozgovi danas leže na policama zatvorenog drvenog ormana, za- motani u papir, poput robe u sovjetskim prodavnicama.33 S obzirom na to da je, istina u nešto izmenjenom obliku, zamisao Behterjeva o sovjetskom Panteonu bila ostvarena, moglo se očekivati da će istraživanja nadahnuta Fogtovom metodologijom tokom tridesetih godina doneti prve oz- biljnije rezultate. Tim pre što su sovjetski istraživači usvojili i drugu Fogto- vu sugestiju prema kojoj je u rešavanju problema identifikovanja veze između strukture i funkcija pojedinačnog mozga važno imati uvid u individualno psiho- loške karakteristike i biografiju njegovog „vlasnika“.34 Stoga je dokumentaci- ona osnova čitavog istraživanja znatno proširena sakupljanim svedočanstava o ličnim osobinama, temperamentu, navikama i svakodnevnom životu osobe čiji je mozak predmet ćelijsko-arhitektonske analize. Za tu svrhu obavljen je niz 32 Glavni argument ministra zdravlja bio je Lenjinov mozak: „U Institutu za proučavanje mozga nalazi se mozak V.- I. Lenjina. Samim tim, suštinski je predodređeno da Panteon bude osnovan u Institutu. Ako bi se on nalazio u Lenjingradu, nužno bi bilo tamo preneti Lenjinov mozak. Transport 30 hiljada preparata bi, samo po sebi, bilo veoma težak posao, povezan sa velikim opasnostima. U Institutu mozak V. I. nalazi se pod stalnom zaštitom OGPU“ (cit. prema Спивак 2010, 43). 33 Videti dokumentarni tv film «Комната №19»: https://www.youtube.com/ watch?v=H-zUAICzBmE Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 35 Cenzurisanje odgovora Krupske je rukovođeno potrebom da se „empirijski Lenjin“ usaglasi sa već stvorenim predstavom o „Vođi revolucije“: Njegov glas nije bio, kao u odgovoru Krupske, „tenor“, već „bariton“; vid mu je bio odličan (iako je, po Krupskoj, na jedno oko slabije video); iz ankete je izbačeno da je voleo laku literaturu (naglašen je interes ka enciklopedijskim izdanjima), itd. Snažna volja, odlučnost i enormna marljivost, dopunjene su isticanjem emocionalnosti, ljudske nežnosti i samokritičnosti. 36 Aleksandar Bogdanov (Богданов, 1873–1928) bio je filozof i ekonomista koga je Lenjin oštro kritikovao zbog „empiriokriticizma“. Po profesiji lekar, poznat Sovjetski „Panteon mozgova“ 2 (2016) 575 Lൾඇඃංඇඈඏ ආඈඓൺ඄ posebno koncipiranih intervjua s osobama iz neposrednog životnog okruženja onih ličnosti čiji su mozgovi bili predmet analize. Tako je Nadežda Krupska tokom 1935. godine u dva navrata odgovarala na pitanja o različitim osobina- ma svog muža – njeni odgovori su sačuvani i nedavno objavljeni (vid. Спивак 2010, 52–58). Dostupna sadržina intervjua zanimljivija je po onome što je u njima ostalo prećutano (npr. skup pitanja iz Upitnika o seksualnosti), kao i po naknadno izvršenim korekcijama odgovora Krupske,35 nego po tome šta je o samom Lenjinu u njemu rečeno. S obzirom na to da je sakralizacija Lenjinovog lika tokom protekle decenije već bila izvedena, to naknadno sakupljanje sve- dočanstava o njegovom karakternim osobinama nije moglo dati nekakva nova saznanja koja bi bila disonantna u odnosu na već uspostavljeni kanon. Zato su u istorijsko-biografskom pogledu znatno zanimljiviji sačuvani materijali koje su saradnici Instituta prikupili o drugim ličnostima uvrštenim u „Panteon mozgo- va“– poput dostupnih intervjua o životima i osobinama Majakovskog, Bjelog i Bagrickog (vid. u: Спивак 2010, 59–243). Nakon čitave decenije istraživanja, 27. maja 1936. godine zamenik direktora Instituta Sarkisov dostavio je Politbirou i Vladi izveštaj o rezultatima proučava- nja Lenjinovog mozga. Uz kratki rezime pisan za laike, on je priložio i opširniji tekst (153 stranice) u kome je objašnjen postupak i tok istraživanja, kao i 12 albuma u kojima je sabrano po pedeset fotografija sačinjenih preparata. Zaklju- čak je formulisan na samom početku dopisa, neposredno posle izlaganja kratke istorije institutskog rada: „Dobijeni rezultati istraživanja mozga V. I. Lenjina daju nam za pravo da govorimo o izuzetno složenoj organizaciji mozga V. I. u pogledu čitavog niza karakteristika koje se tiču kako spoljašnjeg izgleda mozga – moždanih vijuga (gyri) i moždanih žlebova (sulci), tako i niza osobenih karakteristika koje se tiču njegove suptilne strukture vidljive pomoću mikroskopskog istraživanja“ (Источник 1994, 83). 37 Sarkasov je svoje teze numerički ilustrovao: „Tako, na primer, u polju 71 veličina ćelija trećeg sloja dostiže na nekim mestima 56-60 mikrona, a u čitavom tom polju kod Lenjina je srednja vrednost 32,7 mikrona, dok je kod drugih istraženih mozgova 24,3 mikrona (Skvorcov-Stepanov), 27,1 mikrona (Majakovski) i 28,2 mikrona (Bogdanov)“ I u svim drugim kvantifikovanim karakteristikama arhitektonike mozga, Lenjin nadmašuje ostale (vid. Источник 1994, 84-85). Sovjetski „Panteon mozgova“ imao tako visok stepen organizacije da je i tokom bolesti, bez obzira na velika oštećenja, ostao na veoma visokom nivou funkcio- nalnosti“ (Источник 1994, 85). * * * U završnom delu pomenutog izveštaja, Sarkisov je ukazao na pravce daljeg razvoja naučnih istraživanja u Institutu koja nisu u bila u neposrednoj vezi s proučavanjem Lenjinovog mozga. Naučni napredak u različitim oblastima ne- urologije, smatrao je on, omogućiće saradnicima Instituta da se u budućnosti vrate istraživanju mozga zbog koga je ova naučna institucija i osnivana. Ne dovodeći u pitanje uspehe saradnika Instituta koji je tokom decenija menjao organizacionu formu i status, danas možemo tvrditi da ta optimistička očeki- vanja o budućem napretku u proučavanju Lenjinovog mozga nisu ostvarena. U osnovi, dalje od ponavljanja prvih Fogtovih nalaza nije se otišlo o čemu, između ostalog, svedoči činjenica da detaljniji rezultati istraživanja nikada nisu objavljeni.38 Najnoviji rad o rezultatima istraživanja koji je objavljen u ruskom naučnom časopisu 1993. godine, sudeći na osnovu dostupnog rezima, je po pokušaju da se zamenom krvi uspori proces starenja – umro je usled posledica eksperimenta sa primanjem krvi. Ivan Skvorcov-Stepanov (Скворцов-Степанов, 1870–1928) bio je partijski rukovodilac, prevodilac Marksa i direktor Instituta Lenjina. je po pokušaju da se zamenom krvi uspori proces starenja – umro je usled posledica eksperimenta sa primanjem krvi. Ivan Skvorcov-Stepanov (Скворцов-Степанов, 1870–1928) bio je partijski rukovodilac, prevodilac Marksa i direktor Instituta Lenjina. 37 Sarkasov je svoje teze numerički ilustrovao: „Tako, na primer, u polju 71 veličina ćelija trećeg sloja dostiže na nekim mestima 56-60 mikrona, a u čitavom tom polju kod Lenjina je srednja vrednost 32,7 mikrona, dok je kod drugih istraženih mozgova 24,3 mikrona (Skvorcov-Stepanov), 27,1 mikrona (Majakovski) i 28,2 mikrona (Bogdanov)“ I u svim drugim kvantifikovanim karakteristikama arhitektonike mozga, Lenjin nadmašuje ostale (vid. Источник 1994, 84-85). 37 Sarkasov je svoje teze numerički ilustrovao: „Tako, na primer, u polju 71 veličina ćelija trećeg sloja dostiže na nekim mestima 56-60 mikrona, a u čitavom tom polju kod Lenjina je srednja vrednost 32,7 mikrona, dok je kod drugih istraženih mozgova 24,3 mikrona (Skvorcov-Stepanov), 27,1 mikrona (Majakovski) i 28,2 mikrona (Bogdanov)“ I u svim drugim kvantifikovanim karakteristikama arhitektonike mozga, Lenjin nadmašuje ostale (vid. Источник 1994, 84-85). 38 Poslednji put kada je u CK KPSS raspravljano o rezultatima istraživanja, 20. oktobra 1969. godine, zaključeno je: „Bez obzira na to što su rezultati citoarhitektonskog istraživanja mozga V. I. Lenjina od velikog naučnog interesa, treba se uzdržati od njihovog publikovanja…. Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) Sovjetski „Panteon mozgova“ Takav zaključak zasnivao se na citoarhitektonskom proučavanju Lenjino- vog mozga tokom koga je, kao komparativni okvir, analizirano deset hemisfera mozga običnih ljudi i šest hemisfera tri „elitna mozga“ – Majakovskog, Bogda- nova i Skvorcova-Stepanova.36 Oslanjajući se na Fogtovo istraživanje veličine 35 Cenzurisanje odgovora Krupske je rukovođeno potrebom da se „empirijski Lenjin“ usaglasi sa već stvorenim predstavom o „Vođi revolucije“: Njegov glas nije bio, kao u odgovoru Krupske, „tenor“, već „bariton“; vid mu je bio odličan (iako je, po Krupskoj, na jedno oko slabije video); iz ankete je izbačeno da je voleo laku literaturu (naglašen je interes ka enciklopedijskim izdanjima), itd. Snažna volja, odlučnost i enormna marljivost, dopunjene su isticanjem emocionalnosti, ljudske nežnosti i samokritičnosti. 36 Aleksandar Bogdanov (Богданов, 1873–1928) bio je filozof i ekonomista koga je Lenjin oštro kritikovao zbog „empiriokriticizma“. Po profesiji lekar, poznat Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) 576 Mංඅൺඇ Sඎൻඈඍංම piramidalnih ćelija trećeg sloja i prelaznih granica različitih oblasti korteksa, Sarkisov je tvrdio da je Lenjinov mozak znatno složeniji i razvijeniji ne samo od „običnih“, nego i od sva tri proučavana „elitna mozga“.37 Sličan rezultat dobijen je i makro-morfološkim poređenjem s ostalim mozgovima istaknutih pojedinaca koji su se nalazili u Panteonu – u odnosu na sve njih, Lenjinov mozak izdvajao se kako gustinom moždanih vijuga u prednjem režnju (koji je „zadužen“ za više mentalne funkcije), tako i veličinom tog režnja u odnosu na ukupnu površinu mozga. Rečju, oba istraživačka pristupa potvrđivala su Lenjinovu superiornost koju, prema Sarkisovu, ni teška bolest nije mogla da naruši: „Poređenje vodi zaključku da je mozak V. I. imao tako visok stepen organizacije da je i tokom bolesti, bez obzira na velika oštećenja, ostao na veoma visokom nivou funkcio- nalnosti“ (Источник 1994, 85). piramidalnih ćelija trećeg sloja i prelaznih granica različitih oblasti korteksa, Sarkisov je tvrdio da je Lenjinov mozak znatno složeniji i razvijeniji ne samo od „običnih“, nego i od sva tri proučavana „elitna mozga“.37 Sličan rezultat dobijen je i makro-morfološkim poređenjem s ostalim mozgovima istaknutih pojedinaca koji su se nalazili u Panteonu – u odnosu na sve njih, Lenjinov mozak izdvajao se kako gustinom moždanih vijuga u prednjem režnju (koji je „zadužen“ za više mentalne funkcije), tako i veličinom tog režnja u odnosu na ukupnu površinu mozga. Rečju, oba istraživačka pristupa potvrđivala su Lenjinovu superiornost koju, prema Sarkisovu, ni teška bolest nije mogla da naruši: „Poređenje vodi zaključku da je mozak V. I. je po pokušaju da se zamenom krvi uspori proces starenja – umro je usled posledica eksperimenta sa primanjem krvi. Ivan Skvorcov-Stepanov (Скворцов-Степанов, 1870–1928) bio je partijski rukovodilac, prevodilac Marksa i direktor Instituta Lenjina. Sovjetski „Panteon mozgova“ Mnoge anatomsko-fiziološke paralele između strukture mozga i nivoa njegove delatnosti ostaju sporne“ (Источник 1994, 87). ssues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 577 Lൾඇඃංඇඈඏ ආඈඓൺ඄ u osnovi samo ponavlja glavne nalaze koje je još Sarkisov istakao u svom izveštaju.39 u osnovi samo ponavlja glavne nalaze koje je još Sarkisov istakao u svom izveštaju.39 j Razloge upadljivo skromnih rezultata dugog naučnog istraživanja može- mo tražiti u dva osnovna smera. Prvi bi se ticao heurističke vrednosti polazne Fogtove teorijske „paradigme“ i iz nje izvedene istraživačke metodologije. I bez ekspertskog poznavanja razvoja savremene neurologije, očigledno je da danas „citoarhitektonski pristup“ pre predstavlja poglavlje istorije neurologije, nego aktuelni istraživački program. Ipak, za laika u oblasti neurologije, značajniji je drugi razlog za svojevrsni „ćorsokak“ u koji je zapalo prikazano istraživanje Lenjinovog mozga. On se tiče socijalno-istorijske i političke sfere života, tj. činjenice da je vremenom sama potreba za „materijalističkom potvrdom“ Le- njinove genijalnosti bivala sve manja. Naime, uspešnom konsolidacijom kulta Lenjina drugim sredstvima, kao i pretvaranjem lenjinizma u „obavezujuće uče- nje i rukovodstvo za akciju“, smanjena je potreba da se na „klizavom terenu“ prirodnonaučnog istraživanja dokazuje ono u šta se već dovoljno čvrsto veruje. Za potvrdu te vere nije bilo nužno korišćenje komplikovane i laicima nerazu- mljive nauke poput neurologije – pri ruci su bila i znatno jednostavnija sredstva. Tako je, na primer, čak i Lenjinov stomatolog mogao dati svoj „naučni“ dopri- nos toj popularnoj „karakterologiji“: „Sećajući se zuba V. I. Lenjina, pomislio sam – ne može li se o karakteru čoveka suditi na osnovu njegovih zuba?... I ako posebno govorimo o Lenjinovim zubima, onda možemo reći da su oni bili jaki po svojoj konstrukciji, žute boje (po Ašovoj paleti – F5), generalno pravilni po obliku, rasporedu i ugrizu. Gornji prednji zubi su bili široki (širina donje ivice prednjih zuba bila je gotovo jednaka dužini njihove krunice), sa jako razvijenim zagrižajnom ivicom usmerenom unutra (ka nepcu). Nesumnjivo, njegovi zubi su savršeno bili u skladu s opštim utiskom o iskrenosti, čvrstini i snazi njegovog karaktera“ (cit. prema: Петренко 1990, 182). 39 Адрианов О. С., Боголепова И. Н., Блинков С. М., Кукуев Л. А. „Исследование мозга В. И. Ленина“, Успехи физиологических наук, Москва: РАН, 1993. 24 (3): 40–52. Dostupan mi je bio samo engleski rezime ovog rada. 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Milan Subotić Institute of European Studies, Belgrade Lenin`s Brain: An attempt of “materialistically based genius” In this paper, an attempt to confirm the faith in Lenin`s genius by the help of the neurological research of Lenin`s brain has been presented and interpreted. Considering “the cult of personality” as a permanent and significant character- istic of the ‘Soviet-type’ political system, the author has reconstructed an aspect of the posthumous (mis)use of the Lenin`s body undertaken to consolidate the regime and strengthen the legitimacy of Bolshevik power. A frequently thema- Issues in Ethnology and Anthropology, n. s. Vol. 11 Is. 2 (2016) 581 Lൾඇඃංඇඈඏ ආඈඓൺ඄ tized symbolic and political meaning of the embalming and exposing of Lenin`s body in the Mausoleum has in this paper been amended by taking into consider- ation the activities of the Moscow Brain Research Institute which was founded in order to prove the genius of the October Revolution Leader. Regardless of the limited results, the use of science in creating and strengthening the cult of a leader presented, in the author`s opinion, a paradoxical combination of the enlightenment genealogy of the Marxist theory and Soviet political practice. Key words: Lenin, brain, a cult of personality, materialism, neurology, science, politics Le cerveau de Lénine: sur une tentative de « fondement matérialiste de la génialité» Le cerveau de Lénine: sur une tentative de « fondement matérialiste de la génialité» Le cerveau de Lénine: sur une tentative de « fondement matérialiste de la génialité» Ici est présentée et interprétée la tentative de confirmer la croyance dans la génialité de Lénine à l’aide des recherches neurologiques effectuées sur son cerveau. En considérant que le « culte de la personnalité» est une caractéristique durable et importante du système de type soviétique, l’auteur a présenté un as- pect de l’utilisation (abus) posthume du corps de Lénine entreprise afin de con- solider le régime et de renforcer la légitimité du pouvoir bolchévique. Souvent thématisée, la signification symbolique et politique du processus d’embaume- ment et de l’exposition du corps de Lénine dans le Mausolée, a été complétée dans ce travail par une considération de l’activité de l’Institut pour les recherch- es sur le cerveau fondé pour prouver la génialité du chef de la Révolution d’oc- tobre. Même si les résultats en sont limités, cette utilisation de la science pour créer et pour renforcer le culte du chef représentait, selon l’opinion de l’auteur, une alliance paradoxale entre la généalogie éclairée de la théorie marxiste et la pratique soviétique politique. Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) Lenin`s Brain: An attempt of “materialistically based genius” Mots clés: Lénine, cerveau, culte de la personnalité, matérialisme, neurologie, science, politique Mots clés: Lénine, cerveau, culte de la personnalité, matérialisme, neurologie, science, politique Primljeno / Received: 17.03.2016 Prihvaćeno / Accepted: 09.05.2016 Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016) Етноантрополошки проблеми, н. с. год. 11 св. 2 (2016)
https://openalex.org/W4381106267	https://zenodo.org/records/8051536/files/807-812.pdf	Indonesian	null	Motivasi Kinerja Karyawan UMKM Seblak Sadang Cabang Kosambi Ditengah Pandemi Virus Covid-19	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,748	2023 Nanggroe : Jurnal Pengabdian Cendikia Madani: Jurnal Ilmiah Multidisiplin Volume 1, Nomor 5, Juni 2023 e-ISSN: 2986-6340 DOI: https://doi.org/10.5281/zenodo.805 Madani: Jurnal Ilmiah Multidisiplin Volume 1, Nomor 5, Juni 2023 e-ISSN: 2986-6340 DOI: https://doi.org/10.5281/zenodo.8051536 Abstrak Observasi ini bermaksud untuk mengetahui bagaimana UMKM Seblak Sadang Cabang Kosambi di Kabupaten Karawang dalam menghadapi pandemi Covid-19. Wawancara langsung dengan para informan, terutama para pekerja di UMKM Seblak Sadang Kabupaten Karawang cabang Kosambi dilakukan dalam observasi ini sebagai bagian dari metode pendekatan kualitatif. Berdasarkan hasil observasi diketahui bahwa disiplin kerja dan motivasi berpengaruh terhadap kinerja pegawai. Karyawan bertanggung jawab atas pekerjaan mereka dan didorong oleh rasa penghargaan yang mereka terima. Adanya tujuan yang ingin dicapai, keinginan untuk berprestasi dan menerima bonus, adanya peraturan yang fleksibel dan tidak membatasi agar karyawan termotivasi, dan masih banyak faktor lain yang berdampak pada kinerja karyawan. Berdasarkan Observasi UMKM Seblak Sadang kesimpulan yang di ambil adalah sebaiknya menerapkan sistem penilaian dalam kinerja karyawan dilakukan secara individu dengan pokok-pokok standar penilaian kerja, dan selalu memberi motivasi kerja sehingga kompensasi dapat diberikan secara adil sesuai dengan prestasi kerjanya. Untuk para karyawan sebaiknya lebih mengembangkan prestasi dan produktivitas kerjanya, dan bertanggung jawab atas pekerjaannya sehingga berdampak baik untuk UMKM Seblak Sadang. Kata kunci: Motivasi, Kinerja Karyawan, UMKM Seblak Sadang Kata kunci: Motivasi, Kinerja Karyawan, UMKM Seblak Sadang Motivasi Kinerja Karyawan UMKM Seblak Sadang Cabang Kosambi Ditengah Pandemi Virus Covid-19 Rostiani1, Pratiwi Anastasya Putri2, Dwi Epty Hidayaty3, Santi Pertiwi Hari Sandi4 1234Prodi Manajemen, Ekonomi Dan Bisnis, Universitas Buana Perjuangan Karawang Email: mn21.rostiani@mhs.ubpkarawang.ac.id1, mn21.pratiwiputri@mhs.ubpkarawang.ac.id2, dwi.epty@ubpkarawang.ac.id3, santi.pertiwi@ubpkarawang.ac.id4 Keywords: Motivation, Employee Performance, Seblak Sadang MSMEs Abstract This observation intends to find out how the Seblak Sadang UMKM Kosambi Branch in Karawang Regency is dealing with the Covid-19 pandemic. Direct interviews with informants, especially workers at the UMKM Seblak Sadang Karawang branch of the Kosambi branch were carried out in this observation as part of a qualitative approach method. Based on the results of observations it is known that work discipline and motivation affect employee performance. Employees take responsibility for their jobs and are driven by the sense of appreciation they receive. There are goals to be achieved, the desire to achieve and receive bonuses, the existence of regulations that are flexible and not restrictive so that employees are motivated, and there are many other factors that have an impact on employee performance. Based on the Seblak Sadang UMKM Observations, the conclusions drawn are that it is better to apply an appraisal system in employee performance carried out individually with the main points of work evaluation standards, and always provide work motivation so that compensation can be given fairly according to work performance. Employees should further develop their performance and work productivity, and be responsible for their work so that it has a good impact on the Seblak Sadang MSMEs. Keywords: Motivation, Employee Performance, Seblak Sadang MSMEs 807 \| Vol. 1 No. 5 Motivasi Kinerja Karyawan (Rostiani, dkk) 2023 2023 Nanggroe : Jurnal Pengabdian Cendikia PENDAHULUAN Sebagai Kedaruratan Kesehatan Masyarakat Tahun 2019, Coronavirus Disease 2019 (Covid-19) berkembang menjadi isu kesehatan global di awal bulan 2020. Peraturan Pemerintah Nomor 21 Tahun 2020 tentang Pembatasan Sosial Berskala Besar (PSBB) menjadi respon pemerintah Indonesia untuk ini. Di satu sisi regulasi ini sangat membantu, namun di sisi lain juga berdampak pada sektor bisnis atau perekonomian Indonesia. Selain itu, ada pembatasan beberapa kegiatan atau pertemuan sosial yang merugikan perekonomian negara meskipun dimaksudkan untuk menghentikan penyebaran pandemi Covid-19. Selain itu, dampak pandemi Covid-19 terhadap perekonomian sudah terasa di Indonesia. p p Pentingnya UMKM terutama terlihat di Indonesia, di mana mereka mempekerjakan hingga 97% dari angkatan kerja dan menyumbang 60% dari PDB negara. Saat pandemi Covid-19 melanda, UMKM Indonesia terkena dampaknya dari segi produksi, pendapatan, dan penurunan jumlah tenaga kerja. Kenyataannya, situasinya sangat berbeda sekarang. Jumlah UMKM di Indonesia adalah sekitar 62 juta usaha mikro, 700.000 usaha kecil, 50.000 usaha menengah, dan kemudian sekitar 5.000 usaha besar. Terjadi penurunan volume penjualan sebesar 56%, kesulitan mendapatkan uang sebesar 22%, masalah distribusi sebesar 15%, dan kesulitan menemukan pasokan mentah sebesar 4%. Seblak Sadang UMKM merupakan UMKM yang bergerak di bidang makanan dan minuman yang didirikan pada tahun 2017 di daerah Sadang Purwakarta, Seblak Sadang menyediakan berbagai macam makanan dengan image pedas, Saat ini Seblak Sadang memiliki 4 cabang yaitu di Jl. Raya Sadang-Subang, Jl. Raya Cikampek-Parakan No 36, Cikampek Utara, Jl. Raya Kosambi - Telagasari dan Jl. Pinayungan Telukjambe Timur, Karawang. Kinerja karyawan yang berkualitas sangat diperlukan untuk Seblak Sadang. Berbagai strategi dilakukan setiap negara untuk menjaga sektor UMKM dalam upaya memastikan kelangsungan hidupnya dalam menghadapi pandemi Covid-19, mulai dari kebijakan pemerintah hingga undang-undang protokol kesehatan (7). Dalam skenario ini, UMKM Seblak Sadang juga harus berkontribusi mengerahkan seluruh kemampuan, keterampilan, dan keterampilannya dalam menemukan cara bertahan hidup di masa pandemi, selain upaya pemerintah. Para pelaku UMKM perlu diilhami dari dalam untuk mengubah cara hidup mereka. Kami menemukan bahwa individu-individu yang menganggur dan berpenghasilan rendah saat ini harus terus bekerja untuk memenuhi kebutuhan sehari-hari keluarga mereka selama pandemi virus Covid-19. Pelaku UMKM juga harus termotivasi untuk terus bertindak meskipun berbahaya jika menyangkut nilai-nilai kehidupan terutama kebutuhan dasarnya. Sehingga diperlukan strategi yang harus dilakukan mengenai bagaimana tetap memotivasi pelaku UMKM kuliner dalam beradaptasi di tengah pandemi virus Covid-19. KAJIAN TEORI UMKM Dalam perekonomian Indonesia, keberadaan Usaha Mikro, Kecil, dan Menengah memberikan dampak yang sangat menguntungkan bagi penciptaan lapangan kerja, penyediaan barang dan jasa, serta pemerataan pemerataan usaha. Pentingnya UMKM dalam pembangunan perekonomian nasional tidak dapat dilebih-lebihkan mengingat fungsinya sebagai usaha mikro, kecil, dan menengah. Terlepas dari sumber daya yang tersedia, UMKM adalah proses yang berupaya merebut dan memanfaatkan peluang dan membutuhkan keberanian untuk mengambil risiko yang diperhitungkan. UMKM adalah usaha kecil yang memiliki aset selain tanah dan bangunan sama dengan atau kurang dari Rp 200 juta dan memiliki pendapatan tahunan sampai 808 \| Vol. 1 No. 5 808 \| Vol. 1 No. 5 Motivasi Kinerja Karyawan (Rostiani, dkk) 2023 2023 Nanggroe : Jurnal Pengabdian Cendikia Nanggroe : Jurnal Pengabdian Cendikia dengan Rp 1 miliar, menurut Undang-Undang Nomor 9 Tahun 1995 tentang Usaha Kecil. Sebaliknya, bisnis menengah digambarkan sebagai keberadaan Usaha Mikro, Tergantung dari pengertian yang digunakan bangsa tersebut, tidak semua bangsa memiliki pemahaman yang sama tentang UMKM. Akibatnya, setiap negara memiliki cara pandang yang berbeda terhadap UKM. Dalam pengertian yang paling umum mencakup dua aspek yaitu aspek ketenagakerjaan dan aspek pengelompokan dalam hal jumlah tenaga kerja yang terserap dalam kelompok usaha (kisaran jumlah tenaga kerja). Dalam perdebatan UMKM, dua jenis perusahaanindustri kecil menengah (ISKM) dan perdagangan kecil menengah (PSKM) dikelompokkan menjadi satu. Karena kemampuan mengembangkan ISKM dan PSKM lebih diprioritaskan dibanding konsentrasi akhir pengelompokan pada masalah prospek ketenagakerjaan. Motivasi Kinerja Karyawan UMKM Motivasi adalah kapasitas untuk mengambil tindakan atau memasok energi untuk memenuhi kebutuhan. Sementara motivasi adalah keadaan yang dapat memaksa orang untuk mencapai tujuan dari motifnya, yang terkait erat dengan kebutuhan manusia, motif didefinisikan sebagai dorongan untuk melakukan tindakan yang dimulai dari dalam dan kemudian beradaptasi. Untuk menganalisis masalah yang diteliti, penelitian ini menggunakan teori motivasi Maslow. Maslow menegaskan bahwa manusia terdorong untuk memuaskan lima kategori keinginan, yaitu: y 1) Tuntutan Fisiologis, meliputi kebutuhan akan makanan, air, dan udara. 2) Kebutuhan akan rasa aman, meliputi kebutuhan akan kestabilan, rasa aman, dan kebebasan dari bahaya. 2) Kebutuhan akan rasa aman, meliputi kebutuhan akan kestabilan, rasa aman, dan kebebasan dari bahaya. 3) Kebutuhan sosial, termasuk keinginan untuk berteman, cinta, persetujuan, dan keterlibatan dengan orang lain. 4) Kebutuhan untuk dihormati atau dihargai (Esteem need), seperti rasa pencapaian dan diakui oleh orang lain. 5) Kebutuhan aktualisasi diri, seperti kepuasan terhadap diri sendiri atau pencapaian potensi diri. Menurut teori Maslow, seseorang bertindak atau bekerja untuk memuaskan berbagai tingkat kebutuhannya. Yang kedua, ketiga, dan seterusnya, hingga tingkat kelima, akan terwujud jika kebutuhan awal telah terpenuhi. Meskipun kebutuhan manusia sangat bervariasi, sebagian besar pada dasarnya sama. HASIL DAN PEMBAHASAN Salah satu strategi pemerintah Indonesia dalam menghadapi Covid-19 adalah dengan membatasi mobilitas masyarakat dalam hal interaksi sosial, ketenagakerjaan, ibadah, dan pembelajaran jarak jauh. Undang-undang baru ini telah menyebabkan industri kuliner lamban menyajikan makanan di tempat yang padat atau di atas meja. Akibatnya, para pelaku UMKM di industri makanan dituntut untuk terus mendorong kinerja karyawan. Berikut adalah beberapa saran untuk menginspirasi kinerja staf berdasarkan pengamatan: Upaya karyawan perusahaan menentukan seberapa baik kinerjanya di Seblak Sadang. Atasan, bagaimanapun, dapat berkontribusi pada strategi, pelaksanaan, dan manajemen perusahaan. Dalam situasi ini, manajer harus memainkan peran penting dalam upaya menginspirasi dan mengelola staf mereka. Agar UMKM Seblak Sadang berhasil mewujudkan tujuannya, mereka harus mencermati prestasi yang telah dicapai oleh karyawannya dan mengakuinya dengan imbalan (hadiah, imbalan, dan penghargaan) serta dengan mendorong mereka untuk bekerja dengan gembira dan bertanggung jawab. direncanakan dalam konteks COVID 19. Memiliki motivasi kerja yang kuat jelas sangat penting, terutama mengingat pandemi. Motivasi kerja karyawan Seblak Sadang di cabang 3 tidak naik atau turun selama wabah. Masalah pertama adalah bisnis di restoran jauh lebih lambat dari biasanya, yang membuat anggota staf bosan karena tidak ada yang bisa dilakukan. Sebelum pandemi, UMKM di Cabang 3 selalu disibukkan dengan nasabah sehingga membuat pegawai sibuk dengan tamu. Jam kerja karyawan juga terpengaruh oleh hal ini. UMKM Seblak Sadang cabang 3 mau tidak mau harus memangkas jam kerja karyawan demi menghemat biaya operasional karena minimnya kunjungan pembeli dari UMKM Seblak Sadang, ini berakibat pada jam kerja karyawan yang semakin pendek yang secara tidak langsung mempengaruhi jumlah gaji yang diterima oleh karyawan. Karena personel harus memakai masker saat bekerja dan juga harus berinteraksi dengan pelanggan dan ojek online yang menerima pesanan, kendala kedua pandemic juga menimbulkan emosi resah dan tidak nyaman. Terlepas dari kenyataan bahwa langkah-langkah kesehatan telah dilakukan, kecemasan dan ketidaknyamanan masih ada saat ini.. Kesulitan ketiga adalah banyak karyawan yang mengambil pekerjaan lepas dan paruh waktu di tempat lain untuk menambah penghasilan, yang menyebabkan mereka kehilangan fokus dan merasa lelah saat bekerja. Dorongan untuk bekerja lebih keras dan menghasilkan lebih banyak uang tidak berbanding terbalik dengan dorongan untuk berprestasi di tempat kerja. Keberlangsungan usaha UMKM Seblak Sadang terhambat oleh minimnya pengetahuan tentang kapan pandemi akan berhenti. Terlepas dari kenyataan bahwa banyak pekerja masih belum terbiasa bekerja di bawah kondisi wabah COVID-19, karyawan yang senang dan termotivasi untuk bekerja adalah aset organisasi yang signifikan. Nanggroe : Jurnal Pengabdian Cendikia pengamatan adalah Kota Karawang, salah satu dari tiga cabang Seblak Sadang di sekitar Kosambi. Sasaran Pengamatan ini kami tanyakan langsung kepada staf Cabang 3 Seblak Sadang. pengamatan adalah Kota Karawang, salah satu dari tiga cabang Seblak Sadang di sekitar Kosambi. Sasaran Pengamatan ini kami tanyakan langsung kepada staf Cabang 3 Seblak Sadang. METODE PENELITIAN Pengamatan ini menggunakan metode kualitatif deskriptif yaitu penelitian studi kasus. menggunakan data sekunder untuk memperoleh informasi atas penelitian yang dilakukan dari berbagai sumber literatur, antara lain buku, jurnal, dan artikel. Tujuan dari pendekatan kualitatif deskriptif adalah untuk menghasilkan gambaran dan gambaran yang jelas dalam menanggapi artikulasi masalah yaitu metode untuk menjaga motivasi UMKM kuliner tetap tinggi. Untuk menjawab rumusan masalah yaitu strategi melestarikan motivasi UMKM kuliner untuk beradaptasi dalam menghadapi pandemi Covid-19, maka teknik deskriptif kualitatif berupaya menghasilkan gambaran dan gambaran yang jelas. Berdasarkan beberapa isu krusial yang diklarifikasi oleh berbagai penulis, berbagai makalah dan jurnal dikumpulkan dari berbagai referensi untuk menggambarkan keinginan untuk beroperasi dalam situasi berbahaya. Lokasi Motivasi Kinerja Karyawan (Rostiani, dkk) 809 \| Vol. 1 No. 5 2023 Nanggroe : Jurnal Pengabdian Cendikia 810 \| Vol. 1 No. 5 2023 Nanggroe : Jurnal Pengabdian Cendikia bawah kendali perusahaan. Ini mencakup hal-hal yang akan dilakukan bisnis untuk menginspirasi pekerja, seperti: bawah kendali perusahaan. Ini mencakup hal-hal yang akan dilakukan bisnis untuk menginspirasi pekerja, seperti: bawah kendali perusahaan. Ini mencakup hal-hal yang akan dilakukan bisnis untuk menginspirasi pekerja, seperti: 1. Setting yang kondusif untuk bekerja, dimana ruang kerja yang nyaman akan meningkatkan semangat kerja 2 kompensasi yang adil 1. Setting yang kondusif untuk bekerja, dimana ruang kerja yang nyaman akan meningkatkan semangat kerja 2. kompensasi yang adil. 3. Insentif dan prestasi yang sesuai untuk kerja keras karyawan akan memberi mereka rasa pengakuan diri dan meningkatkan motivasi karyawan. Tujuan dasar bekerja adalah untuk memenuhi kebutuhan. 4. Kebutuhan akan penghargaan adalah kebutuhan akan harga diri, kebutuhan untuk dihormati oleh orang lain, dan rasa takut kehilangan pekerjaan atau kemungkinan penurunan jabatan atau demosi, antara lain ancaman terhadap kondisi kerja terkait dengan penurunan kesejahteraan psikologis . 5. Kebutuhan aktualisasi diri adalah dorongan untuk menggunakan kemampuan dan potensi diri serta kebutuhan untuk berdebat dengan mengemukakan pendapat dan memberikan kritik. Yang pertama dari lima keinginan ini, yaitu tuntutan fisik, harus dipenuhi. Ini adalah kebutuhan mendasar untuk mempertahankan kehidupan manusia. Akibat kekhawatiran akan kehilangan pekerjaan selama wabah COVID-19, banyak tuntutan fisiologis karyawan yang tidak terpenuhi, padahal banyak dari mereka yang masih harus menafkahi keluarga. Selain itu, tuntutan mereka akan rasa stabilitas dalam pekerjaan mereka saat ini tidak terpenuhi. Banyak UMKM yang mencoba lari namun kemudian tutup karena biaya yang dikeluarkan melebihi pendapatan yang diterima sehingga membuat karyawan merasa khawatir, takut, kesal, dan tidak jelas tentang keadaan dan keberadaan pekerjaan yang dimiliki saat ini. Selain itu, pekerja harus terus bekerja di luar rumah untuk mengurangi risiko tertular HASIL DAN PEMBAHASAN Menurut Robins dalam Yenni (2019), motivasi adalah yang mendorong orang untuk melakukan berbagai tindakan guna mencapai tujuan tertentu. Prosedur yang juga menetapkan seberapa serius, sengaja, dan gigih orang mengejar tujuan mereka. Menurut Theodora (2015), unsur merangsang, membimbing, memelihara, menunjukkan intensitas, berkelanjutan, dan memiliki tujuan adalah bagian dari motivasi. Dua jenis dasar motivasi adalah artifisial (eksternal) dan intrinsik (interior). Menurut Sutrisno (2010), motivasi eksternal adalah dorongan atau kekuatan yang berada dalam diri seseorang dan dipengaruhi oleh variabel internal yang berada di 810 \| Vol. 1 No. 5 810 \| Vol. 1 No. 5 Motivasi Kinerja Karyawan (Rostiani, dkk) 2023 Motivasi Kinerja Karyawan (Rostiani, dkk) KESIMPULAN Pandemi COVID-19 yang menyebabkan keresahan pada staf UMKM Seblak Sadang memiliki perhatian utama terkait motivasi kerja sebagai berikut: Pandemi COVID-19 yang menyebabkan keresahan pada staf UMKM Seblak Sadang memiliki perhatian utama terkait motivasi kerja sebagai berikut: 1) Karyawan UMKM Seblak Sadang merasa sedikit risih dengan penggunaan gaji karena tidak sepenuhnya menyentuh kehidupan sehari-hari. 1) Karyawan UMKM Seblak Sadang merasa sedikit risih dengan penggunaan gaji karena tidak sepenuhnya menyentuh kehidupan sehari-hari. 2) Karyawan UMKM Seblak Sadang memiliki keinginan dan kemampuan untuk menguasai gajinya sendiri. 2) Karyawan UMKM Seblak Sadang memiliki keinginan dan kemampuan untuk menguasai gajinya sendiri. 3) Restoran karyawan mengalami kesulitan keuangan akibat banyaknya perselisihan perburuhan yang sering diselesaikan oleh manajemen restoran dalam upaya menekan biaya operasional. Karyawan harus multitasking dengan melakukan berbagai pekerjaan secara bersamaan, seperti melayani pelanggan, membuat makanan, dan bekerja sebagai kasir. 4) Ikatan sosial yang positif, termasuk kerja sama antara rekan kerja, atasan, dan pelanggan, berdampak besar pada motivasi karyawan. 4) Ikatan sosial yang positif, termasuk kerja sama antara rekan kerja, atasan, dan pelanggan, berdampak besar pada motivasi karyawan. Melihat hasil tersebut di atas, maka para pelaku usaha UMKM Seblak Sadang cabang 3 Kosambi harus dapat melakukan pengawasan dan disiplin pegawai yang ketat terhadap prosedur yang diterapkan sehingga tercipta suasana kerja yang menyenangkan. Pemilik usaha UMKM di Seblak Sadang harus disiplin menerapkan praktik kesehatan pengunjung. 811 \| Vol. 1 No. 5 Motivasi Kinerja Karyawan (Rostiani, dkk) Motivasi Kinerja Karyawan (Rostiani, dkk) Referensi Surat Edaran Menteri Perhubungan Republik Indonesia Nomor PM 25 Tahun 2020 Tentang Pengendalian Transportaso Selama Masa Mudik Idul Fitri Tahun 1441 Hijriah Dalam Rangka Pencegahan Penyebaran Corona Virus Disease 2019 (COVID-19) g g y World Health Organization (2020). Listings of WHO’s response to COVID-19. Tersedia secara online di https://www.who.int/news/ite m/29-06-2020-covidtimeline Kosakoy, Y. I. (2021). Motivasi Kerja Karyawan Restoran Pada Masa Pandemi Covid-19 Di Bali. Journal of Accounting and Management Innovation, 5(2), 70-76. Pakpahan AK. Covid-19 Dan Implikasi Bagi Usaha Mikro, Kecil, Dan Menengah. J Ilm Hub Int. 2020;0(0):59–64. Hadiwardoyo W. Kerugian Ekonomi Nasional Akibat Pandemi Covid-19. Baskara J Bus Entrep. 2020;2(2):83–92. Islam A. Configuring a Quadruple Helix Innovation Model (QHIM) based blueprint for Malaysian SMEs to survive the crises happening by Covid-19. Emerald Open. 2020;2(May):1–4 UKM KK dan. Perkembangan Data Usaha Mikro, Kecil, Menengah (UMKM) dan Usaha Besar (UB). Vol. 2018. UKM, Koperasi K. Menkop dan UKM paparkan Skema Pemulihan Ekonomi KUKM di Masa dan Pasca Covid-19. 2020. OECD. Covid-19: SME Policy Responses. Tackling coronavirus Contrib to a Glob effort [Internet]. 2020;(March):1–55. Available from: https://oecd.dam- broadcast.com/pm 7379 119 119680- di6h3qgi4x.pdf. Sugiri D. Menyelamatkan Usaha Mikro, Kecil dan Menengah dari Dampak Pandemi Covid-19. Fokus Bisnis Media Pengkaj Manaj dan Akunt. 2020;19(1):76–86. g j j Gibson JL, Ivancevich JM, Donnelly JH, Konopaske R. Organization; Behavior, Stucture, Processes. Fourteenth. United States: Mc Graw Hilll Irwin; 2009. 642 p. Amanda, R., Suherman, E., & Hidayaty, D. E. (2022). Pengaruh Stres Kerja Terhadap Kinerja Karyawan Melalui Kepuasan Kerja Pada Karyawan Imigrasi Kelas I Non TPI Karawang. Jurnal Ilmiah Mandala Education, 8(4). https://ejournal.mandalanursa.org/index.php/JIME/article/view/3931 812 \| Vol. 1 No. 5
https://openalex.org/W2982377894	https://genomebiology.biomedcentral.com/track/pdf/10.1186/s13059-019-1833-x	English	null	Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues	Genome biology	2,019	cc-by	12,967	Hanna et al. Genome Biology (2019) 20:225 https://doi.org/10.1186/s13059-019-1833-x Hanna et al. Genome Biology (2019) 20:225 https://doi.org/10.1186/s13059-019-1833-x Open Access Endogenous retroviral insertions drive non- canonical imprinting in extra-embryonic tissues Courtney W. Hanna1,2* , Raquel Pérez-Palacios3, Lenka Gahurova4,5, Michael Schubert6, Felix Krueger7, Laura Biggins7, Simon Andrews7, Maria Colomé-Tatché6,8,9, Deborah Bourc’his3, Wendy Dean1,10 and Gavin Kelsey1,2* © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post- implantation development remain unexplored. Results: We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast. Conclusions: This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements. Keywords: Genomic imprinting, Histone modifications, Extra-embryonic, Development, Embryo, H3K27me3, Non- canonical imprinting, Long terminal repeats (LTRs), Placenta, Endogenous retroviruses (ERVs) p y ; gavin.kelsey@babraham.ac.uk 1Epigenetics Programme, Babraham Institute, Cambridge, UK Full list of author information is available at the end of the article g y@ 1Epigenetics Programme, Babraham Institute, Cambridge, UK Full list of author information is available at the end of the article * Correspondence: courtney.hanna@babraham.ac.uk; gavin.kelsey@babraham.ac.uk 1Epigenetics Programme, Babraham Institute, Cambridge, UK Full list of author information is available at the end of the article Results Study design To evaluate the allelic regulation of gene expression in the embryo, we assessed the epigenetic modifications in C57BL6/Babr and CAST/EiJ reciprocal hybrid (denoted as B6/CAST and CAST/B6, in which by convention, the maternal strain is indicated first) embryonic day (E) 6.5 epiblast and extra-embryonic ectoderm (ExE). We assayed H3K4me3, H3K36me3, and H3K27me3 using ultra low-input ChIP-seq and DNA methylation using post-bisulphite adaptor tagging (PBAT), as previously de- scribed [22], from a pool of ~ 2500 cells of either epiblast or ExE (Fig. 1a, b; Additional file 1: Figure S1 and S2). We additionally profiled these epigenetic marks in E6.5 hybrid embryos derived from B6 females with a double conditional knockout for Dnmt3a and Dnmt3b in oocytes, driven by Zp3-cre, crossed to CAST males (denoted matDKO/ CAST) (Additional file 1: Figure S1 and S2). Consequently, these matDKO/CAST embryos will inherit no maternal DNA methylation [9] but are able to sufficiently establish DNA methylation post-fertilization [23]. Allelic gene ex- pression was evaluated in E7.5 epiblast and ExE of all hy- brid crosses (Fig. 1a, b; Additional file 1: Figure S3). Details of biological replicates and datasets generated for this study are summarized in Additional file 2: Table S1. Imprinted genes are essential for the regulation of mammalian development, placentation, and fetal growth. It has been proposed that imprinting arose as a conse- quence of the conflict between the paternal and mater- nal genomes within the conceptus in placental mammals to increase or restrict demand for maternal resources, respectively [13]. The barrier between the mother and fetus, the extra-embryonic tissues, perhaps unsurprisingly, has more expressed imprinted genes than most other tis- sues [14, 15]. Furthermore, several observations suggest that imprinted gene regulation in extra-embryonic tissues may be dependent on a unique combination of multiple epigenetic layers, utilizing differential DNA methylation together with or in addition to histone modifications and long non-coding RNAs (lncRNAs) [16, 17]. © 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. Hanna et al. Genome Biology (2019) 20:225 Page 2 of 17 Page 2 of 17 Hanna et al. Genome Biology (2019) 20:225 Background Zfp64, Phf17, Smoc1, Pde10a) appear to have no associ- ated gDMRs, suggesting they may be solely regulated by histone modifications [15, 19, 20]. Indeed, a recent study found that maternally deposited H3K27me3 can confer imprinted gene expression. However, this “non-canon- ical” imprinting appears to be predominantly transient in the early embryo, and the key mechanisms that main- tain this form of imprinting are still unknown [21]. Notably, for the few genes with persistent mono-allelic expression in later development, mono-allelic expression becomes restricted to extra-embryonic lineages, suggest- ing that extra-embryonic tissues may be uniquely permis- sive for this additional form of imprinted gene regulation. Importantly, it remains to be shown whether (1) histone modifications have a unique allelic patterning in extra- embryonic tissues conferring imprinted gene expression and (2) non-canonical imprinting is truly independent of maternally inherited gDMRs. The genetic contributions from both the sperm and oocyte are essential for successful development in mammals. Thirty-five years ago, seminal embryo manipulation experi- ments in mice showed that embryos with either two mater- nal or two paternal genomes die early in gestation [1, 2], and it was postulated that the parental genomes were somehow differentially imprinted during gametogenesis. Shortly thereafter, three genes, Igf2r, H19, and Igf2, were identified to be expressed mono-allelically based on the parent of origin, revealing the first examples of “genomic imprinting” [3–5]. Importantly, the regulation of imprinted mono-allelic expression was found to be due to the asym- metric deposition of an epigenetic mark, DNA methyla- tion, in gametes [6, 7]. The study of imprinted genes has been integral to our understanding of epigenetic regulation of gene expression and has revealed the capacity for intergenerational trans- mission of epigenetic instructions from gametes to a newly formed embryo. Imprinting classically depends on locus- specific differences in DNA methylation established in the gametes [8, 9], with the vast majority of germ line differen- tially methylated regions (gDMRs) being established on maternal alleles during oogenesis [10]. Upon fertilization, despite the widespread epigenetic reprogramming, which includes the erasure of DNA methylation, reallocation of histone modification patterns, and dynamic chromatin re- modeling [11], imprinted gDMRs are protected from these reprogramming events. In the post-implantation embryo, as there is re-acquisition of genomic DNA methylation, gDMRs maintain their inherited mono-allelic status through the protection of the unmethylated allele [12]. Imprinted H3K4me3 is associated with imprinted gene expression Two 10% inputs were taken from each pool of embryos, one for a ChIP-seq input control and the other for low-coverage bisulphite-seq. RNA-seq was done on matched single E7.5 epiblast (N = 3) and ExE (N = 3). b Screenshot of E7.5 gene expression; E6.5 H3K4me3, H3K36me3, and H3K27me3; and E6.5 DNA methylation for B6/CAST epiblast and ExE. H3K4me3 is enriched at gene promoters, H3K36me3 along gene bodies of expressed genes, and H3K27me3 at transcriptionally silent promoters. The epiblast is highly methylated with exception of promoters, while ExE shows the expected lower global levels of DNA methylation. The box highlights the Sfmbt2 gene, which shows tissue-specific expression in ExE. ChIP-seq enrichment (RPKM) is shown for 1-kb running windows, with a 100-bp step (scales in square brackets), while gene expression and DNA methylation are shown using 2-kb running windows, with a 500-bp step Fig. 1 Experimental design and data evaluation. a Schematic of experimental design demonstrating the collection of reciprocal hybrid post-implantation embryos for ultra low-input ChIP-seq, bisulphite-seq, and RNA-seq. Two replicates of H3K4me3, H3K27me3, and H3K36me3 ChIP-seq were each done using a pool of either E6.5 epiblasts (N = 4) or ExE (N = 8), approximating an input of ~ 2500 cells. Two 10% inputs were taken from each pool of embryos, one for a ChIP-seq input control and the other for low-coverage bisulphite-seq. RNA-seq was done on matched single E7.5 epiblast (N = 3) and ExE (N = 3). b Screenshot of E7.5 gene expression; E6.5 H3K4me3, H3K36me3, and H3K27me3; and E6.5 DNA methylation for B6/CAST epiblast and ExE. H3K4me3 is enriched at gene promoters, H3K36me3 along gene bodies of expressed genes, and H3K27me3 at transcriptionally silent promoters. The epiblast is highly methylated with exception of promoters, while ExE shows the expected lower global levels of DNA methylation. The box highlights the Sfmbt2 gene, which shows tissue-specific expression in ExE. ChIP-seq enrichment (RPKM) is shown for 1-kb running windows, with a 100-bp step (scales in square brackets), while gene expression and DNA methylation are shown using 2-kb running windows, with a 500-bp step a significant allelic bias in at least one dataset (Add- itional file 2: Table S2). Thus, imprinted H3K4me3 peaks are strongly predictive of mono-allelic H3K36me3 and gene expression of the nearest genes, as demonstrated by the Peg3 gene (Fig. 2c; Additional file 1: Figure S4). for multiple comparisons). Imprinted H3K4me3 is associated with imprinted gene expression Histone modifications H3K27me3 and H3K9me2/3 have been associated with placental-specific imprinting of distal genes in the Kcnq1/Kcnq1ot1 and Igf2r/Airn clusters; however, this distal mono-allelic silencing is mediated by a non-coding RNA that is regulated by a canonical gDMR [16, 18]. Intriguingly, a number of iso- lated placental-specific imprinted genes (e.g., Sfmbt2, To identify imprinted domains in E6.5 embryos, we called H3K4me3 peaks on autosomes in the epiblast (N = 33,329) and ExE (N = 40,468) of B6/CAST and CAST/B6 embryos. H3K4me3 peaks with a minimum of 20 strain-specific SNP-spanning reads in at least 1 repli- cate of epiblast (N = 15,407) and ExE (N = 15,976) were evaluated for allelic bias using EdgeR (p < 0.05, corrected Hanna et al. Genome Biology (2019) 20:225 Page 3 of 17 Hanna et al. Genome Biology Fig. 1 Experimental design and data evaluation. a Schematic of experimental design demonstrating the collection of reciprocal hybrid post-implantation embryos for ultra low-input ChIP-seq, bisulphite-seq, and RNA-seq. Two replicates of H3K4me3, H3K27me3, and H3K36me3 ChIP-seq were each done using a pool of either E6.5 epiblasts (N = 4) or ExE (N = 8), approximating an input of ~ 2500 cells. Two 10% inputs were taken from each pool of embryos, one for a ChIP-seq input control and the other for low-coverage bisulphite-seq. RNA-seq was done on matched single E7.5 epiblast (N = 3) and ExE (N = 3). b Screenshot of E7.5 gene expression; E6.5 H3K4me3, H3K36me3, and H3K27me3; and E6.5 DNA methylation for B6/CAST epiblast and ExE. H3K4me3 is enriched at gene promoters, H3K36me3 along gene bodies of expressed genes, and H3K27me3 at transcriptionally silent promoters. The epiblast is highly methylated with exception of promoters, while ExE shows the expected lower global levels of DNA methylation. The box highlights the Sfmbt2 gene, which shows tissue-specific expression in ExE. ChIP-seq enrichment (RPKM) is shown for 1-kb running windows, with a 100-bp step (scales in square brackets), while gene expression and DNA methylation are shown using 2-kb running windows, with a 500-bp step Fig. 1 Experimental design and data evaluation. a Schematic of experimental design demonstrating the collection of reciprocal hybrid post-implantation embryos for ultra low-input ChIP-seq, bisulphite-seq, and RNA-seq. Two replicates of H3K4me3, H3K27me3, and H3K36me3 ChIP-seq were each done using a pool of either E6.5 epiblasts (N = 4) or ExE (N = 8), approximating an input of ~ 2500 cells. Imprinted H3K4me3 is associated with imprinted gene expression In epiblast, 85.2% (23/27) of informative genes associated with an imprinted H3K4me3 peak (Additional file 1: Figure S4) showed a sig- nificant allelic bias in at least one dataset (Additional file 2: Table S3). In ExE, 94.4% (51/54) of informative genes asso- ciated with an imprinted H3K4me3 peak (Fig. 2b) showed Imprinted H3K4me3 is associated with imprinted gene expression A consensus set of allelic H3K4me3 peaks was identified for epiblast (N = 329) and ExE (N = 913), as those that were significant in both B6/ CAST and CAST/B6 crosses (Fig. 2a; Additional file 1: Figure S4). The vast majority of allelic H3K4me3 peaks demonstrated strain-specific inheritance patterns (92%), with the remaining 8% of peaks showing parent-of-origin (imprinted) inheritance. In total, we identified 69 imprinted H3K4me3 peaks in ExE and 29 in the epiblast (Fig. 2a; Add- itional file 1: Figure S4; Additional file 2: Tables S2 and S3). The majority (72.4%) of imprinted H3K4me3 peaks were lo- cated at an annotated gene promoter(s), and the remaining were assigned to the nearest gene within 10 kb, where ap- plicable (Additional file 2: Tables S2 and S3). When compared to a list of known (and putative) imprinted genes (Additional file 2: Table S4), known imprinted genes comprised 77.8% and 96.2% of genes associated with an imprinted H3K4me3 peak in ExE and epiblast, respectively. for multiple comparisons). A consensus set of allelic H3K4me3 peaks was identified for epiblast (N = 329) and ExE (N = 913), as those that were significant in both B6/ CAST and CAST/B6 crosses (Fig. 2a; Additional file 1: Figure S4). The vast majority of allelic H3K4me3 peaks demonstrated strain-specific inheritance patterns (92%), with the remaining 8% of peaks showing parent-of-origin (imprinted) inheritance. In total, we identified 69 imprinted H3K4me3 peaks in ExE and 29 in the epiblast (Fig. 2a; Add- itional file 1: Figure S4; Additional file 2: Tables S2 and S3). The majority (72.4%) of imprinted H3K4me3 peaks were lo- cated at an annotated gene promoter(s), and the remaining were assigned to the nearest gene within 10 kb, where ap- plicable (Additional file 2: Tables S2 and S3). When compared to a list of known (and putative) imprinted genes (Additional file 2: Table S4), known imprinted genes comprised 77.8% and 96.2% of genes associated with an imprinted H3K4me3 peak in ExE and epiblast, respectively. We then evaluated whether imprinted H3K4me3 was as- sociated with allele-specific gene expression, using the EdgeR statistical approach to identify genes with allelic bias for H3K36me3 in E6.5 epiblast and ExE, and gene ex- pression in E7.5 epiblast, E7.5 ExE, and E12.5 placenta [15] (Fig. 2b; Additional file 1: Figure S4). Non-canonical vs. canonical imprinted gene regulation Non-canonical vs. canonical imprinted gene regulation To determine which imprinted loci are dependent on ma- ternally inherited gDMRs, we evaluated allelic H3K4me3 in post-implantation matDKO/CAST embryos. Using the EdgeR statistical approach described for the reciprocal hy- brids, we identified H3K4me3 peaks that lost allelic bias in the matDKO/CAST (canonical maternal imprints) and those that remained imprinted (non-canonical imprints and canonical paternal imprints) (Fig. 3a; Additional file 1: Figure S5). In epiblast, there were only 5 imprinted H3K4me3 peaks present in the matDKO/CAST (H19, IG- DMR, Meg3, Slc38a4, and Gab1); the former 3 are regu- lated by paternal gDMRs, thus leaving 2 that could be classified as non-canonical (Additional file 2: Table S3). In ExE, we identified 3 H3K4me3 peaks associated with known paternal gDMRs (H19, Igf2, and Meg3), 17 that we classified as non-canonical (including all 4 previously reported non-canonical imprinted genes [21]), with a remaining 49 canonical maternally regulated imprinted H3K4me3 peaks that were lost in matDKO/CAST (Fig. 3a; Additional file 2: Table S2). These data support previous reports that non-canonical imprinting is largely restricted to the extra-embryonic lineage [21]. We then evaluated whether imprinted H3K4me3 was as- sociated with allele-specific gene expression, using the EdgeR statistical approach to identify genes with allelic bias for H3K36me3 in E6.5 epiblast and ExE, and gene ex- pression in E7.5 epiblast, E7.5 ExE, and E12.5 placenta [15] (Fig. 2b; Additional file 1: Figure S4). In epiblast, 85.2% (23/27) of informative genes associated with an imprinted H3K4me3 peak (Additional file 1: Figure S4) showed a sig- nificant allelic bias in at least one dataset (Additional file 2: Table S3). In ExE, 94.4% (51/54) of informative genes asso- ciated with an imprinted H3K4me3 peak (Fig. 2b) showed Hanna et al. Genome Biology (2019) 20:225 Page 4 of 17 Hanna et al. Genome Biology Fig. 2 Imprinted H3K4me3 peaks are associated with imprinted gene expression in ExE. a Scatter plots of allelic H3K4me3 enrichment at autosomal H3K4me3 peaks (N = 15,976) in B6/CAST E6.5 ExE (top) and CAST/B6 E6.5 ExE (bottom). Peaks with allelically biased H3K4me3 were identified using EdgeR statistic (p < 0.05, corrected for multiple comparisons). Significant peaks were then classified into strain-specific allelic H3K4me3 if their allelic enrichment switched in the reciprocal cross, denoted as B6-specific (green) and CAST-specific (turquoise). Significant peaks were identified as imprinted if the allelic enrichment was consistent between reciprocal crosses, denoted as paternal (blue) or maternal (red). Non-canonical vs. canonical imprinted gene regulation Allelic bias (log2(pat/mat)) for E6.5 ExE H3K36me3, E7.5 ExE gene expression, and E12.5 placenta (P) gene expression is shown for associated nearby genes (Additional file 2: Table S2). Reciprocal hybrids are denoted as B/C (B6/CAST), C/B (CAST/B6), F/C (FvB/CAST), and C/F (CAST/FvB). White boxes indicate where there was insufficient data (ChIP-seq < 20 SNP-spanning reads in all replicates, RNA-seq < 5 SNP-spanning reads in all replicates). ChIP-seq data was quantitated is as in a, RNA-seq data was quantitated as read count over exons. H3K4me3 peaks were excluded if there was no gene within 10 kb or the associated gene was uninformative in all datasets. H3K4me3 peaks overlapping more than one gene promoter are duplicated in the H3K4me3 column. Novel imprinted genes are marked with an asterisk. c Screenshot of allelic enrichment for H3K4me3 and H3K36me3 in E6.5 ExE and gene expression in E7.5 ExE for B6/CAST and CAST/B6 at the known imprinted gene Peg3. Box indicates the location of the maternal gDMR. ChIP-seq data is quantitated using enrichment normalized RPKM for autosomal 1-kb running windows with a 100-bp step (scales in square brackets); paternal (blue) and maternal (red) enrichments are shown on mirrored axes. Gene expression is quantitated as log2(RPKM) for 500-bp running windows with a 50-bp step As ERV LTRs have been implicated in regulating tissue- specific gene expression [24], we identified those ERVK LTRs within non-canonical imprinted H3K4me3 peaks that were transcription initiation sites in extra-embryonic tissues (N = 8 out of 28) (Additional file 1: Figure S5; Add- itional file 2: Table S5). Similar to the genes associated with non-canonical imprinted H3K4me3 peaks (Fig. 2b) [21], ERVK LTR promoters underlying imprinted paternal H3K4me3 peaks showed predominantly imprinted pater- nal expression in E12.5 FvB x CAST hybrid placenta and visceral endoderm [15] (Fig. 3c). Similarly, using publically available RNA-seq data [15], we observed they were also expressed specifically in extra-embryonic lineages during post-implantation development (Fig. 3d). Two non-canonical imprinted H3K4me3 peaks identi- fied in ExE were on the maternal alleles (Pde10a and Cd81) and were localized at the large Igf2r/Airn and Kcnq1/Kcnq1ot1 imprinted clusters, and thus have dis- tinct regulatory mechanisms to non-canonical H3K4me3 peaks on paternal alleles [15]. Thus, all subsequent ana- lyses have been done on the 15 non-canonical imprinted paternal H3K4me3 peaks identified in ExE. Non-canonical imprinted H3K4me3 peaks localize to endogenous retroviral LTRs We evaluated whether canonical and non-canonical imprints in ExE are enriched for similar genomic features. While canonical imprinted H3K4me3 peaks were strongly enriched for CGIs (88%), non-canonical imprinted H3K4me3 peaks were enriched for regulatory sequences of repetitive elements, the most significant of which was the long terminal repeats (LTRs) of endogenous retroviral (ERV) (93%) (Fig. 3b), specifically endogenous retrovirus- K (ERVKs) (Additional file 1: Figure S5). Non-canonical vs. canonical imprinted gene regulation Enrichment is quantitated as read count normalized to library size, correcting for peak length. b Heatmap showing allelic bias (log2(pat/mat)) for E6.5 ExE H3K4me3 at H3K4me3 peaks identified in E6.5 ExE. Allelic bias (log2(pat/mat)) for E6.5 ExE H3K36me3, E7.5 ExE gene expression, and E12.5 placenta (P) gene expression is shown for associated nearby genes (Additional file 2: Table S2). Reciprocal hybrids are denoted as B/C (B6/CAST), C/B (CAST/B6), F/C (FvB/CAST), and C/F (CAST/FvB). White boxes indicate where there was insufficient data (ChIP-seq < 20 SNP-spanning reads in all replicates, RNA-seq < 5 SNP-spanning reads in all replicates). ChIP-seq data was quantitated is as in a, RNA-seq data was quantitated as read count over exons. H3K4me3 peaks were excluded if there was no gene within 10 kb or the associated gene was uninformative in all datasets. H3K4me3 peaks overlapping more than one gene promoter are duplicated in the H3K4me3 column. Novel imprinted genes are marked with an asterisk. c Screenshot of allelic enrichment for H3K4me3 and H3K36me3 in E6.5 ExE and gene expression in E7.5 ExE for B6/CAST and CAST/B6 at the known imprinted gene Peg3. Box indicates the location of the maternal gDMR. ChIP-seq data is quantitated using enrichment normalized RPKM for autosomal 1-kb running windows with a 100-bp step (scales in square brackets); paternal (blue) and maternal (red) enrichments are shown on mirrored axes. Gene expression is quantitated as log2(RPKM) for 500-bp running windows with a 50-bp step Fig. 2 Imprinted H3K4me3 peaks are associated with imprinted gene expression in ExE. a Scatter plots of allelic H3K4me3 enrichment at Fig. 2 Imprinted H3K4me3 peaks are associated with imprinted gene expression in ExE. a Scatter plots of allelic H3K4me3 enrichment at autosomal H3K4me3 peaks (N = 15,976) in B6/CAST E6.5 ExE (top) and CAST/B6 E6.5 ExE (bottom). Peaks with allelically biased H3K4me3 were identified using EdgeR statistic (p < 0.05, corrected for multiple comparisons). Significant peaks were then classified into strain-specific allelic H3K4me3 if their allelic enrichment switched in the reciprocal cross, denoted as B6-specific (green) and CAST-specific (turquoise). Significant peaks were identified as imprinted if the allelic enrichment was consistent between reciprocal crosses, denoted as paternal (blue) or maternal (red). Enrichment is quantitated as read count normalized to library size, correcting for peak length. b Heatmap showing allelic bias (log2(pat/mat)) for E6.5 ExE H3K4me3 at H3K4me3 peaks identified in E6.5 ExE. Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs We then sought to determine (1) whether sequence or genomic features underlie the tissue specificity of extra-embryonic ERVK LTR promoters and (2) why a Page 5 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology Fig. 3 Non-canonical imprinted H3K4me3 in ExE demarcates imprinted ERVK LTR elements with extra-embryonic-specific imprinted expression. a Allelic ratio for H3K4me3 at canonical maternally regulated imprinted H3K4me3 peaks (N = 49) canonical paternally regulated imprinted H3K4me3 peaks (N = 3), and non-canonical imprinted H3K4me3 peaks (N = 17) in B6/CAST, CAST/B6, and matDKO/CAST E6.5 ExE. Informative H3K4me3 peaks were quantitated using read counts corrected for library size, and relative allelic ratios were calculated (allelic ratio = mat/(mat + pat)). b The percentage of non-canonical imprinted H3K4me3 peaks with paternal allelic bias (N = 15) and canonical imprinted H3K4me3 peaks (N = 52) that were overlapping each category of genomic feature, including CpG islands (CGIs) and classes of repetitive elements. Each pair-wise comparison was done using chi-square statistic, with a significance threshold adjusted for multiple comparisons using Bonferroni correction. c Allelic expression of transcribed ERVK LTRs within a non-canonical imprinted paternal H3K4me3 peak (N = 8, Additional file 2: Table S5) is shown in extra-embryonic tissues at E12.5 (placenta and visceral endoderm (VE)). Reciprocal hybrids are denoted as F/C (FvB/CAST) and C/F (CAST/FvB). White boxes indicate where there was insufficient data (< 5 SNP-spanning reads in all replicates). d Heatmap showing expression levels across extra-embryonic and embryonic tissues of transcriptionally active ERVK LTR elements within a non-canonical imprinted paternal H3K4me3 peak (N = 8). The nearest gene is denoted in brackets next to the ERVK LTR identifier Fig. 3 Non-canonical imprinted H3K4me3 in ExE demarcates imprinted ERVK LTR elements with extra-embryonic-specific imprinted expression. a Allelic ratio for H3K4me3 at canonical maternally regulated imprinted H3K4me3 peaks (N = 49) canonical paternally regulated imprinted H3K4me3 peaks (N = 3), and non-canonical imprinted H3K4me3 peaks (N = 17) in B6/CAST, CAST/B6, and matDKO/CAST E6.5 ExE. Informative H3K4me3 peaks were quantitated using read counts corrected for library size, and relative allelic ratios were calculated (allelic ratio = mat/(mat + pat)). b The percentage of non-canonical imprinted H3K4me3 peaks with paternal allelic bias (N = 15) and canonical imprinted H3K4me3 peaks (N = 52) that were overlapping each category of genomic feature, including CpG islands (CGIs) and classes of repetitive elements. Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs Each pair-wise comparison was done using chi-square statistic, with a significance threshold adjusted for multiple comparisons using Bonferroni correction. c Allelic expression of transcribed ERVK LTRs within a non-canonical imprinted paternal H3K4me3 peak (N = 8, Additional file 2: Table S5) is shown in extra-embryonic tissues at E12.5 (placenta and visceral endoderm (VE)). Reciprocal hybrids are denoted as F/C (FvB/CAST) and C/F (CAST/FvB). White boxes indicate where there was insufficient data (< 5 SNP-spanning reads in all replicates). d Heatmap showing expression levels across extra-embryonic and embryonic tissues of transcriptionally active ERVK LTR elements within a non-canonical imprinted paternal H3K4me3 peak (N = 8). The nearest gene is denoted in brackets next to the ERVK LTR identifier LTRs that act as enhancers in extra-embryonic tissues have been reported to be enriched in transcription factor motifs ELF5, EOMES, and CDX2 [25]; however, we did not identify motifs that were enriched among extra- embryonic ERVK LTR promoters using an unbiased approach. Furthermore, we did not find significant en- richment specifically for ELF5, EOMES, or CDX2 motifs. subset is non-canonically imprinted. To evaluate these ques- tions, we identified all ERVK LTRs that fell within ExE H3K4me3 peaks that were active promoters in extra- embryonic tissues (N = 40), which included the 8 non- canonical imprinted ERVK LTRs and 32 ERVK LTRs with- out imprinted expression (Additional file 2: Table S6). Using these 40 extra-embryonic ERVK LTR promoters, we assessed the sequence composition, sequence motifs, proximity to genes and promoters, ERVK LTR classes, and LTR length. subset is non-canonically imprinted. To evaluate these ques- tions, we identified all ERVK LTRs that fell within ExE H3K4me3 peaks that were active promoters in extra- embryonic tissues (N = 40), which included the 8 non- canonical imprinted ERVK LTRs and 32 ERVK LTRs with- out imprinted expression (Additional file 2: Table S6). Using these 40 extra-embryonic ERVK LTR promoters, we assessed the sequence composition, sequence motifs, proximity to genes and promoters, ERVK LTR classes, and LTR length. As non-canonical imprinting has been associated with maternal H3K27me3 inherited from the oocyte [21], we evaluated whether epigenetic marks (H3K4me3, H3K27me3, and DNA methylation) in the maternal oocyte were associ- ated with the transcriptional status of ERVK LTR promoters in extra-embryonic tissues. Non-canonical imprinted ERVK LTR promoters were indeed significantly associated with oo- cyte H3K27me3 (p < 0.001) (Fig. 4c). Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs Remarkably, H3K4me3 in the oocyte significantly differentiated those ERVK LTRs that were transcriptionally active in extra-embryonic tissues compared to inactive (p < 0.001) (Fig. 4c). g p g In contrast to ERVK LTRs genome-wide, we found that extra-embryonic ERVK LTR promoters had rela- tively high CpG content (Fig. 4a) and were more likely to be in close proximity and on the same strand as an annotated transcription start site (TSS) (Fig. 4b). Similar to the majority of ERVK LTRs in the genome, extra- embryonic ERVK LTR promoters were mostly solo LTR elements (417 ± 19 bp) (Additional file 1: Figure S6), which had lost their associated retroviral genes [24]. Solo Page 6 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology Fig. 4 Genomic features and epigenetic patterning in the maternal oocyte are associated with ERVK LTR expression patterns in extra-embryonic tissues. a CpG content was compared between ERVK LTRs that were transcriptionally active in extra-embryonic tissues, including the subset of non-canonically imprinted ERVKs (N = 40, blue dots) and all mappable ERVK LTRs (N = 334,322) (p < 5E−10, Welch two-sample t test). b The proportion of transcriptionally active ERVK LTRs in extra-embryonic tissues (N = 40) within 3 kb of a transcription start site (TSS) on the same or opposing strand was compared to all mappable ERVK LTRs (N = 334,322) (chi-square statistic, p < 0.0001). c The proportion of transcriptionally active non-canonical imprinted ERVK LTRs (N = 8) and extra-embryonic active ERVK LTRs (N = 32) overlapping epigenetic modifications in GV oocytes was compared to a random subset of mappable ERVK LTRs (N = 100) (chi-square statistic, p = 0.0002 and p = 0.0001, respectively) Fig. 4 Genomic features and epigenetic patterning in the maternal oocyte are associated with ERVK LTR expression patterns in extra-embryonic tissues. a CpG content was compared between ERVK LTRs that were transcriptionally active in extra-embryonic tissues, including the subset of non-canonically imprinted ERVKs (N = 40 blue dots) and all mappable ERVK LTRs (N = 334 322) (p < 5E−10 Welch two-sample t test) b The Fig. 4 Genomic features and epigenetic patterning in the maternal oocyte are associated with ERVK LTR expression patterns in extra-embryonic tissues. Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs a CpG content was compared between ERVK LTRs that were transcriptionally active in extra-embryonic tissues, including the subset of non-canonically imprinted ERVKs (N = 40, blue dots) and all mappable ERVK LTRs (N = 334,322) (p < 5E−10, Welch two-sample t test). b The proportion of transcriptionally active ERVK LTRs in extra-embryonic tissues (N = 40) within 3 kb of a transcription start site (TSS) on the same or opposing strand was compared to all mappable ERVK LTRs (N = 334,322) (chi-square statistic, p < 0.0001). c The proportion of transcriptionally active non-canonical imprinted ERVK LTRs (N = 8) and extra-embryonic active ERVK LTRs (N = 32) overlapping epigenetic modifications in GV oocytes was compared to a random subset of mappable ERVK LTRs (N = 100) (chi-square statistic, p = 0.0002 and p = 0.0001, respectively) Non-canonically imprinted ERVK LTR promoters mediate imprinting of nearby protein-coding genes Non-canonically imprinted ERVK LTR promoters mediate imprinting of nearby protein-coding genes H3K27me3 in the oocyte (Fig. 5c). We find that RLTR15 acts as an alternative promoter for the Gab1 gene on the paternal allele specifically in the placenta (Fig. 5d, e), with intron-spanning reads demonstrating that the ERVK LTR is spliced onto exon 2 (Additional file 1: Figure S7). We found examples of non-canonically imprinted ERVK LTR promoters driving transcription of non-coding RNAs, but also mediating imprinting of protein-coding genes. One such example is the non-canonically imprinted ERVK LTR (RLTR15) located in intron 1 of the Gab1 gene. Gab1 shows imprinted paternal expression in E7.5 ExE; yet, the promoter of Gab1 has bi-allelic enrichment for H3K4me3 (Fig. 5a). Rather, the intronic RLTR15 is demarcated by imprinted paternal H3K4me3 (Fig. 5a) and is non-canonically imprinted (Fig. 5b) with enrichment for g Together, these analyses suggest that ERVK LTR ele- ments can directly mediate imprinted gene expression in extra-embryonic lineages. To demonstrate this using a genetic approach, we designed CRISPR/Cas9 sgRNAs to excise RLTR15 in vivo (Additional file 1: Figure S7). We targeted B6/CAST hybrid zygotes which were implanted in foster mothers, and we subsequently collected E12.5 Page 7 of 17 Hanna et al. Genome Biology (2019) 20:225 (2019) 20:225 Hanna et al. Genome Biology Fig. 5 (See legend on next page.) Page 8 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology (See figure on previous page.) Fig. Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs Together, these data suggest that the anno- tated Slc38a4 promoter is predominantly canonically imprinted by DNA methylation in embryonic lineages, while in extra-embryonic lineages, it appears that the non-canonically imprinted upstream ERVK LTRs may modulate the activity of the paternal allele of the Slc38a4 promoter, resulting in non-canonical imprinted gene expression. extra-embryonic tissues (placenta and yolk sac) and whole embryos (N = 42 embryos). We were able to ob- tain one embryo (F4E5) that was targeted on the paternal CAST allele, although genotyping revealed that the dele- tion was mosaic (Additional file 1: Figure S7). Nevertheless, allelic RNA-seq analysis of F4E5 compared with three con- trols (Additional file 1: Figure S7) demonstrated that Gab1 specifically showed a partial loss of imprinting in E12.5 pla- centa and yolk sac (Fig. 5f; Additional file 1: Figure S7). Another intriguing example is imprinted gene Slc38a4. Slc38a4 has a maternal gDMR at its promoter [26] but paradoxically was recently reported to have non-canonical imprinted gene expression in extra-embryonic lineages [21]. Furthermore, we identified a non-canonical imprinted H3K4me3 peak overlying the Slc38a4 gDMR promoter (Additional file 2: Tables S2 and S3), raising questions as to whether the Slc38a4 promoter is regulated canonically or non-canonically. To investigate this further, we assessed allelic RNA-seq patterns in the epiblast and ExE from B6/ CAST, CAST/B6, and matDKO/CAST embryos in detail. B6/CAST and CAST/B6 epiblast and ExE showed the ex- pected imprinted paternal expression (Additional file 1: Figure S8). In the matDKO/CAST epiblast, loss of the ma- ternal DNA methylation at the gDMR resulted in bi-allelic expression (Additional file 1: Figure S8), consistent with canonical imprinting. However, in the matDKO/CAST ExE, while there was an increase in the expression of the maternal allele, there was still a twofold paternal bias in the expression (Additional file 1: Figure S8), an observation consistent with non-canonical imprinting. We evaluated publically available gene expression [27], DNA methylation [28], and H3K4me3 and H3K27me3 histone modifications [22] in GV oocytes to determine whether the germ line pattern of maternal epigenetic modifications across the Slc38a4 locus is consistent with this finding. Indeed, the annotated promoter is fully methylated in GV oocytes, spanned by an oocyte-specific transcript emanating from multiple mammalian appar- ent LTR retrotransposon (MaLR) elements upstream (Additional file 1: Figure S8), as has been previously re- ported [27, 29]. Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs In contrast, the upstream non-canonical imprinted H3K4me3 peaks are enriched for H3K27me3 in GV oocytes (Additional file 1: Figure S8). Thus, it appears that independent ERV LTR insertions upstream of the Slc38a4 locus, one specifically active in oocytes and the other specifically active in extra-embryonic tissues, may have enabled genomic imprinting to have evolved twice at this locus, using both canonical and non-canonical mechanisms. While this finding needs to be confirmed genetically, it would represent, to our knowledge, the first such example of recurrent evolution of imprinting mechanisms reported to date. In ExE, in addition to the non-canonical H3K4me3 peak at the annotated Slc38a4 promoter, there were four up- stream non-canonical H3K4me3 peaks, all of which were located over ERV LTR element insertions (Additional file 1: Figure S8). In particular, one ERVK LTR ~ 75 kb upstream (MLTR31F_Mm) was highly expressed in ExE and showed non-canonical imprinted expression of a spliced transcript from the paternal allele (Additional file 1: Figure S8). How- ever, we found no evidence that this upstream ERVK Genomic and epigenetic features associated with non- canonically imprinted ERVK LTRs 5 A non-canonically imprinted ERVK LTR drives imprinted expression of Gab1 in placenta. a Screenshot of allelic gene expression, H3K4me3, H3K36me3, and H3K27me3 in B6/CAST and CAST/B6 ExE. ChIP-seq data is quantitated using enrichment normalized RPKM for 1-kb running windows with a 100-bp step (scales in square brackets); paternal (blue) and maternal (red) enrichments are shown on mirrored axes. RNA-seq data is quantitated as RPKM for 1-kb running windows with a 100-bp step. The box denotes the location of the non-canonical imprinted H3K4me3 peak associated with the known non-canonical imprinted gene Gab1. b Screenshot of allelic gene expression, H3K4me3, H3K36me3, and H3K27me3 in matDKO/CAST ExE, quantitated as in a. c Screenshot of H3K4me3, H3K27me3, and DNA methylation in GV oocytes. One-kilobase running windows with a 100-bp step were used; ChIP-seq data was quantitated as RPKM (scales in square brackets). d Screenshot showing allelic gene expression in F/CAST and CAST/F E12.5 placenta across the Gab1 locus. The box depicts the non-canonical imprinted paternal H3K4me3 peak containing an imprinted transcriptionally active ERVK LTR element (RLTR15). RNA-seq data is quantitated as log2RPKM for 1000-bp running windows with a 100-bp step. e Read count for maternal (red) and paternal (blue) transcription is shown for the non-canonically imprinted RLTR15 and exon 1 of the Gab1 gene in E12.5 and E16.5 embryonic (Li, liver; He, heart; Br, brain) and extra-embryonic (Pl, placenta; VE, visceral endoderm) tissues. Only intron- spanning reads were used, and two-tailed t test was used to statistically compare the allelic expression (**p < 0.0005). f Barplot shows the allelic gene expression (allelic ratio = mat/(mat + pat)) for the Gab1 gene in B6/CAST E12.5 yolk sac, placenta, and whole embryos. F4E5 carried CRISPR-targeted deletion of non-canonically imprinted RLTR15 on the paternal allele and was compared to wild-type (WT) controls (N = 3). Two-tailed single sample t test was used to compare the F4E5 value to the WT mean (p < 0.05). Error bars show standard deviation LTR was acting an alternative promoter for Slc38a4, as there were no intron-spanning reads extending to the first or second exon of Slc38a4 in E7.5 ExE or E12.5 placenta. Epigenetic regulation of non-canonical imprints in post- implantation embryos It has been shown that non-canonical imprinting in the early embryo is mediated by the inheritance of maternal H3K27me3 from the oocyte [21]. Therefore, we were Page 9 of 17 Page 9 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology (2019) 20:225 surprised to find that non-canonical imprinted EVRK LTRs did not show enrichment for maternal H3K27me3 in E6.5 ExE (Figs. 5a and 6a). Although there was a sub- tle bias of H3K27me3 towards the maternal allele in ExE at non-canonical imprinted H3K4me3 peaks (p = 0.02), when we identified regions with imprinted H3K27me3 in ExE using the EdgeR statistical approach (Additional file 1: Figure S9), only one non-canonically imprinted H3K4me3 peak was associated with imprinted H3K27me3. Further- more, we found that the vast majority of imprinted H3K27me3 in post-implantation ExE was localized to two large imprinting clusters (Kcnq1/Kcnq1ot1 and Igf2r/Airn) and entirely dependent on maternal gDMRs (Additional file 1: Figure S9 and S10). their epigenetic regulation is superseded by secondary imprinted DMRs specifically acquired in extra-embryonic lineages (Fig. 7). Our findings not only reveal that non- canonical imprinting can be mediated by ERVK LTR inser- tions, but uncover the epigenetic mechanisms responsible for their persistence in extra-embryonic tissues. The majority of the non-canonical imprinted H3K4me3 peaks we identified overlaid mono-allelically expressed ERVK LTR promoters, which mediated transcription of non-coding RNAs (e.g., Platr20, upstream of Slc38a4) or acted as alternative promoters to form chimeric mRNAs with nearby genes (e.g., Gab1, Smoc1). At the Gab1 locus, we demonstrated that spliced transcripts from the ERVK LTR are exclusively expressed from the paternal allele, while the upstream canonical exon 1 is transcribed bi-allelically. Furthermore, when we genetically targeted the Gab1 EVRK LTR promoter, despite only obtaining a mosaic deletion, we were able to disrupt the imprinted gene expression of Gab1. Together, these findings demonstrate that ERVK LTRs are a key genomic feature mediating non-canonical imprinting in murine extra-embryonic development. g To determine whether another repressive epigenetic mark replaced maternal H3K27me3 in the post-implantation em- bryo, we assessed the allelic DNA methylation. We gener- ated high coverage bisulphite sequencing data from ExE and epiblast of E7.5 reciprocal B6 x CAST hybrid embryos enab- ling us to obtain sufficient read depth over ERVK LTRs. These data revealed that non-canonical imprinted EVRK LTR promoters become DMRs in ExE, with the maternal al- lele becoming methylated (Fig. Epigenetic regulation of non-canonical imprints in post- implantation embryos 6a), whereas both alleles were methylated in the epiblast (Additional file 1: Figure S11). Using publicly available bisulphite and RNA sequen- cing data from C57BL/6 germ cells and early embryos [30], we demonstrate that these regions are definitively tissue- specific secondary imprints acquired in the post- implantation de novo DNA methylation wave specifically in ExE (Fig. 6b). The acquisition of bi-allelic DNA methylation in the post-implantation epiblast corresponds to the silen- cing of these ERVK LTR promoters (Fig. 6c). y p ERVs have also been reported to function as enhancers specifically in the placenta through the acquisition of binding sites for developmental transcription factors, and it is thought that the uniquely hypomethylated state of the extra-embryonic tissues may enable transcrip- tional regulation by repetitive elements [25, 32]. We did not find any evidence for shared transcription factor binding motifs among active extra-embryonic ERVK LTR promoters; however, we found that they were pre- dominantly CpG-rich solo LTRs. There are several epi- genetic modifiers containing CxxC domains that bind unmethylated CpGs, such H3K4 methyltransferases [33]; thus, high CpG content may be key to their role in tran- scriptional regulation. Solo LTRs, in particular, may be co-opted as transcriptional regulators in development because their lack of viral genes may enable them to escape the KRAB-ZFP silencing [24]. Notably, ERVs genome-wide are under-represented within 5 kb of pro- moters and specifically in the sense orientation [34]. We find that extra-embryonic ERVK LTR promoters are not only in close proximity to TSSs, but in particular in the sense orientation. Together, these findings suggest that promoter activity of ERVK LTRs in extra-embryonic tis- sues may be attributable to sequence composition and opportunistic positioning in the genome. Conversely, using publically available ChIP-seq data [31], we observed the loss of maternal enrichment for H3K27me3 at non-canonical imprints during pre- implantation development (Fig. 6d; Additional file 1: Figure S11). Thus, non-canonical imprints do not main- tain allelic H3K27me3 beyond early pre-implantation em- bryonic development, supporting that the regulation of allele-specific expression of non-canonical imprinted genes is superseded by DNA methylation in post- implantation development. Discussion In this study, we evaluated allelic histone modifications, DNA methylation, and gene expression to investigate the epigenetic regulation of imprinted genes in the post- implantation embryonic and extra-embryonic lineages. We identified non-canonical imprints that are definitively inde- pendent of maternally inherited DNA methylation in ExE and find that these are located preferentially at active ERVK LTR insertions. Furthermore, we find that while non-canonical imprinted genes inherit allelic H3K27me3 from the oocyte, this allelic enrichment is transient and Notably, ERV-derived placental enhancers and oocyte promoters were found to be species-specific [25, 29], and thus, we may expect that non-canonical imprinted regions, similarly co-opting ERV insertions, may also be species-specific. Indeed, preliminary studies in human embryos found five paternally expressed genes that may be regulated by maternal H3K27me3 [35], none of which have been reported to be imprinted in mice. These Page 10 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology Fig. 6 (See legend on next page.) Fig. 6 (See legend on next page.) Page 11 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology (See figure on previous page.) Fig. 6 Non-canonically imprinted ERVK LTRs lose maternal H3K27me3 and acquire secondary imprinted DMRs in post-implantation ExE. a Heatmap showing enrichment for H3K4me3 and H3K27me3 and DNA methylation on the maternal and paternal allele in B6/CAST and CAST/B6 ExE at non-canonically imprinted active ERVK LTRs ± 500 bp (N = 8). DNA methylation of the maternal and paternal allele is shown in B6/CAST and CAST/B6 E7.5 ExE. b Boxplots show DNA methylation at non-canonically imprinted active ERVK LTRs ± 500 bp (N = 8) in germ cells and pre- and post-implantation stage C57BL/6 embryos. c Boxplots show gene expression for non-canonically imprinted active ERVK LTRs ± 500 bp (N = 8) in pre- and post-implantation embryonic stage C57BL6 embryos. d Heatmap showing enrichment for H3K4me3 and H3K27me3 on the maternal and paternal allele across pre-implantation development (e2-cell, early 2-cell embryo; l2-cell, late 2-cell embryo; 8-cell, 8-cell embryo; ICM, inner cell mass) in B6/PWK embryos at non-canonically imprinted active ERVK LTRs ± 500 bp (N = 8) H3K27me3. These findings support the observations from trophoblast stem cells in vitro that have shown lncRNAs regulated by the Igf2r/Airn and Kcnq1/Kcnq1ot1 maternal gDMRs mediate the recruitment of PRC2 and spreading of H3K27me3 in cis, in an X chromosome inactivation-like mechanism of silencing [40–42]. Discussion findings are reminiscent of placental-specific imprinted gDMRs in humans, which were also found to be species- specific [36, 37]. Furthermore, a recent study demonstrated that species-specific gDMRs were a consequence of unique ERV insertions, which initiated gDMR-spanning transcrip- tion in oocytes [29]. Together, these findings support that ERV activity in the placenta and in the oocyte may be a key driver in the recent evolution of non-canonical and ca- nonical imprinting in extra-embryonic tissues. Conclusions Our study of imprinted genes in in vivo post-implantation extra-embryonic development has provided novel insights into non-canonical imprinted gene regulation, which are otherwise masked in bulk genomic data from inbred strains and are difficult to assess in human populations due to the sparsity of genetic polymorphisms. We reveal that the majority of non-canonical imprints are localized to solo ERVK LTR repeats, which act as imprinted tran- scription initiation sites for non-coding RNAs and chimeric mRNAs in extra-embryonic tissues. Importantly, we find that the regulation of non-canonical imprinted regions transition from inherited maternal H3K27me3 to secondary imprinted DMRs specifically in extra- embryonic lineages. These findings highlight the unique mechanisms regulating imprinted gene expression in the placenta and the potential importance of the still unexplored role of these non-canonical imprints in regulating placentation and fetal growth. Non-canonical imprinting is mediated by inheritance of H3K27me3 from the oocyte and was suggested to maintain a few non-canonical imprints into the extra- embryonic development [21]. In our study, we identified all four previously reported non-canonical imprinted genes [21], in addition to several novel domains. Furthermore, we demonstrated conclusively that non-canonical imprinted genes are mono-allelically expressed independent of inher- ited maternal DNA methylation. However, we find that maternal enrichment for H3K27me3 does not persist be- yond pre-implantation development at non-canonically imprinted loci but rather is replaced by maternal DNA methylation in post-implantation ExE. Conversely, non- canonical imprinted ERVK LTRs become bi-allelically si- lenced in embryonic lineages by the acquisition of bi-allelic DNA methylation. The mechanisms underlying the transi- tion in repressive epigenetic states on the maternal allele are unclear, and why the allelic specificity would persist in ExE, but not in the epiblast, remains to be explored. Methods p p Despite the lack of allelic H3K27me3 at non-canonical imprinted loci in post-implantation ExE, we find a role for imprinted H3K27me3 at other genomic regions. We identi- fied four silenced imprinted genes (Plagl1, Slc22a3, Pde10a, and Magel2) where the active allele was demarked by bi- valent chromatin in E6.5 ExE, which subsequently resolved to imprinted gene expression in E12.5 placentae. Thus, bi- valent chromatin may enable the temporal regulation of imprinted gene expression in extra-embryonic develop- ment, similar to that which has been observed in embry- onic lineages [38, 39]. We also find large allelic H3K27me3 domains at the Igf2r/Airn and Kcnq1/Kcnq1ot1 loci in extra-embryonic tissues in vivo and identify a number of novel imprinted genes distal of the Igf2r/Airn cluster. Furthermore, we show that the maternal gDMRs at these loci are required to prevent bi-allelic acquisition of Sample collection Reciprocal natural timed matings were set up between C57BL6/Babr and CAST/EiJ animals (denoted as B6/ CAST and CAST/B6), and embryos were collected on embryonic days 6.5 (E6.5) and 7.5 (E7.5). Natural timed matings were set up between Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3+ve B6/129 females (resulting in ablation of DNA methylation in the oocyte) [9] and CAST males (denoted as matDKO/CAST). The epiblast (Epi) and extra-embryonic ectoderm (ExE) for each em- bryo were manually separated. E6.5 epiblast (N = 4) and ExE (N = 8) samples were pooled (an estimated ~ 2500 cells), washed in PBS, and then flash-frozen in 10 μL of nuclear lysis buffer (Sigma-Aldrich). Single E7.5 epiblast and ExE samples were individually frozen in 10 μL of buffer RLT Plus (Qiagen). Page 12 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology In vivo CRISPR targeting C57BL6/J females were superovulated and crossed with ERVK element (200 ng/μL each) (Additional file 1: Figure S7) and CAS9 protein (500 ng/μL) Embryos were then implanted Fig. 7 Summary of epigenetic regulation of non-canonical imprinting in embryonic development. Schematic diagram showing the allelic epigenetic regulation of a non-canonically imprinted gene by an ERVK LTR element (top) and the dynamic regulation of non-canonically imprinted ERVK LTRs across pre- and post-implantation development (bottom). In the pre-implantation embryo, inherited H3K27me3 from the oocyte silences the maternal allele. In the post-implantation embryo, maternal H3K27me3 transitions to imprinted maternal DNA methylation in extra-embryonic lineages, thereby retaining the imprinted paternal expression of the ERVK LTR. Alternatively, in the embryonic lineages, both the maternal and paternal alleles acquire DNA methylation, consequently silencing the ERVK LTR transcription. The maternal allele is shown in red, and paternal allele is shown in blue. In the allelic enrichment plot, the solid line is the level of H3K27me3, and the dashed line is the level of DNA methylation. Embryonic day (E) is shown on the x-axis for each respective stage of embryogenesis Fig. 7 Summary of epigenetic regulation of non-canonical imprinting in embryonic development. Schematic diagram showing the allelic epigenetic regulation of a non-canonically imprinted gene by an ERVK LTR element (top) and the dynamic regulation of non-canonically imprinted ERVK LTRs across pre- and post-implantation development (bottom). In the pre-implantation embryo, inherited H3K27me3 from the oocyte silences the maternal allele. Sample collection In the post-implantation embryo, maternal H3K27me3 transitions to imprinted maternal DNA methylation in extra-embryonic lineages, thereby retaining the imprinted paternal expression of the ERVK LTR. Alternatively, in the embryonic lineages, both the maternal and paternal alleles acquire DNA methylation, consequently silencing the ERVK LTR transcription. The maternal allele is shown in red, and paternal allele is shown in blue. In the allelic enrichment plot, the solid line is the level of H3K27me3, and the dashed line is the level of DNA methylation. Embryonic day (E) is shown on the x-axis for each respective stage of embryogenesis ERVK element (200 ng/μL each) (Additional file 1: Figure S7) and CAS9 protein (500 ng/μL). Embryos were then implanted into NMRI pseudo-pregnant females. The embryos were dissected on E12.5, and the following tissues were Low-input mRNA sequencing library preparation Low-input mRNA sequencing library preparation Stranded mRNA-seq libraries were generated for E7.5 embryos: B6/CAST Epi (N = 3) and ExE (N = 3), CAST/ B6 Epi (N = 3) and ExE (N = 3), and matDKO/CAST Epi (N = 3) and ExE (N = 3). Total RNA was extracted using a TRIzol extraction method, as previously described [27]. In brief, samples were homogenized in 400 μL of TRIzol (Invitrogen) and phase-separated by adding 80 μL of chloroform:isoamyl alcohol (Sigma-Aldrich), mixed, and centrifuged at 4 °C for 15 min. The aqueous phase was transferred to a new tube, 1 μL GlycoBlue and 300 μL of ice-cold isopropanol were added and mixed. Samples were incubated for 10 min and then centrifuged for 10 min at 4 °C. The pellet was washed once with 75% ethanol, air-dried, and then resuspended in 5 μL of RNase-free water. Twenty microliters of lysis/binding buffer was immediately added to each RNA sample, and oligo (dT)25 capture of mRNA was done using In vivo CRISPR targeting C57BL6/J females were superovulated and crossed with CAST/EiJ males. Zygotes were recovered the next day and electroporated with two sgRNAs for the Gab1 RLTR15 Page 13 of 17 Hanna et al. Genome Biology (2019) 20:225 Dynabeads mRNA DIRECT kit (Life Technologies). The protocol was implemented as per manufacturer’s in- structions including the additional steps for elimination of rRNA contamination. Maxymum Recovery tubes (Axygen) were used, and volumes were adapted for the low amount of starting material: 5 μL of Dynabeads Oligo (dT)25 were used for each sample, mRNA capture was done in a total volume of 50 μL of lysis/binding buf- fer, washes were done using 100 μL Washing Buffer A or 50 μL of Washing Buffer B, and a final elution volume of 5 μL 10 mM Tris-HCl. The total volume of mRNA was then immediately advanced into the library preparation protocol, using the SMARTer Stranded RNA-seq kit (Clontech), which is optimized for as little as 100 pg of RNA. The protocol was completed as per the manufac- turer’s instructions, and 14 amplification cycles were used for all samples. The quantification of all libraries was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Libraries were sequenced using 50 bp single- end on the Illumina HiSeq 2500 RapidRun, multiplexing 12 samples per lane. Libraries were evaluated for quality in SeqMonk using RNA-seq QC and duplication plots, resulting in 1 replicate of B6/CAST ExE being excluded due to high duplication. collected: (1) visceral yolk sac/amnion, (2) placental disc with as much decidua removed as possible, (3) em- bryo, and (4) tail clip for genotyping. Tissue samples were washed in cold PBS and flash-frozen in 50 μL of RLT+ buf- fer (Qiagen). Tissues were collected from a total of 42 E12.5 embryos from 6 females across 2 independent experiments. DNA from tail clippings were genotyped using MyTaq master mix (Bioline) with primers: F - AGCCCAATCT CACAACAGTT, R - CGGACCAGGTGAACATGTTG. Bands corresponding to the wild-type (847 bp) and knockout (320 bp) alleles were gel extracted and sent for Sanger sequencing to identify the targeted allele (Add- itional file 1: Figure S7). One effectively targeted sample (F4E5) and three wild-type controls (F4E1, F4E3, and F5E6) were selected for RNA sequencing. Ultra low-input native chromatin immunoprecipitation Ultra low-input native chromatin immunoprecipitation Ultra low-input native ChIP-seq was done as previously de- scribed [22]. ChIP-seq libraries for H3K4me3, H3K27me3, H3K36me3, and 10% inputs were generated for replicates of pooled E6.5 embryos: B6/CAST Epi (N = 2) and ExE (N = 2), CAST/B6 Epi (N = 2) and ExE (N = 2), and matDKO/CAST Epi (N = 2) and ExE (N = 2). Prior to the immunoprecipitation with antibody-bound beads, each chromatin sample (200 μL) was divided into 5 aliquots: 1 for each antibody (54 μL), 1 10% input (20 μL), and 1 10% input for bisulphite sequencing (20 μL) (Add- itional file 1: Figure S1). For each immunoprecipitation, 250 ng of anti-H3K4me3 (Diagenode K02921004), 125 ng of anti-H3K27me3 (Millipore 07-449), and 250 ng of anti- H3K36me3 (Diagenode C15410192) were used. Library preparation was done using the MicroPlex Library Prepar- ation kit v2 (Diagenode), as per the manufacturer’s recom- mendations, and libraries amplified using 15 amplification cycles. Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75 bp paired-end sequencing on Illumina NextSeq500. mRNA sequencing library preparation q g y p p RNA was extracted from E12.5 yolk sac, placenta, and whole embryo from F4E1 (Gab1 ERVK RLTR15 +/+), F4E3 (Gab1 ERVK RLTR15 +/+), F4E5 (Gab1 ERVK RLTR15 +/−), and F5E6 (Gab1 ERVK RLTR15 +/+) embryos using RNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. RNA was treated with TURBO DNase (Thermo Fisher Scientific), and quality was assessed with RNA Pico Kit (Agilent) on the Agilent 2100 Bioanalyzer (RIN > 8.7 for all samples). mRNA se- quencing libraries were generated using SmartSeq v4 cDNA generation and Nextera XT library preparation, as per manufacturers’ instructions. The quantification of all libraries was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantifi- cation kit (KAPA). Libraries were sequenced using 125 bp paired-end on the Illumina HiSeq 2500 RapidRun, multiplexing 12 samples over 2 lanes. Post-bisulphite adaptor tagging for deep sequencing p p gg g p q g Post-bisulphite adaptor tagging (PBAT) was done on E7.5 epiblast and ExE samples from single embryos (B6/CAST Epi and ExE (N = 2), CAST/B6 Epi and ExE (N = 2)), as previously described [43]. Quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agilent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 150 bp paired- end sequencing on Illumina NextSeq500, multiplexing four samples per lane. ChIP-seq peak calling Peak calling was done for B6/CAST and CAST/B6 epi- blast and ExE H3K4me3, H3K27me3, and H3K36me3 using chromstaR, a multivariate peak-calling approach based on a multivariate hidden Markov model, using the default parameters [47]. Allelic histone enrichment and gene expression analyses Read counts for maternal and paternal H3K4me3 or H3K27me3 were quantitated over B6/CAST and CAST/ B6 H3K4me3 peaks for either epiblast or ExE. H3K4me3 or H3K27me3 peaks were combined and de-duplicated between the B6/CAST and CAST/B6 epiblast or ExE, to generate a complete list of peaks from both hybrid crosses for each tissue. Peaks were then filtered for those with a minimum read count of 20 in at least 1 allelically mapped biological replicate. Peaks with allelically biased H3K4me3 or H3K27me3 were then identified using EdgeR statistic (p < 0.05, corrected for multiple comparisons). Significant peaks were then classified into strain-specific allelic H3K4me3 or H3K27me3 if their allelic enrichment switched in the reciprocal cross. Significant peaks were identified as imprinted if the allelic enrichment Public datasets The following public datasets were used in this manu- script: stranded total RNA-seq from FvB x CAST recipro- cal hybrids for E12.5 placenta, E12.5 visceral endoderm, E12.5 liver, E16.5 brain, E16.5 heart, and E16.5 liver [15]; H3K27me3 ChIP-seq from B6 x PWK hybrid embryos for early and late 2-cell embryos, 8-cell embryos, and E3.5 blastocyst inner cell mass (ICM) [31]; H3K4me3 ChIP-seq from B6 x PWK hybrid embryos for early and late 2-cell embryos, 8-cell embryos, and E3.5 blastocyst inner cell mass (ICM) [44]; RNA-seq and bisulphite-seq from C57BL/6 pre- and post-implantation embryos [30]; bisulphite-seq [28], total RNA-seq [27], and H3K4me3 and H3K27me3 ChIP-seq [22] data from C57BL/6 GV oo- cytes. All raw data files were obtained from Gene Expres- sion Omnibus or the DNA data bank of Japan and were mapped and evaluated using the following pipelines. Low-input post-bisulphite adaptor tagging from ChIP input samples Post-bisulphite adaptor tagging (PBAT) was done on 10% input samples, as previously described [22]. A cor- responding 10% input was taken from each ChIP sample Page 14 of 17 Page 14 of 17 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology (2019) 20:225 Hanna et al. Genome Biology (2019) 20:225 Following alignments, all sequencing data was then split allele-specifically using SNPsplit (v0.3.3) [46]. In brief, sequencing reads were mapped to a Mus musculus (GRCm38)-derived genome, where SNPs between hybrid strains (C57BL6 and CAST/EiJ, or FvB and CAST/EiJ, or C57BL6 and PWK) had been masked by the ambiguity nucleobase N (N-masked genome). Aligned reads were then sorted into one of three BAM files: C57BL6 (genome 1), CAST/EiJ (genome 2), or unassigned. The females car- rying conditional Dnmt3a/Dnmt3b double knockout (matDKO) were predominantly C57BL6; however, there was approximately 15% of 129 alleles remaining in the strain. Therefore, these data were run through a unique pipeline to allow for a complete mapping of the maternal allele. All datasets generated from matDKO/CAST em- bryos were first aligned to a B6/CAST N-masked genome, as above, but with the difference that all SNPs which were in common between 129 and CAST (~ 2 million) had been excluded. The data was then split against C57BL6 and CAST/EiJ, as above. FastQ reads of the unassigned fraction of reads were then recovered from the original FastQ files, and in the second step, these reads were then aligned to a 129S1/CAST genome (generated with SNPsplit_genome_preparation). Alignments were then SNPplit between 129 and CAST/EiJ, and the 129-specific reads were then combined with the C57BL6-specific reads (from step 1) to comprise a complete maternal allelic set. Raw sequencing reads and allelically mapped BAM files have been deposited into the Gene Expression Omnibus database (GSE124216). above, with the addition of replicates, where available: B6/CAST Epi (N = 3) and ExE (N = 2), CAST/B6 Epi (N = 3) and ExE (N = 2), and matDKO/CAST Epi (N = 3) and ExE (N = 3). All input PBAT libraries were amplified using 12 amplification cycles; quantification was done using the High DNA Sensitivity Bioanalyzer 2500 (Agi- lent) and Illumina library quantification kit (KAPA). Samples were multiplexed using 75 bp paired-end se- quencing on Illumina NextSeq500. One replicate of matDKO/CAST epiblast was excluded from the analysis due to < 5% unique mappability. Allelic mapping of sequencing data pp g q g RNA-seq data was subjected to trimming with Trim Galore (v0.4.5) and aligned using HISAT2 (v2.1.0, guided by gene models from Ensembl annotation release 70, op- tions: --dta –sp 1000,1000). ChIP-seq and input material was also trimmed with Trim Galore and aligned using Bowtie2 (v2.3.2, options -X 1200). In addition to adapter and quality trimming, bisulfite sequencing data had the first 9 bp of both read 1 and read 2 removed to reduce biases arising from the 9 N oligo pull-down reaction (Trim Galore options: --clip_r1 9 --clip_r2 0 --paired). The trimmed PBAT data was then aligned using Bis- mark (v0.19.0, options: --pbat) [45]. Page 15 of 17 Page 15 of 17 Hanna et al. Genome Biology (2019) 20:225 for a parental allele was consistent between reciprocal crosses. imprinted paternal H3K4me3 peaks (Additional file 2: Table S5) and 32 were classified as extra-embryonic active ERVK LTRs (Additional file 2: Table S6). The presence and directionality of reads spanning annotated introns, potential introns between annotated and upstream novel exons, and between upstream novel exons were ana- lyzed to determine those ERVK LTRs that were initiating mRNA chimeras or non-coding RNAs (Additional file 2: Tables S5 and S6). Read counts for maternal and paternal H3K36me3 or RNA-seq were quantitated over autosomal genes. Genes were then filtered for those with a minimum read count of 20 in at least 1 allelically mapped biological replicate of B6/CAST and CAST/B6 H3K36me3 or a minimum read count of 5 in at least 1 allelically mapped biological replicate of B6/CAST and CAST/B6 (or F/CAST and CAST/F for E12.5 placenta) RNA-seq. Genes with alleli- cally biased expression were then identified using EdgeR statistic (p < 0.05, corrected for multiple comparisons). Significant genes were filtered for those that were associ- ated with an imprinted H3K4me3 peak. Funding Coordinates for repetitive elements for the mouse GRCm38 genome build were generated using Repeat- Masker. Active ERVK LTRs were identified as those that fell within an H3K4me3 peak in E6.5 ExE, with ≥5 reads on the same strand at the LTR repeat in at least 2 repli- cates of RNA-seq data from E7.5 ExE, E12.5 visceral endoderm [15], and/or E12.5 placenta [15] with at least 1 intron-spanning read indicative of spliced RNA. These were then filtered for those that were sites of transcription initiation with no apparent upstream intron-spanning reads (N = 40), of which 8 were within non-canonically CH is supported by a fellowship from the Centre for Trophoblast Research and LG is supported by a Marie Curie Intra-European Fellowship (708255). Work in GK’s lab is supported by grants from the UK Medical Research Coun- cil (MR/K011332/1, MR/S000437/1) and Biotechnology and Biological Sci- ences Research Council (BBS/E/B/000C0423). Work in DB’s lab was supported by grants from the ERC (ERC-CoG EpiREPRO). Authors’ contributions CH and GK conceptualized and designed the study. CH, WD, and RPP collected the samples and generated the datasets. RPP designed and performed the CRISPR targeting experiments. CH, LG, MS, LB, FK, and SA performed the data analysis. CH, LG, LB, and RPP generated the figures. CH wrote the manuscript. RPP, LG, FK, SA, MCT, WD, DB, and GK contributed to the manuscript. GK and DB financially supported the study. All authors read and approved the final manuscript. Sequence motif analysis of extra-embryonic ERVK LTR promoters DREME (version 5.0.5) was used to identify transcription factor motifs among ERVK LTRs that are transcription- ally active in extra-embryonic tissues (N = 40) compared to a background set with comparable length and class. AME (version 5.0.5) was used to evaluate enrichment for transcription factor binding sites for CDX2, EOMES, and ELF5. Review history h h The review history is available as Additional file 3. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s13059-019-1833-x. Additional file 1. Supplementary figure and table legends, Figures. S1-S11. Additional file 2. Supplementary Tables S1-S6. Additional file 3. Review history. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13059-019-1833-x. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13059-019-1833-x. Additional file 1. Supplementary figure and table legends, Figures. S1-S11. Additional file 2. Supplementary Tables S1-S6. Additional file 3. Review history. Additional file 1. Supplementary figure and table legends, Figures. S1-S11. Additional file 2. Supplementary Tables S1-S6. Additional file 3. Review history. Acknowledgements g We would like to acknowledge the essential contributions of the Biological Support Unit and Kristina Tabbada in the Sequencing Facility at the Babraham Institute, Julian Iranzo from DB’s lab and Fatima El Marjou, and Colin Jouhanneau of the Trangenesis Platform at the Institut Curie. ChIP-seq quantitation For the quantitative display of allelic ChIP-seq data for a single histone mark and allelic ratios, read counts per running window or peak (where applicable) were aver- aged between biological replicates. Read counts were normalized to library size, excluding X, Y, and mito- chondrial chromosomes, using size-factor normalization in SeqMonk. However, for the comparison of allelic en- richment for H3K4me3 and H3K27me3 across pre- implantation development (Additional file 1: Figure S11B and C), raw read counts were used. For this com- parison, correcting for library size is not appropriate, as there is a known discrepant abundance of H3K27me3 between the maternal and paternal alleles [31]. When ChIP-seq data of multiple histone marks are displayed together in a screenshot, enrichment normalized reads per kilobase per million (RPKM) was used. Enrichment normalization performs an initial additive translation of the data based on a low data percentile (40th percentile) representing non-zero but unambiguously unenriched points, followed by a multiplicative expansion of the data to a second high percentile (99th percentile) representing the most highly enriched regions. Enrichment is therefore scaled between these two points, but following the relative enrichment levels seen in the untransformed data. Availability of data and materials All data generated for this study is available on Gene Expression Omnibus (GSE124216) [48]. 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Protection against de novo methylation is instrumental in maintaining parent-of-origin methylation inherited from the gametes. Mol Cell. 2012;47: 909–20. 37. Hanna CW, Penaherrera MS, Saadeh H, Andrews S, McFadden DE, Kelsey G, et al. Pervasive polymorphic imprinted methylation in the human placenta. Genome Res. 2016;26:756–67. 13. Moore T, Haig D. Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet. 1991;7:45–9. 38. Sanz LA, Chamberlain S, Sabourin JC, Henckel A, Magnuson T, Hugnot JP, et al. A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10. EMBO J. 2008;27:2523–32. 14. Babak T, DeVeale B, Tsang EK, Zhou Y, Li X, Smith KS, et al. Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse. Nat Genet. 2015;47:544–9. 39. Maupetit-Mehouas S, Montibus B, Nury D, Tayama C, Wassef M, Kota SK, et al. 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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W1973744696	https://www.scielo.br/j/eins/a/Nyyk5hL4VgzKxzTjkqKKfDL/?lang=en&format=pdf	English	null	Normalization of lymphocyte count after high ablative dose of I-131 in a patient with chronic lymphoid leukemia and secondary papillary carcinoma of the thyroid. Case report	Einstein	2,014	cc-by	3,622	1 Hospital Israelita Albert Einstein, São Paulo, SP, Brazil. 2 Sociedade Brasileira de Diabetes, São Paulo, SP, Brazil. Corresponding author: Anneliese Rosmarie Gertrud Fischer Thom – Hospital Israelita Albert Einstein, Imagem department, Avenida Albert Einstein, 627/701, 1st sub basement floor, building A – Morumbi – Zip code: 05652-900 – São Paulo, SP, Brazil – Phone: (55 11) 2151-0489 – E-mail: afthom@einstein.br Received on: Dec 3, 2012 – Accepted on: Dec 5, 2013 DOI: 10.1590/S1679-45082014RC2657 CASE REPORT CASE REPORT ABSTRACT The authors report the case of a 70-year-old male patient with chronic lymphoid leukemia who presented subsequently a papillary carcinoma of the thyroid with metastases to regional lymph nodes. The patient was treated with surgical thyroidectomy with regional and cervical lymph node excision and radioiodine therapy (I-131). The protocolar control scintigraphy 4 days after the radioactive dose showed I-131 uptake in both axillae and even in the inguinal regions. PET/CT showed faint FDG-F-18 uptake in one lymph node of the left axilla. An ultrasound guided fine needle biopsy of this lymph node identified by I-131 SPECT/CT and FDG-F-18 PET/CT revealed lymphoma cells and was negative for thyroid tissue and thyroglobulin content. The sequential blood counts done routinely after radiation treatment showed a marked fall until return to normal values of leucocytes and lymphocytes (absolute and relative), which were still normal in the last control 19 months after the radioiodine administration. Chest computed tomography showed a decrease in size of axillary and para- aortic lymph nodes. By immunohistochemistry, cells of the lymphoid B lineage decreased from 52% before radioiodine therapy to 5% after the procedure. The authors speculate about a possible sodium iodide symporter expression by the cells of this lymphoma, similar to some other non-thyroid tumors, such as breast cancer cells. Descritores: Linfoma; Contagem de linfócitos; Leucemia; Neoplasias da glândula tireóide/terapia; Radioisótopos do iodo/uso terapêutico; Relatos de casos Keywords: Lymphoma; Lymphocyte count; Leukemia; Thyroid neoplasms/ therapy; Iodine radioisotopes/therapeutic use; Case reports INTRODUCTION It is well known that chronic lymphocytic leukemia/small cell lymphocytic lymphoma (CLL/SCLL) predispose to the occurrence of subsequent neoplasms, mainly Normalization of lymphocyte count after high ablative dose of I-131 in a patient with chronic lymphoid leukemia and secondary papillary carcinoma of the thyroid. Case report Normalização da contagem de linfócitos após dose ablativa de I-131 em um paciente com leucemia linfóide crônica e carcinoma papilífero da tireóide. Relato de caso Anneliese Rosmarie Gertrud Fischer Thom1, Nelson Hamerschlak1, Verônica Goes Teles2, Akemi Osawa1, Fabio Pires de Souza Santos1, Denise da Cunha Pasqualin1, Jairo Wagner1, Lilian Yuri Itaya Yamaga1, Marcelo Livorsi da Cunha1, Guilherme de Carvalho Campos Neto1, Marcelo Buarque de Gusmão Funari1 Anneliese Rosmarie Gertrud Fischer Thom1, Nelson Hamerschlak1, Verônica Goes Teles2, Akemi Osawa1, Fabio Pires de Souza Santos1, Denise da Cunha Pasqualin1, Jairo Wagner1, Lilian Yuri Itaya Yamaga1, Marcelo Livorsi da Cunha1, Guilherme de Carvalho Campos Neto1, Marcelo Buarque de Gusmão Funari1 papilífero da tireóide com metástases para linfonodos regionais. O paciente foi tratado com tireoidectomia total cirúrgica com exérese de linfonodos regionais e cervicais e radioiodoterapia (I-131). A pesquisa de corpo inteiro protocolar de controle 4 dias após a dose radioativa mostrou captação de I-131 em ambas as axilas e mesmo nas regiões inguinais. PET/CT mostrou discreta captação de FDG-F-18 em um linfonodo da axila esquerda. A biópsia por agulha fina guiada por ultrassom deste linfonodo identificado por SPECT/CT com I-131 e PET/CT com FDG-F-18 revelou células linfomatosas e foi negativa para tecido tireoidiano e conteúdo de tireoglobulina. Os hemogramas sequenciais feitos rotineiramente após tratamento com radiações mostraram uma acentuada queda até retorno aos valores normais de leucócitos e de linfócitos (absolutos e relativos), que continuavam normais no último controle 19 meses após a administração do radioiodo. Tomografia computadorizada de tórax mostrou uma redução em tamanho de linfonodos axilares e para-aorticos. Por imunohistoquímica, as células da linhagem linfoide B decresceram de 52% antes da radioiodoterapia para 5% depois do procedimento. Os autores conjeturam sobre uma possível expressão de symporter de iodeto de sódio pelas células deste linfoma, à semelhança de outros tumores não tireoidianos, tais como células de câncer da mama. CASE REPORT I-131 WBS done on January 27, 2010 detected tiny remnants of iodine avid tissue in the anterior cervical region, with 24 hour uptake <1% of the tracer dose. No other definite uptake was seen besides a very faint dubious higher concentration in both axillary regions, which was disregarded (Figure 1 A). I.F., a 70-year-old male patient, was diagnosed as having chronic lymphocytic leukemia (CLL) in July 2009. During the staging workup, a huge substernal multinodular goiter, with an estimated volume of 82cm3 was found by computed tomography (CT). An ultrasound (US) guided fine needle biopsy of one of the nodules was suspicious for thyroid cancer. This suspicion was reinforced by a serum thyroglobulin value of 936.9ng/mL (chemiluminescence immunoassay), measured in October of the same year. A therapeutic dose of 7,400MBq (200mCi) I-131 was administered on February 2, 2010. At the routine post-therapeutic WBS done on February 6, in addition to the expected uptake in the cervical remnants, definite uptake in both axillary regions and even in tiny structures in the inguinal regions was seen (Figure 1B). The planar scintigrams were complemented by a SPECT/CT study of the chest, which clearly showed radioiodine concentration in the axillary lymph nodes. At least one enlarged lymph node in the left axilla could be precisely identified (Figure 2). As for the CLL, no specific treatment had been recommended. However, in view of the above mentioned findings concerning the thyroid, the patient was submitted to total thyroidectomy with regional lymph node (LN) resection in January 5, 2010. A total of 29 LN were excised (5 right paratracheal, 8 from level VI, 5 from level VII on the right side, 7 from level VI and 4 from level VII on the left side). The pathological diagnosis of the excised material reported: “follicular variant of papillary microcarcinoma of the thyroid in the right lobe, confined to the thyroid gland, measuring 1.2mm, without involvement of surgical margins, extrathyroid tissue, or vascular invasion; multinodular goiter with foci of lymphocytic thyroiditis”. Figure 1. Whole-body scintigraphy before (1A) and 4 days after (1B) therapeutic dose of I-131. Uptake in axillae is suspected in the pre-dose scan but is evident in the post-dose scan. Note uptake also in the inguinal regions A B B Metastases were found in 8/29 LN: in all 5 right paratracheal LN and in 3/8 LN of level VI, on the right site. RESUMO Os autores relatam o caso de um paciente de 70 anos com leucemia linfóide crônica que apresentou subsequentemente um carcinoma einstein. 2014;12(1):100-5 Normalization of lymphocyte count after high ablative dose of I-131 101 – CT of the chest and abdomen: enlarged axillary and retroperitoneal LN, not further investigated and attributed to the underlying lymphocytic leukemia. kidney and skin cancers.(1-7) The association with thyroid carcinoma is extremely rare, according to the medical literature.(8, 9) We present the case of a patient with chronic lymphocytic leukemia and secondary papillary thyroid carcinoma whose primary neoplasm had an unusual evolution after a single therapeutic dose of I-131 intended to ablate thyroid remnants after total thyroidectomy. By that time (January 27), his complete blood count showed 4.74x106 erythrocytes, 19,200 leukocytes from which 13,958 (72.7%) were lymphocytes, and 265,000 platelets. Serum values obtained in January 23 were: TSH: 63mIU/L; thyroglobulin: 93.4ng/mL (963.9ng/mL before surgery); anti-thyroglobulin antibodies: absent. CASE REPORT All others were free of disease. The patient was referred for internal radiation therapy with I-131, which was planned for February 2010 and was preceded by a whole-body scintigraphy (WBS) one week before. Preparation for WBS and subsequent treatment was instituted during one month according to the international guidelines. The patient had remained without hormone replacement since the surgery. In the meantime the following follow-up studies were done, listed with the respective results: In the meantime the following follow-up studies were done, listed with the respective results: – Chest X-ray: normal. – Chest X-ray: normal. B B A – US of the cervical region: enlarged LN in levels I, II, and VI bilaterally consistent with an inflammatory reaction. Figure 1. Whole-body scintigraphy before (1A) and 4 days after (1B) therapeutic dose of I-131. Uptake in axillae is suspected in the pre-dose scan but is evident in the post-dose scan. Note uptake also in the inguinal regions einstein. 2014;12(1):100-5 einstein. 2014;12(1):100-5 102 Thom AR, Hamerschlak N, Teles VG, Osawa A, Santos FP, Pasqualin DC, Wagner J, Yamaga LY, Cunha ML, Campos Neto GC, Funari MB Metastatic spread of thyroid carcinoma to the axillary lymphatic chains is a very rare condition and is generally related to advanced disease according to the literature,(10-13) which was not the case of our patient. Another potential mechanism mentioned in the literature is an anomalous lymphatic drainage caused by a complex surgery,(10) which could have been possible in that case. The aspirated material was submitted to cytology, flow cytometry, and dosing of thyroglobulin and anti- thyroglobulin antibodies. Cytology analysis reported the absence of epithelial cells and calcification and was consistent with CLL/ SCLL. Flow cytology/immunophenotypic profile showed CD19+ cells with coexpression of CD5, CD20, CD23 and kappa light chain, being also consistent with CLL/ SCLL. On the other hand, the moderate diffuse and bilateral enlargement of the lymph nodes found upon clinical examination was more consistent with lymphoma. A differential diagnosis was imperative for decision-making between surgical or conservative treatment. Thyroglobulin and anti-thyroglobulin antibodies measured in the biopsied tissue by chemiluminescence immunoassay were undetectable. Based on these results, thyroid carcinoma metastases were ruled out and adenomegaly by CLL/SCLL was reported. A FDG-F-18 PET/CT revealed only faint metabolic activity in both axillae, slightly more pronounced (SUVmax 1.3) in one nodule on the left side, assumed to be the same that showed the highest radioiodine uptake. CASE REPORT As part of the follow-up protocol of radioiodine treatment, sequential blood counts were done weekly between the 3rd and 6th week and eventually at later times after the radioactive dose. Although a transient drop of white blood cells, and particularly lymphocytes, Because the patient refused open biopsy, a SPECT/CT, PET/CT, and ultrasound-guided fine needle aspiration biopsy of the well-defined left axillary LN was performed. Figure 2. SPECT/CT 4 days after I-131 therapy. Beside radioiodine concentration in the surgical remnants of the thyroidectomy, significant uptake is detected in the axillary lymph nodes, mainly in the left axillat axilla Figure 2. SPECT/CT 4 days after I-131 therapy. Beside radioiodine concentration in the surgical remnants of the thyroidectomy, significant uptake is detected in the axillary lymph nodes, mainly in the left axillat axilla einstein. 2014;12(1):100-5 Normalization of lymphocyte count after high ablative dose of I-131 103 of the lymphoid B lineage (lymphoid B cells) dropped from 52% to 5% respectively. always occurs after radiation therapy, an unusual reduction of the number of leucocytes mainly due to the drop of lymphocytes was seen in this patient. There was a remarkable reduction in the absolute as well as in the relative number of lymphocytes, which returned to normal values and remained normal for up to 19 months (September 2011). In January 24, 2012, leukocyte count was 8,590/mm3 (100%) and lymphocytes were 3,800/mm3 (44%), showing a small rise in the relative count beyond the normal upper limit of 40% (Table 1, Figure 3). Follow-up CT of the chest was also done on April 15, 2010, and compared to the CT from September 8, 2009, and showed a decrease in the number and the dimensions of the axillary lymph nodes (Figure 4), as well as absence of mediastinal and hilar lymphadenomegaly. A WBS with I-131 under recombinant TSH (rhTSH) was repeated for follow-up control on February 3, 2011, and was negative for thyroid iodine-avid tissues. Serum TSH, thyroglobulin and antithyroglobulin antibodies measured before the first dose and 48 hours after the second dose of rhTSH yielded, respectively, the following pp ( g ) A flow cytometry of the peripheral blood had been performed on September 8, 2009 (before surgery), and was repeated on April 15, 2010 (2 months after I-131). Cells Figure 3. Graphic representation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radiation therapy with I-131 Table 1. CASE REPORT Erythrocyte, leucocyte, lymphocyte and platelet counts in sequential hemograms from immediately before radioiodine therapy until 19 months after the treatment Date 6 days prior I-131 I-131 therapy Time after I-131 therapy Jan 27, 2010 Feb 2, 2010 Mar 6, 2010 4 week Mar 27, 2010 7 week Jul 6, 2010 5 month Oct 20, 2010 Jan 14, 2011 Sep 17, 2011 Jan 24, 2012 Erythrocytes /mm3 4.74x106 7,400mBq (200 mCi) 4.32x106 3.87x106 4.08X106 4.54x106 4.28x106 4.58X106 4.58X106 Leucocytes/mm3 19,000 5,730 4,800 6,000 5,190 5,450 7,010 8,590 Lymphocytes/mm3 13,958 1,250 1,248 859 1,520 1,150 2,240 3,800 Lymphocytes relative count 72% 21% 26% 14.3% 29.3% 27.7% 32% 44% Platelets/mm3 265,000 104,000 145,000 201,000 198,000 204,000 212,000 Figure 3. Graphic representation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radiation therapy with I-131 Table 1. Erythrocyte, leucocyte, lymphocyte and platelet counts in sequential hemograms from immediately before radioiodine therapy until 19 months after the treatment Date 6 days prior I-131 I-131 therapy Time after I-131 therapy Jan 27, 2010 Feb 2, 2010 Mar 6, 2010 4 week Mar 27, 2010 7 week Jul 6, 2010 5 month Oct 20, 2010 Jan 14, 2011 Sep 17, 2011 Jan 24, 2012 Erythrocytes /mm3 4.74x106 7,400mBq (200 mCi) 4.32x106 3.87x106 4.08X106 4.54x106 4.28x106 4.58X106 4.58X106 Leucocytes/mm3 19,000 5,730 4,800 6,000 5,190 5,450 7,010 8,590 Lymphocytes/mm3 13,958 1,250 1,248 859 1,520 1,150 2,240 3,800 Lymphocytes relative count 72% 21% 26% 14.3% 29.3% 27.7% 32% 44% Platelets/mm3 265,000 104,000 145,000 201,000 198,000 204,000 212,000 Table 1. Erythrocyte, leucocyte, lymphocyte and platelet counts in sequential hemograms from immediately before radioiodine therapy until 19 months after the treatment Table 1. Erythrocyte, leucocyte, lymphocyte and platelet counts in sequential hemograms from immediately before radioiodine thera Figure 3. Graphic representation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radiation therapy with I-131 presentation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radiation therapy with I-13 Figure 3. Graphic representation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radi Figure 3. Graphic representation of the sequential leukocyte and lymphocyte counts before and along 19 months after internal radiation therapy with I-131 A B Figure 4. Computed tomography before (4A) and 9 weeks after (4B) I-131 therapy. DISCUSSION The occurrence of secondary tumors in patients with chronic lymphocytic leukemia is not infrequent and is well-documented. According to the literature, those that occur most often are kidney cancer(3,4) and melanoma, among others. Secondary thyroid tumors seem to be rare, as they are found in the literature only as case reports.(8, 9) Lymphoma cells are sensitive to the energy of the beta emission of I-131.(14) This is the basis of radioimmunotherapy, where the antibody is the carrier of the radioisotope. But no reference to free radioiodine uptake by lymphoma cells was found in the literature. Iodide uptake by thyroid follicular cells and cells of differentiated thyroid carcinoma is mediated by the sodium iodide symporter (NIS), which is responsible for an active co-transport mechanism of the ion across the basolateral membrane.(15) Our patient presented a well-differentiated papillary microcarcinoma follicular variant secondary to (or concomitant with) chronic lymphocytic leukemia with regional LN metastases. The finding of radioiodine uptake in the axillary lymph nodes was perplexing, since this region normally has no anatomical connection with the vessels departing from the thyroid region. References concerning axillary LN metastases of thyroid tumors are equally limited to case reports, in general of patients with advanced disease.(10-13) One possible manner for this unusual spread could have been the large surgery to remove the intrathoracic goiter, as already mentioned. NIS is also known to be expressed by breast cancer(16) and by glioma tumor cells.(17) One may speculate that lymphoma cells eventually might also be able to express NIS and that this might have been a possibility in our patient. The documentation of this case is limited by the absence of a histological evaluation of the axillary lymphnode which had concentrated the radioactive agent. Nevertheless the normalization of the white cell count and the reduction in size of the axillary and paraaortic lymph nodes following the therapeutic dose of I-131 suggest that there was indeed a radiotherapeutic effect of the radioiodine on the lymphoma cells. This might be a subject for further research. As a matter of fact, FDG-F-18 PET/CT was not properly indicated for differential diagnosis because both types of tumors (CLL/SCLL and papillary thyroid carcinoma) have a rather low metabolic activity. It only ruled out a more aggressive process. Ideally, an open biopsy with specimen histology should have been done. CASE REPORT Note the marked reduction of lymph nodes in the left axilla A B A B Figure 4. Computed tomography before (4A) and 9 weeks after (4B) I-131 therapy. Note the marked reduction of lymph nodes in the left axilla einstein. 2014;12(1):100-5 Thom AR, Hamerschlak N, Teles VG, Osawa A, Santos FP, Pasqualin DC, Wagner J, Yamaga LY, Cunha ML, Campos Neto GC, Funari MB 104 results: TSH <0.05mIU/L and 306mIU/L; thyroglobulin, undetectable at both measurements; anti-thyroglobulin antibodies <5U/mL at both determinations. returned to normal values in the sixth-week follow-up after the therapeutic radioiodine dose and remained normal for up to 19 months, becoming only minimally elevated after 24 months; the reduction in size of the axillary and mediastinal LN documented by CT prior to and after radioiodine administration, and the compared blood immunohistochemistry. DISCUSSION Bocian et al.(8) published a case of coinciding metastases of papillary carcinoma and of SCLL in cervical lymph nodes. Reid-Nicholson et al.(9) reported the finding of concurrent papillary thyroid carcinoma and CLL/SCLL in a thyroid nodule. A similar concurrence could explain the radioiodine uptake in the axillary LN of our patient. But one may accept that the fine-needle aspiration biopsy was informative in that it confirmed the presence of lymphoma cells by histology and immunohistochemistry, and was entirely negative for cells of the thyroid lineage as well as for thyroid cell- related substances. einstein. 2014;12(1):100-5 REFERENCES 1. Manusow D, Weinerman BH. Subsequent neoplasia in chronic lymphocytic leukemia. JAMA. 1975;232(3):267-9. 2. Travis LB, Curtis RE, Hankey BF, Fraumeni JF Jr. Second cancers in patients with chronic lymphocytic leukemia. J Natl Cancer Inst. 1992;84(18):1422-7. 3. Mellemgaard A, Geisler CH, Storm HH. Risk of kidney cancer and other second solid malignancies in patients with chronic lymphocytic leukemia. Eur J Haematol. 1994;53(4):218-22. 4. Nishikubo CY, Kunkel LA, Figlin R, Belldegrun A, Rosen P, Elashoff R et al. An association between renal cell carcinoma and lymphoid malignancies. A case series of eight patients. Cancer. 1996;78(11):2421-6. 5. Hisada M, Biggar RJ, Greene MH, Fraumeni JF Jr, Travis LB. Solid tumors after chronic lymphocytic leukemia. Blood. 2001;98(6):1979-81. 5. Hisada M, Biggar RJ, Greene MH, Fraumeni JF Jr, Travis LB. Solid tumors after chronic lymphocytic leukemia. Blood. 2001;98(6):1979-81. Beside the contents of the material of the aspiration biopsy taken from a LN that had stored the radioactive isotope, one may hypothesize that there was, indeed, a radioiodine uptake by the lymphoma cells and a consequent radiotherapeutical action because of three additional observations: the drastic drop in white blood cell count, in particular of the lymphocytes which 6. Dasanu CA, Alexandrescu DT. Risk for second nonlymphoid neoplasms in chronic lymphocytic leukemia. MedGenMed. 2007;9(4):35.Review. 6. Dasanu CA, Alexandrescu DT. Risk for second nonlymphoid neoplasms in chronic lymphocytic leukemia. MedGenMed. 2007;9(4):35.Review. 7. Royle JA, Baade PD, Joske D, Girschik J, Frischi L. Second cancer incidence and cancer mortality among chronic lymphocytic leukaemia patients: a population-based study. Br J Cancer. 2011;105(7):1076-81. 8. Bocian A, Kopczynski J, Rieske P, Piaskowski S, Stuszniak J, Kupnicka D et al. Simultaneous occurrence of medullary and papillary carcinomas of the thyroid gland with metastases of papillary carcinoma to the cervical lymph Normalization of lymphocyte count after high ablative dose of I-131 105 nodes and the coinciding small B-cell lymphocytic lymphoma of the lymph nodes – a case report. Pol J Pathol. 2004:55(3):23-30. nodes and the coinciding small B-cell lymphocytic lymphoma of the lymph nodes – a case report. Pol J Pathol. 2004:55(3):23-30. metastasis as a late manifestation of papillary thyroid carcinoma. Thyroid. 2009;19(4):417-9. 14. Kaminski MS, Zelenetz AD, Press OW, Saleh M, Leonard J, Fehrenbacher L, et al. Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-Cell non-Hodgkin’s lymphomas. J Clin Oncol. 2001;19(19):3918-28. 9. Reid-Nicholson M, Moreira A, Ramalingam P. REFERENCES Cytologic features of mixed papillary carcinoma and chronic lymphocytic leukemia/small lymphocytic lymphoma of the thyroid gland. Diagn Cytopathol. 2008;36(11):813-7. 10. Koike K, Fujii T, Yanaga H, Nakagawa S, Yokoyama G, Yahara T, et al. Axillary lymph node recurrence of papillary thyroid microcarcinoma: report of a case. Surg Today. 2004;34(5):440-3. 15. Dohán O, De la Vieja A, Paroder V, Riedel C, Artani M, Reed M, et al. The sodium/iodide Symporter (NIS): characterization, regulation and medical significance. Endocr Rev. 2003;24(1):48-77. 11. Ers V, Galant C, Malaise J, Rahier J, Daumerie C. Axillary lymph node metastasis in recurrence of papillary thyroid carcinoma: a case report. Wien Klin Wochenschrift. 2006;118(3-4):124-7. 16. Renier C, Yao C, Goris M, Grosh M, Katznelson L, Noless K, et al. Endogenous NIS expression in triple-negative breast cancers. Ann Surg Oncol. 2009;16(4):962-8. 12. Nakayama H, Wada N, Masudo Y, Rino Y. Axillary lymph node metastasis from papillary thyroid carcinoma: report of a case. Surg Today. 2007;37(4):311-5. 17. Carlin S, Akabani G, Zalutsky MR. In vitro cytotoxicity of (211) at-astatide and (131) I-iodide to glioma tumor cells expressing the sodium/iodide symporter. J Nucl Med. 2003;44(11):1827-38. 13. Kepenekci I, Demirkan A, Cakmac A, Tug T, Ekinci S. Axillary lymph node einstein. 2014;12(1):100-5 einstein. 2014;12(1):100-5
https://openalex.org/W4294032298	https://periodicorease.pro.br/rease/article/download/6647/2539	Portuguese	null	A IMPORTÂNCIA DA ASSISTÊNCIA DO ENFERMEIRO NA CONSULTA PRÉ-NATAL	Revista Ibero-Americana de Humanidades, Ciências e Educação	2,022	cc-by	7,165	doi.org/10.51891/rease.v8i8.6647 doi.org/10.51891/rease.v8i8.6647 doi.org/10.51891/rease.v8i8.6647 1 Faculdade JK- Brasília. E-mail: antonia_amaral@hotmail.com. 2 Faculdade JK- Brasília. E-mail: gisre08@hotmail.com. 3 Orientador do curso de Enfermagem da Faculdade JK- Brasília. E-mail:ronaldo.nunes@facjk.com.br. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 2 Faculdade JK- Brasília. E-mail: gisre08@hotmail.com. 1 Faculdade JK- Brasília. E-mail: antonia_amaral@hotmail.com. 3 Orientador do curso de Enfermagem da Faculdade JK- Brasília. E-mail:ronald A IMPORTÂNCIA DA ASSISTÊNCIA DO ENFERMEIRO NA CONSULTA PRÉ-NATAL THE IMPORTANCE OF NURSES' PERFORMANCE IN PRENATAL CONSULTATIONS Antônia Amaral Lima1 Mirian Mota Carlos2 Ronaldo Nunes Lima3 RESUMO: Introdução A importância ao pré-natal deve ser iniciada assim que a mulher ter a certeza de que está grávida a consulta de pré-natal caracteriza-se como atividade essencial frente a assistência prestada à gestante durante período gravídico, Objetivo: orientar quanto a importância do pré-natal para que ocorra uma gestação saudável e quais são os fatores associados à qualidade da assistência prestada destacando a importância do enfermeiro nesse processo. Materiais e  Métodos: O desenvolvimento desse artigo foi baseado na pesquisa bibliográfica por meio de revisão integrativa de literatura, observando a relevância do tema e a busca do saber sob o olhar de alguns autores, este tipo de pesquisa me permite manipular entre as variáveis. Resultados: tomando por embasamento em 12 artigos analisados e levando em consideração os seus aspectos semelhantes foram possíveis apontar questões centrais e importantes em relação ao pré-natal, onde relata sobre a importância do pré-natal para que ocorra uma gestação saudável, visando analisar a importância sobre a assiduidade nas consultas é essencial, pois é através delas que serão verificados alguns tipos de doenças e evitar uma gravidez que apresenta risco para a mãe e o feto. CONCLUSÃO O material bibliográfico analisado aponta que a atuação dos enfermeiros na educação e na promoção de saúde no pré-natal tem sido essencial de forma satisfatória, com estabelecimento de vínculos consistentes entre enfermeiro gestante e a comunidade ao longo da assistência prestada. 636 Palavras-chaves: Cuidados de enfermagem. Acompanhamento pré-natal. Enfermagem pré-natal. ABSTRACT: Prenatal care is the assistance in the area of nursing and medicine provided to pregnant women during the nine months of pregnancy, it is a preparation for childbirth, it is monitoring each week of the baby and his health in order to improve and avoid problems for the mother and the fetus. Nurses are fundamental in prenatal care, as they are qualified to intervene with health promotion strategies, disease prevention and use humanization in the care provided. The role of the nurse as a caregiver is to fully seek the health of these women, the objective of the article is Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. A IMPORTÂNCIA DA ASSISTÊNCIA DO ENFERMEIRO NA CONSULTA PRÉ-NATAL THE IMPORTANCE OF NURSES' PERFORMANCE IN PRENATAL CONSULTATIONS ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE to guide the importance of prenatal care for a healthy pregnancy and what are the factors associated with the quality of care provided, highlighting the importance of the nurse in this process. Keywords: Nursing care. Prenatal care. Prenatal nursing. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 INTRODUÇÃO A importância ao pré-natal deve ser iniciada assim que a mulher ter a certeza de que está grávida a consulta de pré-natal caracteriza-se como atividade essencial frente a assistência prestada à gestante durante período gravídico, o enfermeiro precisa realizar consultas humanizada e qualificada, dados revelam as taxas de morbimortalidade materno-infantil ainda são preocupantes, principalmente relacionados às condições obstétricas das mulheres como um problema de saúde pública. As gestantes devem buscar apoio, ações e estratégias para minimizar os índices relacionados a essas condições para aumentar a qualidade de vida durante a gestação (SILVA et al, 2018). O enfermeiro é fundamental na assistência pré-natal, por ser qualificado para intervir com estratégias de promoção da saúde, prevenção de doenças e utilizar a humanização nos cuidados prestados. Durante as consultas deve-se realizar ações educativas para elaborar o plano de assistência na consulta de acompanhamento pré- natal, conforme as necessidades identificadas e priorizadas, estabelecendo as intervenções orientações e encaminhando para outros serviços, promovendo e disciplinando sob cada ações (SEHNEM., et al 2019). 637 A grávida quando não dá início ao pré-natal e resolve começar tardio reduzindo assim os números de consultas, está contribuindo para um desfecho desfavorável da gestação, uma vez que estão diretamente relacionados ao diagnóstico precoce de situações que colocam em risco a saúde do feto ou até mesmo óbito. O pré-natal deve ser iniciado logo após que a mulher descobrir a gravidez para garantir que a mulher tenha uma gestação e um parto saudáveis e sem nenhuma complicação o pré-natal além de prevenir e diagnosticar precocemente doenças e problemas também orienta a gestante sobre ações importantes referente a maternidade, favorecendo um acompanhamento e continuidade do cuidado (BALCELLS et al., 2018). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. INTRODUÇÃO ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE A qualidade da assistência pré-natal representa papel fundamental na prevenção e detecção precoce de patologia tanto para a mãe e para o feto, assim permitindo um desenvolvimento saudável do bebe e reduzindo os riscos da gestante, uma vez que contribui para a redução das taxas de morbimortalidade materno e infantil é imprescindível instituir medidas que facilitem o acesso, ampliem a cobertura e qualifique o acompanhamento pré-natal, principalmente em países como o Brasil, com proporções continentais, amplas desigualdades sociais, econômicas, culturais e regionais, de acesso aos serviços de saúde, sobretudo os de alta complexidade (MAYOR et al, 2018). Dessa forma, o objetivo deste estudo é orientar quanto a importância do pré- natal para que ocorra uma gestação saudável e quais são os fatores associados à qualidade da assistência prestada destacando a importância do enfermeiro nesse processo. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 MATERIAIS E   MÉTODOS O desenvolvimento desse seminário artigos foi baseado na pesquisa bibliográfica por meio de revisão integrativa de literatura, observando a relevância do tema e a busca do saber sob o olhar de alguns autores, este tipo de pesquisa me permite manipular entre as variáveis. 638 Como procedimento metodológico selecionou-se material já publicado constituído de artigos on-line nas plataformas google académico, scientific electronic library online, (Scielo), biblioteca virtual em saúde(BVS),foram utilizado como auxiliares nas pesquisas operadores booleanos AND e OR, com as palavras chaves: cuidado de enfermagem, acompanhamento pré-natal, enfermagem pré-natal, onde foram analisados 50 publicações científicos, sendo selecionados 25 periódicos, os quais tinham mais ênfase no tema escolhido, o levantamento bibliográfico e de construção desse estudo foi realizado no período de fevereiro a junho 2022. As pesquisas foram realizadas em banco de fatores de inclusão nacionais de língua portuguesa por considerar mais acessíveis este tipo de publicação entre 2017 a 2022. Foram excluídos os periódicos publicados antes de 2017, e que fogem do objetivo proposto. Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Assim, após a seleção do material bibliográfico, foi promovida uma leitura, oportunidade em que foi produzido o texto final, visando atingir o objetivo pré- estabelecido para o presente trabalho e análise para interpretação do material Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. , ç , g ISSN - 2675 – 3375 DESENVOLVIMENTO O ministério da saúde afirma que, a gravidez é um evento resultante da fecundação do óvulo ovócito pelo espermatozóide. Habitualmente, ocorre dentro da trompa uterina e é responsável pela geração de um novo ser. Este é um momento de grandes transformações para a mulher, para seu parceiro e para toda a família. Durante o período da gestação, o corpo vai se modificar lentamente, preparando-se para o parto e para a maternidade. A gestação é um fenômeno fisiológico e, por isso mesmo, sua evolução se dá, na maior parte dos casos, sem intercorrências. Em alguns casos, o bebê pode não conseguir se desenvolver de forma adequada. Por isso, é importante o pré- natal (BRASIL, 2020). O pré-natal é a assistência na área da enfermagem e da medicina prestada à gestante durante os noves meses de gravidez, é um preparatório para o parto, é acompanhar cada semana do bebê e a saúde dele visando melhorar e evitar problemas para a mãe eo feto, ao compreender a importância do pré-natal para as grávidas o profissional de saúde que a acompanha consegue identificar o que esse cuidado significa para cada uma delas, o que auxilia no direcionamento da assistência social que cada mulher está inserida, sua percepção do mundo e suas particularidades, as quais precisam ser consideradas e assistidas, proporcionando um cuidado mais efetivo e integral, assegurando o desenvolvimento de uma gestação saudável (PEREIRA et al., 2018). 639 639 Sendo assim, a assistência ao pré-natal é uma etapa primordial para uma gestação e parto humanizado, o enfermeiro juntamente com a equipe deve desenvolver assistência integral a grávida essa acessória envolve respeito aos sentimentos, emoções, necessidades, valore culturais- incluindo toda a família; essa assistência se dá também “por meio de ações e procedimentos técnicos e científicos, assegurando uma gestação sem intercorrências ou minimizando os agravos e desconforto que podem surgir no decorrer da gestação”(PAULA, BARBOSA, 2019). Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Embasado na lei 7.498 de 1996, que regulamenta o exercício de enfermagem decreto e resoluções que trata do mesmo assunto, o enfermeiro possui aptidão para desenvolver atividades de atenção à enfermagem e toda a rotina pré-natal. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 , ç , g ISSN - 2675 – 3375 DESENVOLVIMENTO O enfermeiro tem autonomia para prescrever alguns tipos de medicamentos na consulta de pré-natal como o ácido fólico que é recomendado para mulher em idade fértil e dois meses antes de engravidar e nos primeiros meses de gestação, também existe o sulfato ferroso que deve começar a partir do conhecimento da gravidez até o terceiro mês após o parto. Tendo em vista isso, o enfermeiro destaca-se como um profissional capacitado para a prestação do cuidado pré-natal de qualidade, uma vez que demonstra resolutividade e competência em sua assistência. (LEAL et al., 2018) Conforme protocolos, já na primeira consulta de pré-natal o enfermeiro é responsável por realizar ações educativas para a gestante e sua família, e acompanhar a gestação de baixo risco, solicitar exames de rotina e orientar o tratamento de acordo com o protocolo que o enfermeiro é habilitado e respaldado, também coletar exame cito patológico, as consultas de pré-natal devem ser intercaladas uma com o enfermeiro e outra com o médico, assim, sendo podemos considerar a assistência de enfermagem importante e confiável para o acompanhamento da gravidez de baixo risco (SILVA et al., 2018) 640 No pré-natal de baixo risco, a maioria das consultas é realizada pelos enfermeiros, porque não é necessário intervenções de maior complexidade, o total de consultas deverá ser no mínimo 6 intercaladas entre médico e enfermeiro, para uma gestante sem risco estabelece no mínimo 2 consultas realizadas pelo médico uma no início do pré-natal, e outra entre a 29° e a 32° semanas e o intervalo entre as consultas devem ser de 4 semanas, após a 36° semana deverá ser acompanhada a cada 15 dias para avaliar pressão, edema, altura uterina, movimento do feto e batimento cardio fetal, deve acontecer prioritariamente na UBS, a fim de contribuir para melhores desfechos maternos e perinatais. O pré-natal de risco habitual, é onde a mãe e filho não apresentam fatores de risco individual que está relacionado à doença ou agravo que possam interferir negativamente na evolução da gravidez, e são atribuições do enfermeiro no acompanhamento (OLIVEIRA et al.,2017). Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. DESENVOLVIMENTO 2022 , ç , g ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE A gestação de alto risco são os casos mais complexos de assistência durante a gravidez, em que há maior probabilidade de alcançar resultados desfavoráveis e nocivos, tanto para a mãe quanto para o feto. Onde existem as Condições como, idade acima de 35 anos, pois são mais vulneráveis algumas doenças, obesidade, doença previa crônicas, aquelas que tiveram gestação anterior de alto risco, hipertensão, diabetes, deslocamento prévio da placenta, pré-eclâmpsia ou até mesmo infecção viral ou bacteriana, idades menores que 15 anos porque o útero não está preparado, pois há maior chance do bebe nascer com baixo peso, prematuro e a mulher sofrer aborto espontâneo, devem ser acompanhadas pelo profissional medico de forma pontual para que não haja aumento do risco à saúde do binômio mãe e filho decorrentes do processo gestacional (SONCINI, et al., 2019; GADELHA, et al., 2020). Enfim, o papel do enfermeiro como cuidador é buscar integralmente a saúde destas mulheres, seja prescrevendo cuidados de enfermagem e medicamentos previstos em programas de saúde e protocolos das instituições de saúde, durante o pré-natal a gestante deve receber informações sobre seus direitos hábitos saudáveis de vida, também tem de receber informações sobre sinais de risco em cada etapa da gravidez, é muito importante que a gestante aproveite o momento da consulta para colocar suas dúvidas, preocupações, experiências a fim de ampliar o diálogo com os profissionais de saúde mantendo esquemas terapêuticos, solicitados exames complementares e fortalecendo o vínculo entre a gestante e sua equipe. (REIS; RACHED, 2017). 641 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ç g ISSN - 2675 – 3375 O Gráfico 1 - tomando por embasamento em 12 artigos analisados e levando em consideração os seus aspectos semelhantes foram possíveis apontar questões centrais e importantes em relação ao pré-natal, onde relata sobre a importância do pré-natal para que ocorra uma gestação saudável, visando analisar a importância sobre a assiduidade nas consultas é essencial, pois é através delas que serão verificados alguns tipos de doenças e evitar uma gravidez que apresenta risco para a mãe e o feto e consequentemente você vai ter um bom parto seja parto normal ou Cesário, dos artigos selecionados, sendo elas de acordo com os autores em estudo, assiduidade nas Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE evista Ibero- Americana de Humanidades, Ciências e Educação- REASE consultas com 70%, orientação à gestantes 75% para garantir que a mulher e o bebe tenham uma gestação e um parto saudáveis e sem nenhuma complicação e o que fazer para que ocorra um parto sem intercorrência, habilidade nas consultas com 67%, onde a competência pode ser definida como habilidade de desempenhar uma tarefa específica de modo a produzir resultados desejáveis a habilidade nas consultas e o principal marco do desenvolvimento profissional de prestar o bom atendimento onde compete a função do enfermeiro nesse cuidado. Gráfico 1 - Orientação da importância do pré-natal para que ocorra uma gestação saudável. Gráfico 1 - Orientação da importância do pré-natal para que ocorra uma gestação saudável. Fonte: Autores com embasamentos em ANDRADE et al.,2020, FERREIRA et al.,2019, QUEIROZ et al.,2018, ARAÚJO et al.,2022, GADELHA et al.,2020, BALCELLS et al.,2018, GONÇALVES et al.,2018, SOUSA et al., 2021, LADEIRA et al.,2021, CONRADO et al.,2021, SILVA 2018, SENA et al.,2021. Gráfico 2 fatores associados à qualidade da assistência prestada 642 Fonte: Autores com embasamentos em ANDRADE et al.,2020, FERREIRA et al.,2019, QUEIROZ et al.,2018, ARAÚJO et al.,2022, GADELHA et al.,2020, BALCELLS et al.,2018, GONÇALVES et al.,2018, SOUSA et al., 2021, LADEIRA et al.,2021, CONRADO et al.,2021, SILVA 2018, SENA et al.,2021. Gráfico 2 – fatores associados à qualidade da assistência prestada. Gráfico 2 – fatores associados à qualidade da assistência prestada. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 O Gráfico Fonte: Autores com embasamentos em SILVESTRI et al.,2021, BALSELLS et al., 2018, SOUSA et al.,2019, MEDEIROS 2019, MACHADO et al.,2021, SILVA 2018, CAVALCANTE et al.,2022, MENEZES et al.;2021, MENDES et al.,2022, VIANA et al.,2021. Quadro 1- Publicações selecionadas acerca da importância da atuação do enfermeiro na consulta ao pré- natal, estudo que fazem parte dos resultados e discussão. AUTOR/ANO TÍTULO OBJETIVO ANDRADE et al.,2020 O papel do enfermeiro na assistência, educação e promoção da saúde no pré- natal Investigar por meio de uma revisão integrativa da literatura a atuação dos enfermeiros na educação e na promoção de saúde no pré- natal FERREIRA et al.,2019 Pré-natal e a assistência de enfermagem à gestante de alto risco Discutir a partir de achados na literatura a importância do pré- natal e a assistência de enfermagem à gestante de alto risco. QUEIROZ et al.,2018 Fatores associados à qualidade da assistência de enfermagem durante o pré-natal: um referencial teórico Analisar quais os fatores associados à qualidade da assistência pré-natal correta do pré-natal é necessário qualidade da assistência, permitindo um desenvolvimento saudável e os riscos da gestante. Fonte: Autores com embasamentos em SILVESTRI et al.,2021, BALSELLS et al., 2018, SOUSA et al.,2019, MEDEIROS 2019, MACHADO et al.,2021, SILVA 2018, CAVALCANTE et al.,2022, MENEZES et al.;2021, MENDES et al.,2022, VIANA et al.,2021. Q d P bli õ l i d d i tâ i d t ã d f i lt é correta do pré-natal é necessário qualidade da assistência, permitindo um desenvolvimento saudável e os riscos da gestante. Fonte: Autores com embasamentos em SILVESTRI et al.,2021, BALSELLS et al., 2018, SOUSA et al.,2019, MEDEIROS 2019, MACHADO et al.,2021, SILVA 2018, CAVALCANTE et al.,2022, MENEZES et al.;2021, MENDES et al.,2022, VIANA et al.,2021. 644 ro 1- Publicações selecionadas acerca da importância da atuação do enfermeiro na consulta ao pré- estudo que fazem parte dos resultados e discussão. Quadro 1- Publicações selecionadas acerca da importância da atuação do enfermeiro na consulta ao pré- natal, estudo que fazem parte dos resultados e discussão. O Gráfico Os resultados elencados neste gráfico mediante de 13 artigos selecionados permitiram discutir as medidas sob fatores associados à qualidade da assistência prestada ao pré-natal deve ser organizada da melhor forma para atender as reais necessidades das gestantes através da utilização de conhecimentos técnico-científicos 54%, por meio das ações educativas espera-se que a gestante adquira maiores Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE evista Ibero- Americana de Humanidades, Ciências e Educação- REASE conhecimento percebendo sua importância e interesse em participar do pré-natal 62%, uma boa realização correta da anamnese torna-se possível um elo de confiança entre o enfermeiro e a gestante 62% que seja promovidas precisam estar voltadas para cobertura de toda a população de gestante da área de abrangência da unidade de saúde, possibilitando a continuidade no atendimento, o acompanhamento e a avaliação dessas assistência prestada. Fonte: Autores com embasamentos em, PESSOA et al.,2021, FERNANDES et al.,2021, DOURADO et al.,2021, SEHNEM et al.,2019, OLIVEIRA et al.,2019, TAVARES et al.,2021, REIS et al.,2017, GUIMARÃES et al.,2018, PAULA 2019, SILVA et al.,2019, JUSTINO et alt.,2020, NASCIMENTO et al.,2019, GADELHA et al.,2019. Fonte: Autores com embasamentos em, PESSOA et al.,2021, FERNANDES et al.,2021, DOURADO et al.,2021, SEHNEM et al.,2019, OLIVEIRA et al.,2019, TAVARES et al.,2021, REIS et al.,2017, 643 Fonte: Autores com embasamentos em, PESSOA et al.,2021, FERNANDES et al.,2021, DOURADO et al.,2021, SEHNEM et al.,2019, OLIVEIRA et al.,2019, TAVARES et al.,2021, REIS et al.,2017, GUIMARÃES et al.,2018, PAULA 2019, SILVA et al.,2019, JUSTINO et alt.,2020, NASCIMENTO et al.,2019, GADELHA et al.,2019. Gráfico 3 – A importância do enfermeiro na consulta de pré-natal. Ainda neste estudo, foram selecionados 10 artigos, visando analisar a importância do enfermeiro na consulta de pré-natal, sendo que é essencial o acompanhamento do enfermeiro nesse processo é de 50%, para isso a gestante deve receber atenção suficiente e na intensidade desejada de acordo com suas necessidades básicas, para ser realizado um pré-natal adequado é preciso uma assistência integral de 90%, evitando assim morte fetal e complicações durante a gravidez, 70% da realização Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE correta do pré-natal é necessário qualidade da assistência, permitindo um desenvolvimento saudável e os riscos da gestante. O Gráfico AUTOR/ANO TÍTULO OBJETIVO ANDRADE et al.,2020 O papel do enfermeiro na assistência, educação e promoção da saúde no pré- natal Investigar por meio de uma revisão integrativa da literatura a atuação dos enfermeiros na educação e na promoção de saúde no pré- natal FERREIRA et al.,2019 Pré-natal e a assistência de enfermagem à gestante de alto risco Discutir a partir de achados na literatura a importância do pré- natal e a assistência de enfermagem à gestante de alto risco. QUEIROZ et al.,2018 Fatores associados à qualidade da assistência de enfermagem durante o pré-natal: um referencial teórico Analisar quais os fatores associados à qualidade da assistência pré-natal destacando a importância do enfermeiro nesse processo. ARAÚJO et al.,2022 Consulta de enfermagem no pré-natal: atendimento à gestante com sífilis Identificar as ações do enfermeiro durante a consulta pré-natal às gestantes com sífilis Rev Revista Ibero Americana de Humanidades, Ciências e Educação REASE SILVESTRI et al.,2021 Os significados do pré-natal atribuído por gestantes realizado por enfermeiros Compreender os significados atribuídos por gestantes ao pré- natal realizado por enfermeiros. BALCELLS et al.,2018 Avaliação do processo na assistência pré-natal de gestantes com risco habitual Avaliar a qualidade do cuidado quanto ao processo no pré-natal de gestantes com risco habitual. GONÇALVES et al.,2018 Pré-natal: preparo para o parto na atenção primária à saúde no sul do Brasil Avaliar a relação entre assistência pré-natal e orientações para o parto na Atenção Primária à Saúde SOUSA et al., 2021 Consulta de pré-natal de risco habitual na atenção primária à saúde. Relatar as atividades e experiências de acadêmicos do curso de graduação em enfermagem durante o acompanhamento às consultas de pré-natal de risco habitual em uma unidade básica de saúde. LADEIRA et al.,2021 O desafio da atuação do enfermeiro frente a ausência paterna no acompanhamento pré-natal: estratégias e intervenções Incluir a figura paterna nos cuidados pertinentes ao ciclo gravídico é um desafio, já que o mesmo é visto como uma responsabilidade materna e poucos serviços de saúde promovem a integração paterna. CONRADO et al., 2021 Estratégias utilizadas por enfermeiras durante a consulta de pré-natal de mulheres vítimas de violência sexual: revisão integrativa Discutir estratégias utilizadas por enfermeiras durante a consulta de pré-natal de mulheres vítimas de violência sexual. O Gráfico SILVA et al., 2018 O pré-natal e a assistência de enfermagem à gestante de alto risco Objetivo discutir a partir de achados na literatura a importância do pré-natal e a assistência de enfermagem à gestante de alto risco. SENA et al.,2021 Gestação de alto risco: epidemiologia e cuidados, uma revisão de literatura Compreender o perfil epidemiológico das gestantes acompanhadas no pré- natal de alto risco, assim como os cuidados voltados às mesmas. PESSOA et al.,2021, Conhecimento da Gestante Sobre A Importância da Consulta Pré-natal: Revisão Integrativa A assistência pré-natal está voltada aos cuidados com a mulher, durante o período gestacional, com a finalidade de identificar riscos, agir antecipadamente em possíveis intercorrências, garantir qualidade na saúde, evitar morte e danos físicos à gestante e ao feto 64 645 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Rev FERNANDES et al,2021 A atenção do enfermeiro na assistência ao pré-natal de baixo risco O objetivo do trabalho foi realizar um levantamento bibliográfico sobre a atenção do enfermeiro na assistência ao pré-natal de baixo risco, assim como a abordagem frente às dificuldades encontradas e sucesso para um bom parto. DOURADO et al., 2021 Assistência de enfermagem ao pré- natal: Relato de experiência. Observar a atuação do enfermeiro nas consultas de pré-natal de baixo risco. SEHNEM et al.,2019 Consulta de pré-natal na atenção primária à saúde: fragilidades e potencialidades da intervenção de enfermeiros brasileiros Conhecer as fragilidades e potencialidades da intervenção do enfermeiro na consulta de pré-natal OLIVEIRA et al.,2019, Importância do pré-natal na opinião das usuárias de uma unidade básica de saúde da família em porto velho, Rondônia Estudar o nível de conhecimento das pacientes atendidas na Unidade Básica de Saúde Oswaldo Piana sobre o pré-natal de baixo risco TAVARES et al.,2021 Conhecimento Da Gestante Sobre A Importância Da consulta Pré-natal: Revisão Integrativa Garantir qualidade na saúde, evitar morte e danos físicos à gestante e ao feto. REIS; RACHED, 2017, O papel do enfermeiro no acompanhamento de pré-natal de baixo risco utilizando a abordagem centrada na pessoa – gestante. O pré-natal é o acompanhamento da evolução da gestação que visa cuidar da saúde da mulher e do seu bebê até que o parto ocorra, também é o momento que a gestante vivencia diferentes sentimentos, por isso o estabelecimento de relação com a enfermagem se faz imprescindível. GUIMARÃES et al. O Gráfico 2018, Acesso e qualidade da atenção pré-natal na Estratégia Saúde da Família: infraestrutura, cuidado e gestão. Investigação do acesso e da qualidade do cuidado pré-natal na Estratégia Saúde da Família no Brasil e na Região Norte, mediante avaliação de aspectos de infraestrutura nas unidades de saúde, da gestão e oferta do cuidado prestado pelas equipes, sob o prisma das desigualdades regionais e estaduais. PAULA et al. 2019. cuidado-pré-natal-em-uma-unidade-de- saúde-de-cachoeira-alta O acompanhamento pré-natal é um cuidado da saúde de gestantes. É uma poderosa estratégia para reduzir a morbimortalidade materna, perinatal e neonatal. 646 JUSTINO et alt.,2020, Estratégias de educação em saúde durante o pré-natal como agente promotor de qualidade de vida. objetivo de avaliar as estratégias de educação em saúde desenvolvidas na assistência ao pré-natal de risco habitual como agente promotor de qualidade de vida. SILVA et al.,2019, Importância do pré-natal na opinião das usuárias de uma unidade básica de saúde da família em porto Velho, Rondônia O presente trabalho pretende estudar o nível de conhecimento das pacientes atendidas na Unidade Básica de Saúde Oswaldo Piana sobre o pré-natal de baixo risco. NASCIMENTO et al.,2019 Principais fatores associados ao tardiamente do pré-natal: Identificar os principais fatores associados ao início tardio do pré-natal entre gestantes GADELHA et al 2020 Qualidade de vida de mulheres com gravidez de alto risco durante o cuidado pré-natal Analisar a qualidade de vida de gestantes de alto risco SOUSA et al., 2019 O cuidado pré-natal humanizado na atenção primária em saúde: uma revisão de literatura O cuidado pré-natal prestado às gestantes na atenção primária em saúde e as condutas que tornam essa assistência humanizada. MACHADO et al.,2021 Qualidade de vida de gestantes atrelada a assistência do enfermeiro no pré- natal. elencar as evidências científicas sobre a qualidade de vida entre gestantes em assistência pré- natal. CAVALCANTE et al.,2022 Atuação do enfermeiro na gestação de alto risco: uma revisão sistemática Identificar na literatura científica as formas de atuação do enfermeiro à gestantes de alto risco VIANA et al.,2021, Qualidade da assistência pré-natal realizada por enfermeiros das unidades básicas de saúde de Cáceres-MT / Qualidade da assistência pré-natal realizada por enfermeiros nas unidades básicas de saúde de Cáceres-MT . Objetivo: avaliar a qualidade da assistência prestada no pré- natal de baixo risco pelos enfermeiros das Unidades Básicas de Saúde de Cáceres- MT. O Gráfico MENDES et al.,2020 Avaliação da qualidade do pré-natal a partir das recomendações do Programa de Humanização no Pré-natal e Nascimento O objetivo deste estudo foi analisar a qualidade do pré- natal no estado de Sergipe a partir das recomendações do PHPN. MENEZES A assistência do enfermeiro no pré- natal Objetivo: O estudo teve como objetivo conhecer, entender e verificar como se dá a assistência ao pré-natal realizada pelo enfermeiro na atenção básica. 647 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE DISCUSSÃO Percebe-se a imensa importância da consulta de enfermagem no processo de pré-natal, pois é abordada desde os programas de saúde da mulher, pré-natal, parto e puerpério e continua com o desenvolvimento do feto, assim diminuindo os riscos para a mãe e o bebê que são afastados no atendimento dos enfermeiros junto às gestantes. Nas consultas de enfermagem, o enfermeiro não necessita apenas de sua competência técnica, mas também necessita da escuta qualificada, ouvindo suas queixas, preocupações e angústia, criando assim uma relação mais próxima com a gestante, sua família e comunidade, visando a necessidade de resultados positivos o presente estudo permitiu orientar e analisar a importância da atuação do enfermeiro na consulta do pré-natal. Tomando como fundamento os 12 artigos analisados para o estudo no gráfico 1, nos foi permitido concluir que a orientação da importância do pré-natal para que ocorra uma gestação saudável, visando analisar a importância sobre a assiduidade nas consultas, orientação a gestantes e habilidades na consulta, são os itens que não podem acontecer falhas durante o atendimento com a gestante, são apresentados que 70% estão relacionados a assiduidade nas consultas, verificamos que evitar uma gravidez que apresenta risco para a mãe e o feto é através da orientação à gestantes 75%, para garantir que a mulher e o bebe tenha uma gestação e um parto saudáveis e sem nenhuma complicação é aplicada sobre habilidade nas consultas com 67%, através de boa prática e atenção à gestante. 648 Conforme os artigos analisados sobre fatores associados à qualidade da assistência prestada, podem ser alcançadas de acordo com análise dos indicadores apontados no gráfico 2, pois o pré-natal é um acompanhamento do início da gestação que cuida da saúde da mulher e do seu bebê até que o parto aconteça. A consulta de enfermagem é uma atividade que proporciona ao enfermeiro condições para atuar de forma direta e independente caracterizando dessa forma, conhecimento específica de 54%, o pré-natal busca garantir a assistência à saúde da gestante, promovendo ações educativas de 62%, a qualidade no atendimento e prevenção de possíveis complicações durante a gestação é através da realização correta da anamnese de 62%, no atendimento durante as consultas. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. DISCUSSÃO ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Os resultados obtidos no gráfico 3, mostra a importância do enfermeiro na consulta de pré-natal e suas atribuições de forma mais efetivas, 50% é essencial para preparar a mulher para a maternidade, não devendo ser encarada como simples assistência de enfermagem e sim como trabalho de prevenção de intercorrências e complicações gestacional. A atenção à saúde da mulher deve ser organizada de forma que atenda as existentes necessidades das mulheres durante a gestação através da utilização da assistência integral 90%, dos conhecimentos específicos adequados e dos meios de recursos humanos e físicos disponíveis e apropriados para cada caso assegurando uma qualidade da assistência 70%, e dessa forma reduzindo a morbimortalidade materna e infantil. Os resultados da revisão de literatura têm a importância da atuação dos profissionais enfermeiros junto às gestantes e famílias ao longo do período do pré-natal e quando ele é realizado com qualidade e exerce um cuidado integral à gestante e a consolidação do vínculo, que proporciona o bem-estar e o nascimento do bebê. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. CONCLUSÃO 649 O material bibliográfico analisado aponta que a atuação dos enfermeiros na educação e na promoção de saúde no pré-natal tem sido essencial de forma satisfatória, com estabelecimento de vínculos consistentes entre enfermeiro gestante e a comunidade ao longo da assistência prestada. Ainda existe um caminho a ser percorrido para que os enfermeiros atuem plenamente em prol da qualificação da assistência ao pré-natal, é necessário que várias ações educativas sejam colocadas em práticas, para uma assistência de qualidade, onde o enfermeiro é capaz de realizar uma anamnese corretamente, conhecimento específico e assistência integral. A assiduidade nas consultas e a participação nas atividades educativas, para que sejam identificadas precocemente as possíveis complicações e assim a equipe possa intervir e proporcionar um parto e puerpério com desfecho favorável, assegurando a saúde da mulher e de seu bebê. O enfermeiro é a referência principal para as gestantes tratando-se das atividades educativas e ações de promoção e prevenção à saúde durante esse período, além de ser habilitado e preparado para exercer cargos de liderança em diversos serviços de saúde, tendo assim, papel primordial no processo de assistência pré-natal, desde a orientação e acompanhamento nas mudanças necessárias para a , ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE melhoria na assistência prestada e de qualidade. Tendo em vista os aspectos observados no pré-natal compreende-se que o enfermeiro tem um papel fundamental e significante na qualidade e cuidado às gestantes tanto na unidade básicas intra e extra-hospitalar, observa-se um relevante e satisfatória no acompanhamento do pré- natal evidenciado por equipe multidisciplinar e comprometimento na assistência integral. Espera-se que este estudo venha contribuir para a reflexão da importância do enfermeiro na consulta de pré-natal para que a gestante tenha um parto saudável e sem intercorrência REFERÊNCIA 1 - BRASIL. Ministério da Saúde. Gravidez, 2020 https://www.gov.br/saude/pt- br/assuntos/saude-de-a-a-z/g/gravidez 1 - BRASIL. Ministério da Saúde. Gravidez, 2020 https://www.gov.br/saude/pt- br/assuntos/saude-de-a-a-z/g/gravidez 2 - ALVES Thaynara Oliveira Alves; Nunes Raynara Laurinda Nascimento; Sena Luis Henrique Alves De; Alves Fernanda Gonçalves; Souza; Aline Gomes Silva De; Salviano Arianny Moreira; Oliveira Bruna Renata Duarte; Gestação De Alto Risco: Epidemiologia E Cuidados, Uma Revisão De Literatura. High Risk Pregnancy: Epidemiology And Care, A Literature Review. Doi:10.34119/Bjhrv4n4-040. Recebimento Dos Originais: 09/06/2021. 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Ações do enfermeiro no pré-natal e a importância atribuída pelas gestantes Nurse Assistance In Prenatal Care Asistencia De Enfermería En La Atención Prenatal RECEBIDO: 22/10/2021 \| REVISADO: 27/10/2021 \| ACEITO: 29/10/2021 \| PUBLICADO:01/11/2021 https://www.epublicacoes.uerj.br/index.php/sustinere/art icle/view/31722/25719 5 – BARBOSA Mende Rosemar; JESUS Santos José Marcos de; PRADO Daniela Siqueira; QUEIROZ Gurgel Rosana; Bezerra Felipa Daiana; QUEIROZ Gurgel Ricardo. Avaliação da qualidade do pré-natal a partir das recomendações do programa de humanização no pré-natal e nascimento https://www.scielo.br/j/csc/a/cdtVRDQYnSdzTNCGFjSZCJr/?lang =pt Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 6 - BALSELLS Marianne Maia Dutra; Oliveira Tyane Mayara Ferreira De; Bernardo Elizian Braga Rodrigues; Aquino Priscila De Souza; Damasceno Ana Kelve De Castro; Castro Régia Christina Moura Barbosa; Lessa Paula Renata Amorim; Pinheiro Ana Karina Bezerra. REFERÊNCIA Avaliação Do Processo Na Assistência Pré-Natal De Gestantes Com Risco Habitual Artigo Original • Acta Paul Enferm 31 (3) • MAY- JUN 2018 Https://Www.Scielo.Br/J/Ape/A/Kvhnqddlrvtmdb5tr4cksjr/?Lang=Pt 7 - CRISTIANE Dos Santos Viana; Danyella Rodrigues De Almeida; Adryelle Lemes De Campos; Aline De Almeida Silva; Carolina Sampaio De Oliveira; Naudia Da Silva Dias; Samira Michel Garcia; Thaís Martins Dos Santos Qualidade Da Assistência Pré-Natal Realizada Por Enfermeiros Das Unidades Básicas De Saúde De Cáceres-Mt/ Qualidade Da Assistência Pré-Natal Realizada Por Enfermeiros Nas Unidades Básicas De Saúde De Cáceres-Mt Publicação 10/02/2021 Https://Www.Brazilianjournals.Com/Index.Php/BJHR/Article/View/ 24877/19835 8 - DANIELLA Da Silva Nascimento; Danielle Da Silva Nascimento; Valdeluce Freitas De Araujo Silva; Camilla Mirela Viana Belarmino; Vivian Conceição Alves Leite Pereira Do Lago; Revista De Casos E Consultoria, Qualidade De Vida De Gestantes Atrelada A Assistência Do Enfermeiro No Pré- Natal: Uma Revisão Integrativa Quality Of Life Of Pregnant Women Linked To Nurses' Care In Prenatal: An Integrative. Publicação 27/01/2021 File:///C:/Users/User-PC/Downloads/7219-Artigo-76741-1-10-20210426.Pdf 651 9 - DOURADO Gisele Gomes; CARVALHO Brenda Da Rocha; Duarte Iago Araújo;SANTOS Taise Rocha; VIEIRA Nandiara; OLIVEIRA Marina Matos De; MATOS Iandra Gabriela De Almeida; BARBOSA Jaciara Profiro; ROCHA Giovana Alecrim; PONTES Adelia Matos. Assistência De Enfermagem Ao Pré-Natal: Relato De Experiência Prenatal Nursing Care: Experience Report Cuidado De Enfermería Prenatal: Informe De Experiencia Recebido: 12/07/2021 \| Revisado: 16/07/2021 \| Aceito: 20/07/2021 \| Publicado: 28/07/2021. File:///C:/Users/User-PC/Downloads/18140- Article-227188-1-10-20210728%20(1).Pdf 10 - FRANCISCO Lucas Leandro De Sousa; PASSOS Alane Dionizio; Caroline Taiane Santos Da Silva; Consulta De Pré-Natal De Risco Habitual Na Atenção Primária À Saúde: Um Relato De Experiência Https://Doity.Com.Br/Media/Doity/Submissoes/Artigo- 2021 Https://Doity.Com.Br/Media/Doity/Submissoes/Artigo- f19b1b8645ecb9dcb42d09c4ec016cb09a1fe89a-Segundo_Arquivo.Pdf 11 - GADELHA Ivyna Pires;AQUINO Priscila De Souza;BALSELLS Marianne Maia Dutra; DINIZ Flaviane Fabrício; PINHEIRO Ana Karina Bezerra; RIBERIRO Samila Gomes; CASTRO Régia Christina Moura Barbosa. Qualidade De Vida De Mulheres Com Gravidez De Alto Risco Durante O Cuidado Pré-Natal • Rev. Brasil. Enferma.73 (Suppl 5) • 2020 Https://Www.Scielo.Br/J/Reben/A/YZ5QftCZvqHvF5WVrXKS5gv/Abstr act/?Lang=Pt 11 - GADELHA Ivyna Pires;AQUINO Priscila De Souza;BALSELLS Marianne Maia Dutra; DINIZ Flaviane Fabrício; PINHEIRO Ana Karina Bezerra; RIBERIRO Samila Gomes; CASTRO Régia Christina Moura Barbosa. Qualidade De Vida De Mulheres Com Gravidez De Alto Risco Durante O Cuidado Pré-Natal • Rev. Brasil. Enferma.73 (Suppl 5) • 2020 Https://Www.Scielo.Br/J/Reben/A/YZ5QftCZvqHvF5WVrXKS5gv/Abstr act/?Lang=Pt Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. REFERÊNCIA ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 12 - GOMES Celma Barros De Araújo; DIAS Rosane Da Silva; SILVA Walisson Grangeiro Bringel; PACHECO Marcos Antônio Barbosa; SOUSA Francisca Georgina Macedo De; LOYOLA Cristina Maria Douat. Consulta De Enfermagem No Pré-Natal: Narrativas De Gestantes E Enfermeiras Https://Www.Scielo.Br/J/Tce/A/3pldtxnvjlgjwdffhm3fqbv/?Lang=Pt 13 - GUIMARÃES,  Wilderi Sidney Gonçalves; PARENTE,Rosana Cristina Pereira; GARNELO, Thayanne Louzada Ferreira Guimarães Luiza. Acesso e qualidade da atenção pré-natal na Estratégia Saúde da Família: infraestrutura, cuidado e gestão. Artigo, cad. Saúde pública 34, maio 2018. https://www.scielo.br/j/csp/a/9CMWjGgNGcLLYRjpCQQrymh/?lang=pt 14 - JOSÉ William Araújo Do Nascimento; Ana Carla Macedo Da Silva; Natália Borba Cavalcanti Dos Santo; Dhenifer Cristiane Macedo Gonçalves; Ana Carolina Gonçalves Da Silva; Andréa De Oliveira Ribeiro Cavalcanti; Ana Ravely Da Silva Candéas; Érica Lays Leite Pires; Andreza Pereira Dos Santos; Karoliny Alves Pereira. Research, Society And Development, Atuação Do Enfermeiro Na Gestação De Alto Risco: Uma Revisão Sistemática The Role Of Nurses In High-Risk Pregnancy: A Systematic Review El Papel De Las Enfermeras En El Embarazo De Alto Riesgo: Una Revisión Sistemática File:///C:/Users/Usuario-PC/Downloads/24616-Article-292368-1-10- 14 - JOSÉ William Araújo Do Nascimento; Ana Carla Macedo Da Silva; Natália Borba Cavalcanti Dos Santo; Dhenifer Cristiane Macedo Gonçalves; Ana Carolina Gonçalves Da Silva; Andréa De Oliveira Ribeiro Cavalcanti; Ana Ravely Da Silva Candéas; Érica Lays Leite Pires; Andreza Pereira Dos Santos; Karoliny Alves Pereira. Research, Society And Development, Atuação Do Enfermeiro Na Gestação De Alto Risco: Uma Revisão Sistemática Candéas; Érica Lays Leite Pires; Andreza Pereira Dos Santos; Karoliny Alves Pereira. Research, Society And Development, Atuação Do Enfermeiro Na Gestação De Alto Risco: Uma Revisão Sistemática The Role Of Nurses In High-Risk Pregnancy: A Systematic Review El Papel De Las Enfermeras En El Embarazo De Alto Riesgo: Una Revisión Sistemática File:///C:/Users/Usuario-PC/Downloads/24616-Article-292368-1-10- 20220104%20(6).Pdf The Role Of Nurses In High-Risk Pregnancy: A Systematic Review El Papel De Las Enfermeras En El Embarazo De Alto Riesgo: Una Revisión Sistemática File:///C:/Users/Usuario-PC/Downloads/24616-Article-292368-1-10- 20220104%20(6).Pdf 15 - JUSTINO Jessica Micaele Rebouças; NOGUEIRA Cintia Mikaelle Cunha De Santiago; LIRA Cindy Damaris Gomes; MARTINS Remerson Russel; FILHO Ana Virgínia De Melo Estratégias De Educação Em Saúde Durante O Pré-Natal Como Agente Promotor De Qualidade De Vida / Health Education Strategies During Prenatal As An Agent Promoting Quality Of Life. REFERÊNCIA Publicação 13/10/2021 Https://Brazilianjournals.Com/Index.Php/Brjd/Article/View/18436 Https://Www.Brazilianjournals.Com/Index.Php/BRJD/Article/View/18436/14855 652 16 - MENDE Rosemar Barbosa; Santos José Marcos De Jesus; Prado Daniela Siqueira Prado; Gurgel Rosana Queiroz; Bezerra Felipa Daiana; Gurgel Ricardo Queiroz Avaliação Da Qualidade Do Pré-Natal A Partir Das Recomendações Do Programa De Humanização No Pré-Natal E Nascimento Https://Www.Scielo.Br/J/Csc/A/Cdtvrdqynsdztncgfjszcjr/?Lang=Pt 17 - NASCIMENTO José William Araújo Do; Barbosa Lucas Mariano Da Silva; Lima; Sara Sue Helen Da Silva; Lucena Myrella Lins De; Andrade Débora Valéria Da Silva. Principais Fatores Associados Ao Tratamento Do Pré-Natal: Uma Revisão Sistemática Main Factors Associated With Late Prenatal Care: A Systematic Review. Publicação 20/12/2021File:///C:/Users/User-Pc/Downloads/41575-104062-1-Pb.Pdf 18 - PAULA, Michelle Rodrigues Souza De; BARBOSA, Valquíria Vicente Da Cunha Relato De Experiência: Cuidado Pré-Natal Em Uma Unidade De Saúde Da Família De Cachoeira Alta, Goiás Report Of Experience: Prenatal Care In A Family Health Unit Of The City Of Cachoeira Alta, GoiásHttps://Docs.Bvsalud.ORG/BIBLIOREF/2020/12/1140093/Cuidado-Pre- Natal-Em-Uma-Unidade-De-Saude-De-Cachoeira-Alta.Pdf Https://Www.Revista.Esap.Go.Gov.Br/Index.Php/Resap/Article/View/109/126 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 19 – QUEIROZ Kariny Tabosa; CARVALHO Keliane Beltrão; SIQUEIRA Maria Deiza Barros; QUEIROZ Carla Ketelem Tabosa; PINHEIRO Carlos José Moreira. Fatores Associados À Qualidade Da Assistência De Enfermagem Durante O Pré- Natal: Um Referencial Teórico 2018. Https://Downloads.Editoracientifica.Org/Articles/210504647.Pdf 20 – REIS, Rachel Sarmento; RACHED, Chennyfer Dobbins Abi. O Papel Do Enfermeiro No Acompanhamento De Pré-Natal De Baixo Risco Utilizando A Abordagem. International Journal of Health Management Review. V, 3, N. 2, Maio 2017. Https://Ijhmreview.Org/Ijhmreview/Article/View/125 21 - PESSOA Wedja Gleicy Souza; LIMA Manuela Maria De França Bezerra De; TAVARES Lucidia De Medeiros. Conhecimento Da Gestante Sobre A Importância Da Consulta Pré-Natal: Revisão Integrativa.Vol.7-Nª 01- Setembro,2021 Https://Reer.Emnuvens.Com.Br/Reer/Article/Viewfile/554/251 22 - SEHNEM Graciela Dutra; SALDANHA Laísa Saldanha de; ARBOIT Jaqueline; RIBEIRO Aline Cammarano; PAULA Francielle Morais de. Consulta de pré-natal na atenção primária à saúde: fragilidades e potencialidades da intervenção de enfermeiros brasileiros Revista de Enfermagem Referência, vol. V, núm. 1, pp. 1-7, 2020. Escola Superior de Enfermagem de Coimbra https://www.redalyc.org/journal/3882/388263105017/html/ 653 23 - SILVA Ana Alice Bueno Da; ANDRADE Claudiane. Research, Society and Development, V. 9, N. 10, E9989109477, 2020 (Cc By 4.0) \| Issn 2525-3409 \| Doi: Http://Dx.Doi.Org/10.33448/Rsd-V9i10.94771 O Papel Do Enfermeiro Na Assistência, Educação E Promoção Da Saúde No Pré- Natal The Role Of Nurses In Prenatal Care, Education And Health Promotion El Papel De Las Enfermeras En La Atención Prenatal, La Educación Y La Promoción De La Salud 20/10/2020. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 REFERÊNCIA Https://Rsdjournal.Org/Index.Php/Rsd/Article/View/9477/8455 24 - SILVA Dulcinéia Pinho; SILVESTE Grasiela Cristina Silva Botelho; CASTELLI Laíza Strinta; Silva Fabiane Verônica; Vicente Jefferson Tennesse Da Silva; Faria Nemora Barros; Rocha Roseany Patrícia Silva. Research, Society And Development, V. 10, N. 6, E44210615937, 2021 (Cc By 4.0) \| Issn 2525-3409 \| Doi: Http://Dx.Doi.Org/10.33448/Rsd-V10i6.159371 The Meanings Of Prenatal Assigned By Pregnant Women Performed By Nurses Los Significados De Prenatal Asignado Por Mujeres Embarazadas Realizado Por Enfermeras. Https://Rsdjournal.Org/Index.Php/Rsd/Article/View/15937/14276 25 - SOUSA Larissa Aguiar De; VERGARA Lilian Maureira; Revista Científica Da Umc O Cuidado Pré-Natal Humanizado Na Atenção Primária Em Saúde: Uma Revisão De Literatura Humanized Prenatal Care In Primary Health Care: A Literature Review Http://Seer.Umc.Br/Index.Php/Revistaumc/Article/View/1536 25 - SOUSA Larissa Aguiar De; VERGARA Lilian Maureira; Revista Científica Da Umc O Cuidado Pré-Natal Humanizado Na Atenção Primária Em Saúde: Uma Revisão De Literatura Humanized Prenatal Care In Primary Health Care: A Literature Review Http://Seer.Umc.Br/Index.Php/Revistaumc/Article/View/1536 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.8.n.08. ago. 2022. ISSN - 2675 – 3375
https://openalex.org/W4362433438	https://figshare.com/articles/journal_contribution/Data_Supplement_from_Efficient_Identification_of_Mutated_Cancer_Antigens_Recognized_by_T_Cells_Associated_with_Durable_Tumor_Regressions/22454372/1/files/39905480.pdf	English	null	Data Supplement from Efficient Identification of Mutated Cancer Antigens Recognized by T Cells Associated with Durable Tumor Regressions	null	2,023	cc-by	3,019	Supplementary Material Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions Yong-Chen Lu1, Xin Yao1, Jessica S. Crystal1, Yong F. Li1, Mona El-Gamil1, Colin Gross1, Lindy Davis1, Mark E. Dudley1, James C. Yang1, Yardena Samuels2, Steven A. Rosenberg1, and Paul F. Robbins1 Authors’Affiliations: Authors’Affiliations: 1Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. 1 1 Supplementary Fig. S1. Identification of dominant HLA-restriction element. Mel 2359 cells (A) or Mel 2591 cells (B) were pre-incubated with HLA-B,C (B1.23.1) or HLA-A,B,C (W6/32) blocking antibodies for 3 hr, followed by co-cultured overnight with TIL 2359 T cells, C07- restricted MAGE-A12 TCR-transduced T cells, TIL 2591 T cells or A02-restricted MART-1- specific T cells, respectively. The secretion of IFN- by T cells was determined by ELISA. Supplementary Fig. S1. Identification of dominant HLA-restriction element. Mel 2359 cells (A) or Mel 2591 cells (B) were pre-incubated with HLA-B,C (B1.23.1) or HLA-A,B,C (W6/32) blocking antibodies for 3 hr, followed by co-cultured overnight with TIL 2359 T cells, C07- restricted MAGE-A12 TCR-transduced T cells, TIL 2591 T cells or A02-restricted MART-1- specific T cells, respectively. The secretion of IFN- by T cells was determined by ELISA. 2 2 Supplementary Fig. S2. TIL 2591 T cells recognize multiple known antigens. COS-7 cells were transfected with a panel of previously described antigens, together with HLA cDNA constructs. These transfected cells were then co-cultured overnight with TIL 2359 T cells (A) or 2591 T cells (B, C). The secretion of IFN- was determined by ELISA. A positive control of GFP was included to demonstrate the effectiveness of the transfection. Supplementary Fig. S2. TIL 2591 T cells recognize multiple known antigens. COS-7 cells were transfected with a panel of previously described antigens, together with HLA cDNA constructs. These transfected cells were then co-cultured overnight with TIL 2359 T cells (A) or 2591 T cells (B, C). The secretion of IFN- was determined by ELISA. A positive control of GFP was included to demonstrate the effectiveness of the transfection. 3 3 Supplementary Fig. S3. Gene structure of SERPINE2, DUSP12 and SLC24A5. (A) SERPINE2 transcript variants. Exon 7B of transcript variant 2 (TV2) is 3 nucleotides (5’-CAG-3’) shorter than Exon 7A, resulted in amino acid substitution from Thr-Gly to Arg. (B) DUSP12 WT mRNA and transcript variant. The DUSP12 transcript variant contains 11 nucleotides (5’-GTCTCTTTT TCAG-3’) prior to the WT exon 2 and induces early termination of DUSP12 protein synthesis. (C) SLC24A5 WT mRNA and transcript variant. This variant contains a prolong exon 2, which translates into a new in-frame amino acid sequence: CKWLESCPVTFWESVPHRYDCL. Supplementary Fig. S3. Authors’Affiliations: Gene structure of SERPINE2, DUSP12 and SLC24A5. (A) SERPINE2 transcript variants. Exon 7B of transcript variant 2 (TV2) is 3 nucleotides (5’-CAG-3’) shorter than Exon 7A, resulted in amino acid substitution from Thr-Gly to Arg. (B) DUSP12 WT mRNA and transcript variant. The DUSP12 transcript variant contains 11 nucleotides (5’-GTCTCTTTT TCAG-3’) prior to the WT exon 2 and induces early termination of DUSP12 protein synthesis. (C) SLC24A5 WT mRNA and transcript variant. This variant contains a prolong exon 2, which translates into a new in-frame amino acid sequence: CKWLESCPVTFWESVPHRYDCL. Supplementary Fig. S3. Gene structure of SERPINE2, DUSP12 and SLC24A5. (A) SERPINE2 transcript variants. Exon 7B of transcript variant 2 (TV2) is 3 nucleotides (5’-CAG-3’) shorter than Exon 7A, resulted in amino acid substitution from Thr-Gly to Arg. (B) DUSP12 WT mRNA and transcript variant. The DUSP12 transcript variant contains 11 nucleotides (5’-GTCTCTTTT TCAG-3’) prior to the WT exon 2 and induces early termination of DUSP12 protein synthesis. (C) SLC24A5 WT mRNA and transcript variant. This variant contains a prolong exon 2, which translates into a new in-frame amino acid sequence: CKWLESCPVTFWESVPHRYDCL. 4 Supplementary Fig. S4. SLC24A5 WT expression in normal tissue. A human normal tissue cDNA panel was obtained from Clontech (A) or OriGene (B). The copy numbers of SLC24A5 WT cDNA from normal tissue samples were determined by quantitative PCR. Supplementary Fig. S4. SLC24A5 WT expression in normal tissue. A human normal tissue cDNA panel was obtained from Clontech (A) or OriGene (B). The copy numbers of SLC24A5 WT cDNA from normal tissue samples were determined by quantitative PCR Supplementary Fig. S4. SLC24A5 WT expression in normal tissue. A human normal tissue cDNA panel was obtained from Clontech (A) or OriGene (B). The copy numbers of SLC24A5 WT cDNA from normal tissue samples were determined by quantitative PCR. 5 5 Supplementary Fig. S5. Gene structure and DNA sequence chromatogram results of KIF2C and POLA2. (A) Gene structure of KIF2C. The point mutation is located at Exon 1. (B) DNA sequence chromatogram results for KIF2C. Genomic DNA samples were obtained from autologous PBMC and Mel 2359 cells, and cDNA sample was obtained from Mel 2359 cells. (C) Gene structure of POLA2. The point mutation is located at Exon 14. (D) DNA sequence chromatogram results for POLA2. Genomic DNA samples were obtained from autologous PBMC and Mel 2591 cells, and cDNA sample was obtained from Mel 2591 cells. Authors’Affiliations: Minigene ID Tandem Minigene ID Gene Symbol Mut AA Position Mutated Minigene 1 1 EPHB2 489 GAGCTCAGTGAGTACAACGCCACAGCCATAAAAAGCTCCACCAACACGGTCACCGTGCAGGGCCTCAAAGCCGGC 2 1 KIF2C 16 GACTCGTCGCTTCAGGCCCGCCTGTTTCCCGGTCTCACTATCAAGATCCAACGCAGTAATGGTTTAATTCACAGT 3 1 SLC44A5 512 CCACTTTTTACTGCATTTGGACGAGCCATACGATATTACACAGGATCCCTAGCATTTGGATCTTTAATTATTGCA 4 1 ABCA4 18 CAGATACAGCTTTTGCTCTGGAAGAACTGGACCCTGCAGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTG 5 1 DENND4B 41 ACGTGGGTTCCTGAACCCAGTGGGCCCCTGCGCCCTTCCCGGCCAGCTGAGCCCATCACAGATGTGGCAGTCATC 6 1 EPRS 913 GCGAAAGTACTTTTTGACAAAGTAGCTTCTCAAGGGGGAGTAGTTCGGAAACTTAAAACTGAAAAAGCCCCTAAG 7 2 DNAH14 2593 ATATACCATCAAGTACGTCAGAATATGTTACCAACTTCAACAAAATGTCACTACATGTTTAATCTTCGAGATATG 8 2 OR14C36 264 TCATGCTCTTCTGTGTACCTCAGGCCACCTGCGATATCTGCAGCCACCCAGGATCTGATCCTTTCTGGTTTTTAT 9 2 TRAPPC12 104 GCTGAGCCCGGAGGGGAAGGCGACCCAGGCCCGGAGCTCGCGGGCACCCCGAGTCCCAGCGGCGAGGCCGACGGC 10 2 SLC5A7 251 TCTTGGCTTGATAGTTTTCTGTTGTTGATGCTGGGTAGAATCCCATGGCAAGCATACTTTCAGAGGGTTCTCTCT 11 2 SLC5A7 4 ATGCTGGGTAGAATCCCATGGCAAGCATACTTTCAGAGGGTTCTCTCT 12 2 LRP2 2131 GCATCTGATAATGCGATCCGTAGAATTAAACCAGATAGATCTTCTCTGATGAACATTGTGACACATGGAATAGGA 13 3 FANCD2 405 ACTCAGACAAAGAAGTACATTGACAGGGTGCTAAGAAGTAAGATTCGATCAGGCTGCATTCAAGAACAGCTGCTC 14 3 SCN10A 1665 TGCCTCTTCCAGATTACCACGTCGGCCGGCTGGGATAGCCTCCTCAGCCCCATCCTCAACACAGGGCCCCCCTAC 15 3 TBC1D23 227 CAGGCAATATGGGATGGATATCTACAACAAGCAGATCAATTTTTTATTTATTTCTTAATGTTAATTATCCTTGTT 16 3 TNIK 207 GCAAGAAAGATTTCGGTGGTAAATGTAAACCCAACCAGCATTCGGCCTCATAGCGACACACCAGAAATCAGAAAA 17 3 SGMS2 108 ATGATCACAGTTGTACATGAGAGGGTCCCTCCCAAGAAGCTTAGCCCTCCACTCCCAGACAAGTTTTTTGATTAC 18 3 SPATA4 201 AAAGATAACATTAGGTTATCAGAATTACTAAGCAATCTCAACATGCTGACCAATGAACTTAAAGCGGAGTTCCTC 19 4 KLKB1 25 TTGTTTGCTACAGTTTCCTGTGGATGTCTGACTCAATTCTATGAAAACGCCTTCTTCAGAGGTGGGGATGTAGCT 20 4 THBS4 319 GGCTTCCAGTGTGGGCCCTGCCCCGAGGGCTACACAAGAAACGGGATCACCTGTATTGATGTTGATGAGTGCAAA 21 4 PCDHA13 317 AAAGGCAAACTAGATTTCGAAGAAAAGAAATTATATAAAATATCCGTGGAGGCAGTTGACAAAGGAAATATTCCA 22 4 PCDHB8 199 AGTGATGGCAGGAAATACCCAGAGCTGGTGCTGGACAACGCGCTGGACCGAGAGGAAGAAGCTGAGCTCAGGTTA 23 4 ZNF354C 398 TGCTTGGAATGTGGGAGAACCTTCACACGTATTGTAATCCTTATCGAACATCAGCGAATTCACACTGGACAAAAA 24 4 GPR116 506 TCAATGACTTTTAAGAACAATAGCCCTTCAGGCGGCAAAACGAAGTGTGTCTTCTGGAACTTCAGGCTTGCCAAC 25 5 ARG1 305 GTGAACACAGCAGTTGCAATAACCTTGGCTTGTTTCAGACTTGCTCGGGAGGGTAATCACAAGCCTATTGACTAC 26 5 BCLAF1 356 AAGAATCTGGATGCACGAGAAAAGTCTACCTTCAGAAAGGAAAGCCCACTTAGGATCAAAATGATAGCGAGTGAT 27 5 KIAA1244 2039 ACCGAATACAAAAAGAGGAAACAGCAGCACAACCTGTTCGCGTTCCCCAAAGAGGTCAAAGTGGAGAAGAAAGGA 28 5 EIF3B 577 ATCATAGCCTTTGCCTGGGAACCAAATGGAAGTAAGCTTGCTGTGCTGCACGGAGAGGCTCCGCGGATATCTGTG 29 5 TBRG4 207 TGCAGCGTACTTCTGGCTTTTGCGCGTCTGAACTTCTATCCAGACCAAGAGGATCAGTTCTTCAGCCTGGTACAT 30 5 CLIP2 615 ACCAGCGAGAACATGGGGCTAATGGACAACTGGAAATTCAAGCTGGACTCGCTGGCCTCGGACCACCAGAAGTCC 31 6 CLIP2 9 ATGGGGCTAATGGACAACTGGAAATTCAAGCTGGACTCGCTGGCCTCGGACCACCAGAAGTCC 32 6 SAMD9L 1205 AACACAGCTTGTTTCTTGGGTGAAATAGAAGTTGGTTTTTACACTATCCAGATTCTTCAGCTCACTCCCTTTTTC 33 6 C9orf72 196 ATGGAACTGCTTTCATCTATGAAATCACACAGTGTTCTTGAAGAAATAGATATAGCTGATACAGTACTCAATGAT 34 6 ZNF782 432 AAACCATACAAATGTGATGGATGTGATAAAGCTTTCGGTGCAAAGTCAGGCCTAAGAATACACCAGAGAACCCAC 35 6 ABO 63 TTCTGCATGGCTGTTAGGGAACCTGACCATCTGCAGCGTGTCTCGTTGCCAAGGATGGTCTACCCCCAGTCAAAG 36 6 CACNA1B 165 GGCTTTGTCTTCCACAAGGGCTCTTACCTGCGGAACGTCTGGAACGTCATGGACTTCGTGGTCGTCCTCACAGGG 37 7 PCDH15 1419 TTTAAAGTACGTCAAGCTGAGTGTACAAAGACTGCACAAATTCAGGCCGCATTACCCGCGGCTAAACCAGCAGTG 38 7 PCDH15 1389 TACAGACAACGTCAAGCTGAGTGTACAAAGACTGCACAAATTCAGGCCGCATTACCCGCGGCTAAACCAGCAGTG 39 7 CYP2C18 23 CTCTCCTGTTTGTTTCTCCTTTCACTCTGGAGGCAGAACTCTGGAAGAGGGAGGCTCCCGTCTGGCCCCACTCCT 40 7 MUC6 1587 ACACACTCCCCACCTACAGGGAGCAGTCCCTTCTCTCCCACAGGTCCCATGACGGCAACATCCTTCAAGACCACC 41 7 MUC6 1562 ACTAGTACACGCACCAGAACCCCTGTGGCCCACACCACCTCAGCCACCAGCAGCAGGCCACCACCACCCTTCACC 42 7 MUC6 1555 ACCACGATTACTCCCAACCCCACTAGTACACGCACCGGAACCCCTGTGGCCCACACCAACTCAGCCACCAGCAGC 43 8 PIK3C2A 217 CCCTTTCATCCACAAGGAAGCTTACCTATCTATCGTCTAGTAGTCAGTACTGACATGGCAAAACTATTTGACAAA 44 8 OR4C3 157 TATGACCGCTATGTGGCCATCTGTAAGCCCCTGCACTATACTACCATCATGACCAGGCATCTCTGTGCCATGCTT 45 8 OR4C3 157 TATGACCGCTATGTGGCCATCTGTAAGCCCCTGCACAGTACTACCATCATGACCAGGCATCTCTGTGCCATGCTT 46 8 PPP1CA 134 AAGATCAAGTACCCCGAGAACTTCTTCCTGCTCCGTAGGAACCACGAGTGTGCCAGCATCAACCGCATCTATGGT 47 8 SUV420H1 448 ATTCCTCTTCCTCCAGCTAAGCGCTTGAGGTTAATAGCTGGAAAAGACTCTATAGATATTGACATTTCTTCAAGG 48 8 ODZ4 184 TTCGACAAGAATGACCGCCTCTCTTCTGTGACGATGTCCAACGTGGCGCGGCAGACACTAGAGACCATCCGCTCA 49 9 CNTN5 70 TCATTAGGAACACTGAGTGCTTCTTCACCCAGCTGGCGAGGGGCAGCTCAGAATTATTATTCCCCCATCAATCTT 50 9 TRPC6 156 GAGCATCTGGAAATTACAGAACTTCTTCTCAAGAAAAAAAACCTCTCTCGAGTTGGGGATGCTTTGCTTCTAGCT 51 9 EEA1 626 GACCGTGTCCTTTCCCTAGAAACTAGTGTCAATGAATTCAATAGTCAATTAAATGAAAGCAAGGAGAAGGTCTCC 52 9 EP400 2796 ATGCAGAAGCAGAAACTGCAGATGCCCCCGCAGCCCTCACCGCCACAGGCCCAGTCTGCGCCCCCGCAGCCAACA 53 9 ZAR1L 308 CATCGACAGGAACTGTGTGGTCGCTGCAAAGACAAGATATTCTCCTGTGGCAATATTTACAGCTTTAAATATGTG 54 9 DOCK9 309 GAAGATGATGAACAAAGCAAATTGGAAGGTTCTGGTTTCGGTTTAGATAGCTACCTGCCGGAACTTGCCAAGAGT 55 10 COL4A1 1174 GCAGGAGAGAAGGGTGAACCAGGTCTACCAGGAAGAAGATTCCCAGGGTTTCCAGGGGCCAAAGGAGACAAAGGT 56 10 PLEKHG3 699 TCCTACATCCCCAAAGGACTGGTAAGAAACTCCATCTTCAGGTTCAACAGCCTTCCCCGGCCAGACCCAGAGCCA 57 10 LRRK1 1938 GGAGATGTTATCGTCATTGGCCTGGAGAAGGATTCTGACGCCCAGCGGGGCCGAGTCATTGCCGTCTTAAAAGCC 58 10 TMEM204 207 GAGGACTGCATGGCCCCCCGGGTGATTGTCATCAGCCACTCCCTGACAGCGCGCTTTCGCCGTGGGCTGGACAAT 59 10 ITGAL 269 AAAGTGCTTATCATCATCACGGATGGGGAGGCCACTAACAGTGGCAACATCGATGCGGCCAAAGACATCATCCGC 60 10 CNOT1 316 TTAACAGATGGCATTCCATTACAGAGTATTTCTGCTCTGGGCAGTGGGATCTGGAGTGATGGGAAAGATAAAAGT 61 11 DNAH9 2583 GGCCACTGGTATGATCGGAGCAAGCTGTCCCTAAAGAAGATCACAAATGTACAGTATGTTTCCTGTATGAACCCC 62 11 ERBB2 355 CACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCTCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTA 63 11 TRIM25 191 TGCCTGGTGGAGCATAAGACCTGCTCTCCCGCGTCCATGAGCCAGGCCAGCGCCGACCTGGAGGCCACCCTGAGG 64 11 TMEM241 131 GAGAAAACATCTCCTGCAAAGATCTGTAGTGCCCTCTTCCTCCTGGCCGCAGCAGGATGCCTTCCCTTCAATGAC 65 11 PTPRS 141 CAGCTGCCCTCTGGCTTCCCCAACATCGACATGGGCTCACAGTTGAAGGTGGTGGAGCGGACACGGACAGCCACC 66 11 OR2Z1 253 TGCTCCTCGCACATCACGGTAGTGGGGCTCTTTTATGATGCCGCCGTGTTCATGTACATGGTGCCTTGCGCCTAC 67 12 RYR1 103 TCATCCCAGGGCGGGGGACACAGGACGCTCCTGTATAGCCATGCCATCCTGCTCCGGCATGCACACAGCCGCATG 68 12 C20orf26 975 ATTGATACCAACTTCCACACCAACGACATAGCCATCGGAGCTGCTGGCTCCCTCACCAAATTCTCCAATAGATAC 69 12 XKR7 491 ACTACAGGTGCTGAGCGGGATGGGGCCTCGGCGGGAAAGCGTGCAGGGACCCCCACCCCACCTGTCTTCCAGGTG 70 12 TTN 1491 TCTGGGGAATGGACTGTGGTTGCCCAAAACAGGGCAAGCAGATCTTCAATTTCAGTGATTTTAACTGTGGAAGCT 71 12 MUC16 11792 ACAGAGACAAGTAAAACATTTCCTGCTTCAACTGTGCTTCCTCAAGTATCAGAGACCACAGCCTCACTCACCATT Supplementary Table S1. Tandem minigene constructs for Mel 2359. Authors’Affiliations: DNA sequence chromatogram results of KIF2C and e point mutation is located at Exon 1. (B) DNA C. Genomic DNA samples were obtained from DNA sample was obtained from Mel 2359 cells. (C) ation is located at Exon 14. (D) DNA sequence c DNA samples were obtained from autologous e was obtained from Mel 2591 cells. Supplementary Fig. S5. Gene structure an POLA2. (A) Gene structure of KIF2C. T sequence chromatogram results for KIF autologous PBMC and Mel 2359 cells, and Gene structure of POLA2. The point mu chromatogram results for POLA2. Genom PBMC and Mel 2591 cells, and cDNA sam Supplementary Fig. S5. Gene structure and DNA sequence chromatogram results of KIF2C and POLA2. (A) Gene structure of KIF2C. The point mutation is located at Exon 1. (B) DNA sequence chromatogram results for KIF2C. Genomic DNA samples were obtained from autologous PBMC and Mel 2359 cells, and cDNA sample was obtained from Mel 2359 cells. (C) Gene structure of POLA2. The point mutation is located at Exon 14. (D) DNA sequence chromatogram results for POLA2. Genomic DNA samples were obtained from autologous PBMC and Mel 2591 cells, and cDNA sample was obtained from Mel 2591 cells. 6 6 Supplementary Table S1. Tandem minigene constructs for Mel 2359. Authors’Affiliations: Minigene ID Tandem Minigene ID Gene Symbol Mut AA Position Mutated Minigene 1 1 EPHB2 489 GAGCTCAGTGAGTACAACGCCACAGCCATAAAAAGCTCCACCAACACGGTCACCGTGCAGGGCCTCAAAGCCGGC 2 1 KIF2C 16 GACTCGTCGCTTCAGGCCCGCCTGTTTCCCGGTCTCACTATCAAGATCCAACGCAGTAATGGTTTAATTCACAGT 3 1 SLC44A5 512 CCACTTTTTACTGCATTTGGACGAGCCATACGATATTACACAGGATCCCTAGCATTTGGATCTTTAATTATTGCA 4 1 ABCA4 18 CAGATACAGCTTTTGCTCTGGAAGAACTGGACCCTGCAGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTG 5 1 DENND4B 41 ACGTGGGTTCCTGAACCCAGTGGGCCCCTGCGCCCTTCCCGGCCAGCTGAGCCCATCACAGATGTGGCAGTCATC 6 1 EPRS 913 GCGAAAGTACTTTTTGACAAAGTAGCTTCTCAAGGGGGAGTAGTTCGGAAACTTAAAACTGAAAAAGCCCCTAAG 7 2 DNAH14 2593 ATATACCATCAAGTACGTCAGAATATGTTACCAACTTCAACAAAATGTCACTACATGTTTAATCTTCGAGATATG 8 2 OR14C36 264 TCATGCTCTTCTGTGTACCTCAGGCCACCTGCGATATCTGCAGCCACCCAGGATCTGATCCTTTCTGGTTTTTAT 9 2 TRAPPC12 104 GCTGAGCCCGGAGGGGAAGGCGACCCAGGCCCGGAGCTCGCGGGCACCCCGAGTCCCAGCGGCGAGGCCGACGGC 10 2 SLC5A7 251 TCTTGGCTTGATAGTTTTCTGTTGTTGATGCTGGGTAGAATCCCATGGCAAGCATACTTTCAGAGGGTTCTCTCT 11 2 SLC5A7 4 ATGCTGGGTAGAATCCCATGGCAAGCATACTTTCAGAGGGTTCTCTCT 12 2 LRP2 2131 GCATCTGATAATGCGATCCGTAGAATTAAACCAGATAGATCTTCTCTGATGAACATTGTGACACATGGAATAGGA 13 3 FANCD2 405 ACTCAGACAAAGAAGTACATTGACAGGGTGCTAAGAAGTAAGATTCGATCAGGCTGCATTCAAGAACAGCTGCTC 14 3 SCN10A 1665 TGCCTCTTCCAGATTACCACGTCGGCCGGCTGGGATAGCCTCCTCAGCCCCATCCTCAACACAGGGCCCCCCTAC 15 3 TBC1D23 227 CAGGCAATATGGGATGGATATCTACAACAAGCAGATCAATTTTTTATTTATTTCTTAATGTTAATTATCCTTGTT 16 3 TNIK 207 GCAAGAAAGATTTCGGTGGTAAATGTAAACCCAACCAGCATTCGGCCTCATAGCGACACACCAGAAATCAGAAAA 17 3 SGMS2 108 ATGATCACAGTTGTACATGAGAGGGTCCCTCCCAAGAAGCTTAGCCCTCCACTCCCAGACAAGTTTTTTGATTAC 18 3 SPATA4 201 AAAGATAACATTAGGTTATCAGAATTACTAAGCAATCTCAACATGCTGACCAATGAACTTAAAGCGGAGTTCCTC 19 4 KLKB1 25 TTGTTTGCTACAGTTTCCTGTGGATGTCTGACTCAATTCTATGAAAACGCCTTCTTCAGAGGTGGGGATGTAGCT 20 4 THBS4 319 GGCTTCCAGTGTGGGCCCTGCCCCGAGGGCTACACAAGAAACGGGATCACCTGTATTGATGTTGATGAGTGCAAA 21 4 PCDHA13 317 AAAGGCAAACTAGATTTCGAAGAAAAGAAATTATATAAAATATCCGTGGAGGCAGTTGACAAAGGAAATATTCCA 22 4 PCDHB8 199 AGTGATGGCAGGAAATACCCAGAGCTGGTGCTGGACAACGCGCTGGACCGAGAGGAAGAAGCTGAGCTCAGGTTA 23 4 ZNF354C 398 TGCTTGGAATGTGGGAGAACCTTCACACGTATTGTAATCCTTATCGAACATCAGCGAATTCACACTGGACAAAAA 24 4 GPR116 506 TCAATGACTTTTAAGAACAATAGCCCTTCAGGCGGCAAAACGAAGTGTGTCTTCTGGAACTTCAGGCTTGCCAAC 25 5 ARG1 305 GTGAACACAGCAGTTGCAATAACCTTGGCTTGTTTCAGACTTGCTCGGGAGGGTAATCACAAGCCTATTGACTAC 26 5 BCLAF1 356 AAGAATCTGGATGCACGAGAAAAGTCTACCTTCAGAAAGGAAAGCCCACTTAGGATCAAAATGATAGCGAGTGAT 27 5 KIAA1244 2039 ACCGAATACAAAAAGAGGAAACAGCAGCACAACCTGTTCGCGTTCCCCAAAGAGGTCAAAGTGGAGAAGAAAGGA 28 5 EIF3B 577 ATCATAGCCTTTGCCTGGGAACCAAATGGAAGTAAGCTTGCTGTGCTGCACGGAGAGGCTCCGCGGATATCTGTG 29 5 TBRG4 207 TGCAGCGTACTTCTGGCTTTTGCGCGTCTGAACTTCTATCCAGACCAAGAGGATCAGTTCTTCAGCCTGGTACAT 30 5 CLIP2 615 ACCAGCGAGAACATGGGGCTAATGGACAACTGGAAATTCAAGCTGGACTCGCTGGCCTCGGACCACCAGAAGTCC 31 6 CLIP2 9 ATGGGGCTAATGGACAACTGGAAATTCAAGCTGGACTCGCTGGCCTCGGACCACCAGAAGTCC 32 6 SAMD9L 1205 AACACAGCTTGTTTCTTGGGTGAAATAGAAGTTGGTTTTTACACTATCCAGATTCTTCAGCTCACTCCCTTTTTC 33 6 C9orf72 196 ATGGAACTGCTTTCATCTATGAAATCACACAGTGTTCTTGAAGAAATAGATATAGCTGATACAGTACTCAATGAT 34 6 ZNF782 432 AAACCATACAAATGTGATGGATGTGATAAAGCTTTCGGTGCAAAGTCAGGCCTAAGAATACACCAGAGAACCCAC 35 6 ABO 63 TTCTGCATGGCTGTTAGGGAACCTGACCATCTGCAGCGTGTCTCGTTGCCAAGGATGGTCTACCCCCAGTCAAAG 36 6 CACNA1B 165 GGCTTTGTCTTCCACAAGGGCTCTTACCTGCGGAACGTCTGGAACGTCATGGACTTCGTGGTCGTCCTCACAGGG 37 7 PCDH15 1419 TTTAAAGTACGTCAAGCTGAGTGTACAAAGACTGCACAAATTCAGGCCGCATTACCCGCGGCTAAACCAGCAGTG 38 7 PCDH15 1389 TACAGACAACGTCAAGCTGAGTGTACAAAGACTGCACAAATTCAGGCCGCATTACCCGCGGCTAAACCAGCAGTG 39 7 CYP2C18 23 CTCTCCTGTTTGTTTCTCCTTTCACTCTGGAGGCAGAACTCTGGAAGAGGGAGGCTCCCGTCTGGCCCCACTCCT 40 7 MUC6 1587 ACACACTCCCCACCTACAGGGAGCAGTCCCTTCTCTCCCACAGGTCCCATGACGGCAACATCCTTCAAGACCACC 41 7 MUC6 1562 ACTAGTACACGCACCAGAACCCCTGTGGCCCACACCACCTCAGCCACCAGCAGCAGGCCACCACCACCCTTCACC 42 7 MUC6 1555 ACCACGATTACTCCCAACCCCACTAGTACACGCACCGGAACCCCTGTGGCCCACACCAACTCAGCCACCAGCAGC 43 8 PIK3C2A 217 CCCTTTCATCCACAAGGAAGCTTACCTATCTATCGTCTAGTAGTCAGTACTGACATGGCAAAACTATTTGACAAA 44 8 OR4C3 157 TATGACCGCTATGTGGCCATCTGTAAGCCCCTGCACTATACTACCATCATGACCAGGCATCTCTGTGCCATGCTT 45 8 OR4C3 157 TATGACCGCTATGTGGCCATCTGTAAGCCCCTGCACAGTACTACCATCATGACCAGGCATCTCTGTGCCATGCTT 46 8 PPP1CA 134 AAGATCAAGTACCCCGAGAACTTCTTCCTGCTCCGTAGGAACCACGAGTGTGCCAGCATCAACCGCATCTATGGT 47 8 SUV420H1 448 ATTCCTCTTCCTCCAGCTAAGCGCTTGAGGTTAATAGCTGGAAAAGACTCTATAGATATTGACATTTCTTCAAGG 48 8 ODZ4 184 TTCGACAAGAATGACCGCCTCTCTTCTGTGACGATGTCCAACGTGGCGCGGCAGACACTAGAGACCATCCGCTCA 49 9 CNTN5 70 TCATTAGGAACACTGAGTGCTTCTTCACCCAGCTGGCGAGGGGCAGCTCAGAATTATTATTCCCCCATCAATCTT 50 9 TRPC6 156 GAGCATCTGGAAATTACAGAACTTCTTCTCAAGAAAAAAAACCTCTCTCGAGTTGGGGATGCTTTGCTTCTAGCT 51 9 EEA1 626 GACCGTGTCCTTTCCCTAGAAACTAGTGTCAATGAATTCAATAGTCAATTAAATGAAAGCAAGGAGAAGGTCTCC 52 9 EP400 2796 ATGCAGAAGCAGAAACTGCAGATGCCCCCGCAGCCCTCACCGCCACAGGCCCAGTCTGCGCCCCCGCAGCCAACA 53 9 ZAR1L 308 CATCGACAGGAACTGTGTGGTCGCTGCAAAGACAAGATATTCTCCTGTGGCAATATTTACAGCTTTAAATATGTG 54 9 DOCK9 309 GAAGATGATGAACAAAGCAAATTGGAAGGTTCTGGTTTCGGTTTAGATAGCTACCTGCCGGAACTTGCCAAGAGT 55 10 COL4A1 1174 GCAGGAGAGAAGGGTGAACCAGGTCTACCAGGAAGAAGATTCCCAGGGTTTCCAGGGGCCAAAGGAGACAAAGGT 56 10 PLEKHG3 699 TCCTACATCCCCAAAGGACTGGTAAGAAACTCCATCTTCAGGTTCAACAGCCTTCCCCGGCCAGACCCAGAGCCA 57 10 LRRK1 1938 GGAGATGTTATCGTCATTGGCCTGGAGAAGGATTCTGACGCCCAGCGGGGCCGAGTCATTGCCGTCTTAAAAGCC 58 10 TMEM204 207 GAGGACTGCATGGCCCCCCGGGTGATTGTCATCAGCCACTCCCTGACAGCGCGCTTTCGCCGTGGGCTGGACAAT 59 10 ITGAL 269 AAAGTGCTTATCATCATCACGGATGGGGAGGCCACTAACAGTGGCAACATCGATGCGGCCAAAGACATCATCCGC 60 10 CNOT1 316 TTAACAGATGGCATTCCATTACAGAGTATTTCTGCTCTGGGCAGTGGGATCTGGAGTGATGGGAAAGATAAAAGT 61 11 DNAH9 2583 GGCCACTGGTATGATCGGAGCAAGCTGTCCCTAAAGAAGATCACAAATGTACAGTATGTTTCCTGTATGAACCCC 62 11 ERBB2 355 CACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCTCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTA 63 11 TRIM25 191 TGCCTGGTGGAGCATAAGACCTGCTCTCCCGCGTCCATGAGCCAGGCCAGCGCCGACCTGGAGGCCACCCTGAGG 64 11 TMEM241 131 GAGAAAACATCTCCTGCAAAGATCTGTAGTGCCCTCTTCCTCCTGGCCGCAGCAGGATGCCTTCCCTTCAATGAC 65 11 PTPRS 141 CAGCTGCCCTCTGGCTTCCCCAACATCGACATGGGCTCACAGTTGAAGGTGGTGGAGCGGACACGGACAGCCACC 66 11 OR2Z1 253 TGCTCCTCGCACATCACGGTAGTGGGGCTCTTTTATGATGCCGCCGTGTTCATGTACATGGTGCCTTGCGCCTAC 67 12 RYR1 103 TCATCCCAGGGCGGGGGACACAGGACGCTCCTGTATAGCCATGCCATCCTGCTCCGGCATGCACACAGCCGCATG 68 12 C20orf26 975 ATTGATACCAACTTCCACACCAACGACATAGCCATCGGAGCTGCTGGCTCCCTCACCAAATTCTCCAATAGATAC 69 12 XKR7 491 ACTACAGGTGCTGAGCGGGATGGGGCCTCGGCGGGAAAGCGTGCAGGGACCCCCACCCCACCTGTCTTCCAGGTG 70 12 TTN 1491 TCTGGGGAATGGACTGTGGTTGCCCAAAACAGGGCAAGCAGATCTTCAATTTCAGTGATTTTAACTGTGGAAGCT 71 12 MUC16 11792 ACAGAGACAAGTAAAACATTTCCTGCTTCAACTGTGCTTCCTCAAGTATCAGAGACCACAGCCTCACTCACCATT 7 7 Supplementary Table S2. Candidate binding peptides for mutated KIF2C. Authors’Affiliations: Amino acid position Mutated Peptide Predicted HLA-A0205 binding affinity (nM) Co-culture result [IFN- (pg/mL)] 10-19 RLFPGLTIKI 55.21 10690 10-17 RLFPGLTI 132.35 121.5 15-25 LTIKIQRSNGL 251.33 31.5 7-17 LQARLFPGLTI 293.83 27 7-16 LQARLFPGLT 1549.33 24 Supplementary Table S2. Candidate binding peptides for mutated KIF2C. 8 8 Supplementary Table S3. Tandem minigene constructs for Mel 2591. Authors’Affiliations: Minigene ID Tandem Minigene ID Gene Symbol Mut AA Position Mutated Minigene 1 1 TAS1R3 88 ATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGAATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTT 2 1 TARDBP 201 AGAAGCAGAAAAGTGTTTGTGGGGCGCTGTACAGAGTACATGACTGAGGATGAGCTGCGGGAGTTCTTCTCTCAG 3 1 KIAA1026 603 AGCAGGGAGGCCCTCCAGGAGCGCCGGGCCCGCTGCAAGACGCAGAACATTGACCCCGTGGTGTGGACCAACCAG 4 1 DNAJB4 304 AGAATTATTGGATATGGGCTGCCATTTCCAAAAAATTCTGACCAACGTGGTGACCTTCTAATAGAATTTGAGGTG 5 1 OLFM3 178 GATGCTAAGTTAATCACCCAGTTCAAGGAGGAAATAAAGAATCTGTCTGCTGTCCTCACTGGTATTCAGGAGGAA 6 1 CLCC1 131 GGCGATATGCATTATGATGCTGAGATTATCCTTAAAAAAGAAACTTTGTTAGAAATACAGAAGTTTCTCAATGGA 7 2 CELSR2 2719 CTGGAAGGTCAAGACCAGCAGCATGATCCTGACACGAACTCCGACAGTGACCTGTCCTTAGAAGACGACCAGAGT 8 2 NES 1015 ACAGAGGAGGTCTGGATCCCAGGCGAGGGGCACCCAAAGAGCCCTGAGCCCAAAGAGCAGAGAGGCCTGGTTGAG 9 2 SH2D2A 360 CAGGACAGAGGACAGGCATGGCTTCCCCTTGGGCCTCTTCAGTAG 10 2 SPTA1 818 GAGGCCTGGATCCAAGAGACTGAACCCTCAGCTACTTTCACCTACCTTGGAAAGGACCTGATTGCTTCCAAAAAG 11 2 TNN 134 ATGAAGGAACAGTGTAGTGCCCAGCGCTGCTGCCAGAGAGTCACTGATCTAAGCCGCCACTGCAGCGGCCACGGG 12 2 PAPPA2 1380 CCCAGTAACTGCATCTCAGAGGACGAGGGGCAGAATTATCAGGGACAGAGCTGTATCCATCGGCCCTGTGGGAAG 13 3 NPHS2 109 GGCTTAGGGGCCTGTGAGTGGCTTCTTGTCCTCATTTTCCTGCTCTTCATCATCATGACCTTCCCTTTTTCCATC 14 3 CENPF 678 AGAACGCTGGAGATGGACAGAGAAAACCTAAGTGTCAAGATCAGAAACCTTCACAACGTGTTAGACAGTAAGTCA 15 3 HEATR1 3 ATGACGTTCTTAGCCCAGCAGCTGCAACGACTCGCCCTCCCTCAA 16 3 AKT3 51 AAAGAGAAACCTCAAGATGTGGATTTACCTTATCCCCGCAACAACTTTTCAGTGGCAAAATGCCAGTTAATGAAA 17 3 TFB2M 21 CCTCGGCGGCTGAGGCTCTCCGCCTTGGCGGGCGCTGATCGCTTTTGCATTTTAGGGTCTGAAGCGGCGACGCGA 18 3 FAM171A1 664 GGCACCCGGGAGTGGAGCCCTCAGAACGCATCCATGTTGGAGTCTCTCTCCATCCCAGCTTCCCTGAACGACGCG 19 4 FAM171A1 663 GCGGGCACCCGGGAGTGGAGCCCTCAGAACGCATCCATTTCGGAGTCTCTCTCCATCCCAGCTTCCCTGAACGAC 20 4 MPP7 325 TCCAACAGGAAATCATCTGGTTTTAGAAAAAGTTTTTGTCTTAGTAGAAAAGATAAGAAAACAAATAAATCCATG 21 4 P4HA1 65 TTAGAACAAATAAAAAAATGGGCAGAGAAGTTAGATTGGCTAACTAGTACAGCGACAAAAGATCCAGAAGGATTT 22 4 LRIT1 575 GACTCCACTGAGGCCACAGTTACCTACGTCAACCTAAAGAGACTGGGCTACAGCGAGGACGGCTTGGAGGAGCTG 23 4 SLIT1 296 GCCATGTGCACCTGCAGCAATGGCATCGTGGACTGTTGTGGAAAAGGCCTCACTGCCATCCCGGCCAACCTGCCC 24 4 PPRC1 281 AGCAGTATCCCGGACTTCCCCATGCATTTGGCCTGCTCTGAGGAGGAAGATAAAGCAACAGCAGCAGAGATGGCA 25 5 OR52N1 151 ACTAATTCAGTCATTGCTAAAGCTGGGTTCCTCACTTGTCTTAGGGGTGTGATGCTTGTTATCCCTTCCACTTTC 26 5 DNHD1 384 TCCTTTACCTGTTGGAAGAAGAATGTGAGATTACAGAGGCTGCATCGACTCCAGAAATTCCTAGAGAATCATCTG 27 5 OR10A3 269 ATGACTTATTTACAACCCAAATCTGGCTACTCACCCAAAACCAAGAAACTGATCTCATTGGCTTACACGTTGCTT 28 5 BBOX1 173 GAAGTTTCAAAACTTGGGAAAAGGATGGGTTTCCTCTGTCTCACATTTTATGGACATACTTGGCAAGTGCAAGAC 29 5 CCDC73 432 TATAATACTGAGCAAGAAATAAGGGAAGAAAATATGAAGAATTTTTGTTCAGATACTGAATACAGAGAAAAAGAA 30 5 QSER1 936 AATCAGGTTACTGTGAACCTTTCACCAGTACCTGCCCCTCAGTCAAAAATGACTCTTGATCAACAGCACATTGAA 31 6 POLA2 420 ACAATTATTGAAGGCACAAGAAGCTCCGGCTCCCACTTTGTCTTTGTCCCGTCATTGAGAGATGTGCACCATGAG 32 6 PDGFD 54 GACTTGTACCGAAGAGATGAGACCATCCAGGTGAAAGAAAACGGCTACGTGCAGAGTCCTAGATTCCCGAACAGC 33 6 MLL 3174 TTACATTCCTTCCCTGCAGCTACTCAAAGTAGTTTCCTACCAAACATCAGCAATCCTCCTTCAGGCCTGCTTATT 34 6 GPR19 42 GAAACAGCCACACCTCTGCCAAGCCAATACCTGATGAAATTAAGTGAGGAGCACAGTTGGATGAGCAACCAAACA 35 6 SFRS2IP 482 AAAAAGCCTCGTACTCGAAGATCTAGATTTCATTCTTCATCTACAACTTGGTCACCCAACAAAGACACTCCACAA 36 6 COL2A1 238 AATCCTGGTGAACCTGGTGAACCTGGTGTCTCTGGTCTCATGGGTCCCCGTGGTCCTCCTGGTCCCCCTGGAAAG 37 7 RHEBL1 124 GTGCCAGTGGTTCTAGTGGGGAACAAGGCAGATCTCTTTCCAGAGAGAGAGGTACAGGCAGTTGAAGGAAAGAAG 38 7 SUOX 223 AACCCTATCTTCTTCACCCGGAACCATCTGCCTGTATCTAACCTGGATCCAGACACCTATCGCTTACACGTAGTA 39 7 PTPRB 1067 ACGGTGAACTGGACTCCTGGTGGGGGAGACGTTGATTTCTACACGGTGTCGGCATTCAGGCACAGTCAAAAGGTT 40 7 MYL2 10 ATGTTCGAACAGACCCAAATCCAGGAAATTAAGGAGGCCTTCACTATCATGGACCAGAACAGGGAT 41 7 MYL2 29 GTGTTCTCCATGTTCGAACAGACCCAAATCCAGGAAATTAAGGAGGCCTTCACTATCATGGACCAGAACAGGGAT 42 7 CCDC60 406 GCCCAGGAGGCTGGCTTCTGCCTGCAGGACAAGATGAAAATCCTCATGAAGCGCCAAGAAGAGAGAGGTATCCAG 43 8 HNF1A 353 TTAGTGACAGTGTCTACACCCCTCCACCAAGTGTCCCTCACGGGCCTGGAGCCCAGCCACAGCCTGCTGAGTACA 44 8 KNTC1 1517 CTGACGAGCACAAAAGATTTGGTCATCAGTCTTAGTGAAATACTACATAAGTTGGATCCTTATGACTATGAAATG 45 8 SBNO1 299 GGCTGGTTATCAGCATTGCAGCTTGAGGCAATTACACATGCAGCCCAGCAACATGAAACTTTCCTACCTAATGGA 46 8 ZNF268 40 CTCCAAGGTCAGGAATCCATCTTGGGCCAAGGGACTTCTGGTCTGCAACCTCTCCCTGGAACACCCAGGCAGAAG 47 8 RNF17 1333 GACATTCACATTATGGAGTTACCTAAAAATCCATGGAAGAAATTGTCTATTCACCTCTATTTTGATGGAATGTCA 48 8 CSNK1A1L 243 CAAAAATATGAAAAGATTAGTGAGAAGAAGATGTCCATCCCTGTTGAAGTTTTATGTAAGGGGTTTCCTGCAGAA 49 9 G2E3 670 GTGGATTTTCCTGTTGGAAACAAGTGTAATAACTGTGTAGCAATTCCCATCACCAATACATATAAAGAGTTTCAA 50 9 GARNL1 1201 AATACAATCATCAAACACTGCTCACCTCAATTTTTTTTACTTGGTTTGCCTGGTGCCACAATGCTTATTATGGAT 51 9 TTC6 152 CGAGGGCTTTGTAAAGTGAAACTCCACAAGGATAGCTTGATTCTGGATTTTAATCGTGCAATTACCCTCAATCCA 52 9 SIPA1L1 359 ATTATGAACAGGCACAATGTTATTAAGAGGAGAAACATCACCACTGGAGCTTCCGCAGCTGCCGTGGCATCCTTG 53 9 YLPM1 1342 TCTCTTCCACCTTTACCGCCCCTCCCACCTCTTCCACTTTTGGATAGATATCGGGATGATAGATGGAGAGAAGAA 54 9 TRIP11 80 AATGAAAGGCTTAAGAAACTTTGTACTGATCTAGAAAAGAAACATGAAGCATCAGAGATTCAAATAAAGCAGCAA 55 10 SERPINA9 58 AGTGCATACCCCCGCCCTTCCTCCACAAAGAGCACCTCTGCCTCACAGGTGTATTCCCTCAACACCGACTTTGCC 56 10 GABRG3 91 GTTAACAGCATTGGTCCTGTGTCATCAATAAACATGAAATACCAAATTGACATATTTTTTGCTCAGACCTGGACA 57 10 ADAL 240 GACAGAATCGGGCATGGAACATTTCTCAACTCCGGTAAGGGAGGATCCCTGGATCTGGTGGACTTTGTGAGGCAA 58 10 TRPM7 961 TTTGCAAATGCATATGATAATCATGTTTTTGTGGCTGAAAGATTAATTTACTGTCTTAACATAATATTTTGGTAT 59 10 LCTL 174 CTCCAGGTCAAATACGGTGGGTGGCAGAATGTGAGCATAGCCAACTACTTCAGAGACTACGCCAACCTGTGCTTT 60 10 IQCH 282 CGCATCAGGACCTCCAGGAGGACTATTATCCATATCCTATCATTAGGTATAACACTTTTGCCTGCAAATCTATCA 61 11 IQCH 455 CGCATCAGGACCTCCAGGAGGACTATTATCCATATCCTATCATTAGGGTATTCCCAGCCTGTGAGAGAACATATT 62 11 MAN2C1 202 GTGGATCTGGAGCTGCTGCTGGGCATAGCCAAGGGCTTCGGGAAGGACAACCAGCGCAGCTTCCAGGCCCTGTAC 63 11 CLDN9 171 GAGCTGGGGGCCTCCCTCTACCTGGGCTGGGCGGCGACTGCACTGCTTATGCTGGGCGGGGGGCTCCTCTGCTGC 64 11 CLDN6 188 CTGGGTGGGGGGTTGCTGTGCTGCACTTGCCCCTCGAGGGGGTCCCAGGGCCCCAGCCATTACATGGCCCGCTAC 65 11 MKL2 314 AAGTTAAAGTACCACCAATACATTCCACCAGATCAGGAGGGTGAGAAGAATGAGCCGCAGATGGACTCTAACTAC 66 11 EXOD1 362 AGATTAATGGTTTTGAAAGAATTGGAAATGTCAAGTTATGAAAACTTTGGAGACATAGAGGAAACTCCTCAAAAA 67 12 XPO6 265 CTGGCCCATCTCTTCAGTTGGATTCCTCTGTCTGCCGGCATCACCCCATCCCTCCTTACCACCATCTTCCACTTT 68 12 HYDIN 2425 AAGGAAGAGCTAAATAAGAAGAAAAGGAACATGGGCAATGTCAGCATGCATGGGCTTCCTCTTGTCCAGGACCAA 69 12 WDR59 58 AGTGGGAGCCCCACTCGCAGCGAGAAAGAGCAGGTCTTCATCAGCTCCTTCTACTACAAGGAGCGGAAATCAAGA 70 12 HSD17B2 199 CAATGCATGGCCGTGAACTTCTTTGGAACTGTGGAGATCACAAAGACGTTTTTGCCTCTTCTTAGAAAATCCAAA 71 12 FOXC2 125 AAGCAGGGCTGGCAGAACAGCATCCGCCACAACCTCTTGCTCAACGAGTGCTTCGTCAAGGTGCCCCGCGACGAC 72 12 CYB5D2 37 CTTATGGGCTGGTGGGGTCCCCGCGCTGGCTTTCGCTTTTTCATACCGGAGGAGCTGTCTCGCTACCGCGGCGGC 73 13 XPO6 255 GAGTATATCTGTTCCCTGGCTTTGGAGTGCCTGGCCCAGCTCTTCAGTTGGATTCCTCTGTCTGCCAGCATCACC 74 13 KIF1C 40 GTCAGCATGCAGGGCAACACCACCTCCATCATCAATCTTAAACAGAGCAAGGATGCCCCCAAAAGCTTCACCTTT 75 13 PITPNM3 111 TTGAGAAGACAGAGGTTCCCAGCCCAGGGAAGCATCAAGATCCACGAAGACAGCGAGGAAGGCTGCCCGCAGCGC 76 13 SLC13A5 453 CACGCAGTGCCCCCGGCAGCCATCACCTTGATCTTGTTCTTGCTCGTTGCCGTGTTCACTGAGTGCACAAGCAAC 77 13 ACADVL 307 CCCCCTGAGAAGAAGATGGGCATCAAGGCTTCAAACAGAGCAGAGGTGTTCTTTGATGGAGTACGGGTGCCATCG 78 13 DNAH2 2828 TCCTTCATTTTTGTGGACACCCAAATAGCTGATGAGTTCTTCCTAGAGGACATCAACAACATCCTCAGCTCAGGC 79 14 DNAH9 1138 ATAAAGAAGAGTGAGAGCGGCTTACTCAAGAAAGTTAAAAAAGGAGATTTCCAAGGCTTGGTTGAGATCATGGGA 80 14 LRRC37B 202 AAGAACCTGAAGAAAGATCTAGCTGAACGTTGGAGCTTTCCTGAGATTGTTGGGATTCCACACCAATTATCCAAA 81 14 KPNB1 140 GAACTCATTCCTCAGCTGGTGGCCAATGTCACAAACCTCAACAGCACAGAGCACATGAAGGAGTCGACATTGGAA 9 82 14 QRICH2 948 CTCTTTGACAGTCATGATTCAATGTATCCTGGTTATTGTGGCCCAGGGTATCTAAGTGCTGATCAGCATGGCCAG 83 14 ASPSCR1 303 CCCCAGCAGGAGCAGGAGCAGGAGCGGGAGCGGGATACCCAGCAGGAGCAGGAGCGGGAGCGGCCCGTGGACCGG 84 14 SLC14A2 339 GGGCTGCTAGCAGCCCTGTCAGTGGCCACACCCTTCAAGACCATCTACACAGGCCTCTGGAGCTACAACTGCGTC 85 15 SLC14A1 23 GACAGCCCCACTATGGTTAGGGGTGAAAACCAGGTTTTGCCATGTCAAGGGAGAAGGTGCTTCCCCAAAGCTCTT 86 15 LIPG 67 ACCTCCAAGGACCCAGAGCATGAAGGATGCTACCTCTTCGTCGGCCACAGCCAGCCCTTAGAAGACTGCAGTTTC 87 15 WDR7 829 GATGAAGTTTGCCTGGATCGCCTTGGAATGCTGAAACTCCACTGCACCGTATCGTTTGGCCTCTTGTCAAGAGGA 88 15 RAD23A 209 TATCTGCTCACGGGAATTCCTGGGAGCCCCGAGCCGAAACACGGTTCTGTCCAGGAGAGCCAGGTATCGGAGCAG 89 15 AKAP8L 511 CAGAAGCATCTGAAGACCATGGATCACAACCGGAACTGCAGGCTCATGATGGAGCAGTCCAAGAAGTCCTCCCTC 90 15 CYP2F1 102 GACCAGGGAGAGGAGTTTAGTGGCCGCGGTGACTACTCTGCCTTTTTCAACTTTACCAAGGGCAATGGCATCGCC 91 16 SIGLEC12 258 CCTCCTCAGAACTTGACCATGACTGTCTTCCAAGGAAATGGCACAGCATCCACAACCTTGAGGAATGGCTCGGCC 92 16 LILRA3 366 ACTTTCCTTTTGACCAAGGAGGGGGCAGCTGATTCCTCGCTGCGTCTAAAATCAAAGCGCCAATCTCATAAGTAC 93 16 LILRA2 76 AACAAATCAGCATCCTGGGTTAGACGGATACAAGAGCTTGGGAAGAATGGCCAGTTCCCCATCCCATCCATCACC 94 16 NLRP13 140 GCAGCAGGGAATATGCAGACCCAGGGATGCCAAGATTCAAACCAAGAAGAACTAGACGAGCTAGAAGAAGAAACA 95 16 USP29 88 TTGAAAAACAACGTGTTCTTGTTTATTGACAAATTATTCTACAGAGATGCTAAACAGTTGAATATGTTCCTGGAC 96 16 SNTG2 99 AGTATAAAGGGAGGTTCTGAGCACAACGTCCCTGTCGCCATATCAAAAATATTCGAAGACCAAGCAGCTGACCAG 97 17 ATP6V1C2 189 GATTCTGAATATCTCGTCACACTTCTGGTCATCGTCCTCAAACCAAACTACTCACAATGGCAAAAAACCTACGAA 98 17 DDX1 206 TGTTACCTGGATATAGATAAGGGACATGTCAAGTTCTTCAAAAATGGAAAAGATCTTGGTCTGGCATTTGAAATA 99 17 C2orf16 1402 TATTATCCAAAACAAAATGCCAGGGACTATTGCTTACTAAGCAGTATCAAAAGAGACAAGAGGTCAGCTGACAAG 100 17 SRD5A2 9 ATGCAGGTTCAGTGCCAGCAGAGCTCAGTGCTGGCAGGCAGCGCCACTTTGGTCGCCCTTGGG 101 17 LTBP1 427 TCAAGGGACAAATGTCAGTGCCCTCCAAATTTCACAGAAAAACTTTGTCAGATCCCAGTCCATGGTGCCAGCGTG 102 17 PELI1 117 GTAGGGTTCAACACACTAGCATTTCCTAGTATGAAGAGCAAAGACGTTGTAGATGAAAAACAACCATGGGTATAT 103 18 ADD2 333 TCCAGTGCCGGGGGAGTGGAGAACCTCATCCTCCTGAAGCAGGAGAAGCACCGGCCCCATGAGGTGGGCTCCGTG 104 18 NPAS2 790 TCGCTACTTCTCTCCACCTACTCACAACAGCCAGGGATCCTGGGCTACCCCCAACCACCCCCAGCACAGCCCCAG 105 18 SULT1C3 2 ATGGGGAAGATTGAGAAAAACGCTCCCACGATGGAAAAAAAG 106 18 IL1F7 137 GATAAAGGACAAAGTCATCCATCCCTTCAGCTGAAGAATGAGAAACTGATGAAGCTGGCTGCCCAAAAGGAATCA 107 18 PSD4 450 AGGAGCCCAGCTTCTTCTCCAGAGCCTAGCAGCCCAAAATCTGAGAGCAGAGGCCCTGGTCCCAGGCCCAGCCCT 108 18 ZRANB3 99 CCTTCGTCTCTGAGGTACCCTTGGACAGAAGAAATTAAAAAATGGATCCCAGAGCTAAGTCCAGAAGAAATCAAT 109 19 PSD4 421 ACTCTTAACTCCCAGGACAGAGAGCCTAGCAGCCCAAAATCTGAGAGCAGAGGCCCTGGTCCCAGGCCCAGCCCT 110 19 BAZ2B 313 GTGCTATTACATGGTATTTCAGACCCAAAAGCAGATAGACAGAAAGCAACTGAAAAAGCCCAGGAAAAAAGAATA 111 19 XIRP2 2019 GGCAATTTAGTAGAAGAAAGAACTGAGGTTAATCTTTCAAAAGCCCCCAAAGGCACTGTAAAGATTGTCATAGAT 112 19 CALCRL 109 CCATCAGAAAAAGTTACAAAGATCTGTGACCAAGATGAAAACTGGTTTAGACATCCAGCAAGCAACAGAACATGG 113 19 HIBCH 310 GTAATTAATAAAATGTCTCCAACATCTCTAAAGATCTCACTAAGGCAACTCATGGAGGGGTCTTCAAAGACCTTG 114 19 DNAH7 818 GGATTATATAAACTGGAGAAAACCTTTCATGATTCTCTATATGCATTGGCAATGACAAAAAAAGTAAGATCAAAG 115 20 ZDBF2 108 AAGGTTGAGGATGAGGATGCTACCGAAGAGAGACCATTCGAGGTTTCAGAACCTATTGAAGAGTTACATTCCAGA 116 20 FN1 1501 AGTGGGAGACCTCGAGAAGATCGGGTGCCCCACTCTTGGAATTCCATCACCCTCACCAACCTCACTCCAGGCACA 117 20 ALPP 29 CTACAGCTCTCCCTGGGCATCATCCCAGTTGAGGAGAAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCC 118 20 TRPM8 278 GGCTGTCATGGACATCCCACTGTCGAAGCAAAGCTCTGGAATCAGCTAGAGAAGTATATCTCTGAGCGCACTATT 119 20 CD93 204 GCCCTGGGGGGCCCAGGTCAGGTGACCTACACCACCTCCTTCCAGACCACCAGTTCCTCCTTGGAGGCTGTGCCC 120 20 BAGE2 76 ATCCTTGTGCTGCAGGAGCCGACACCTTTCAGGATTTTAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACC 121 21 TTC3 608 CTCAATCACTTTGAGAAAGCAAGAACCTTGATTTATTGTCTTCCTGGAGTGTTAACTTGGCCCACGAGTAATGTG 122 21 IGSF5 263 TTATCAAGTTTACCGAGTTTAGGTTTTTCATTGCCTATTTGGGGCAAAGTTGGACTTGGACTAGCAGGCACCATG 123 21 PCNT 176 CACCCACCAGAACAGCGTGGGATGTTCACAATCAGTAACCACCAACCGGAACAGCGTGGGATGTTCACAGTCAGT 124 21 MYO18B 193 TCTCCCCCCGCCACAGATACTGGAAAGGAAAAGAAAAGGGAGACCTCTAGGACTCCTTGTGGCTCCCAGGCCAGC 125 21 SEZ6L 90 GAAGTGCTGGGCGAGCTGGTGCTGGATGGGACCGCACTCTCTGCACATCACGACATCCCAGCCCTGTCACCGCTG 126 21 PATZ1 530 CTGAGGCAGGGCTGGACCACCCCAGAGGGCAGCAGGGTCTTTACCCAGTGGCCTGTTGGCTAG 127 22 NCF4 232 CCTCTCTCCTTCGTGAAGATCCTCAAAGACTTCCCTAAGGAGGACGACCCCACCAACTGGCTGCGTTGCTACTAC 128 22 SEPT3 193 ACAGGACACTCCTTGCGACCTCTGGATCTTGAGTTCATTAAACACCTCAGCAAGGTTGTGAACATCATCCCTGTC 129 22 TCF20 502 TCATCTTCCAAGAAAGCAGATAGCTGCACAAATTCTAAAGGCTCCTCACAACCTGAAGAACAGCTGAAGTCCCCT 130 22 SEC13 126 TTTGCATCAGGTGGCTGTGACAACCTCATCAAGCTGTCGAAGGAGGAGGAGGACGGCCAGTGGAAGGAGGAGCAG 131 22 ATP2B2 1103 TTCCTCAAGGAGGCAGGCAGGCTCACACAGAAGGAGAAGATCCCGGAGGAGGAGCTCAACGAGGACGTGGAGGAG 132 22 CMC1 92 GAATACCTGAAGGAAAGGGAAGAATTCAGAAAAACTGAAATTCCTACAAAGAAAAGGCTACAGAAGCTTCCAACA 133 23 CTNNB1 387 CAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAAACTTTCAGATGCTGCAACTAAACAGGAAGGGATGGAA 134 23 STAB1 1124 CACATCCTCAGCCAGGTCTTACTGCCCCCCCGAGGGAATGTGCCCGGTGGGCAGGGGTTGCTGCAGCAGCTGGAC 135 23 HHLA2 184 TGGAAAATGGACAACACACCTATCTCTGAAAACAACATAGAAGAAACAGGGTCTTTGGATTCTTTTTCTATTAAC 136 23 HGD 99 GAAGTTGATCCTGATCCTAACCAGCTTAGATGGAAACTATTTGAGATTCCAAAAGCATCTCAGAAGAAAGTAGAC 137 23 STXBP5L 789 AAAGTAAATCGCTGGGGTCCTGGAAGACCACCATTTCAAAAGGCCCAGTCAGCAGCCTGCATGGAGATTTCTTTA 138 23 MLF1 198 GATGAGGAGTGGCAAAGTGAGGTTTTGAAGTACAAATCAGGACGACACAATCTAGGAAACACTAGAATGAGAAGT 139 24 ATP13A5 159 CGGTTTCAGAAAGTTGGGTTGCTAGAAGACAGCAATTTCTGCTCTGACATCCATCAGACATTTGGATTGGGTCTG 140 24 MAN2B2 78 CGCGGCCAGCAGCGCCGGTTCATCGCTGTGGAGCAGAAGTTTTTCCGGCTGTGGTGGGATGGCGTCGCCTCGGAC 141 24 TBC1D1 441 ATTTTTGAAGAGGTTCAGAAATTGAGACCGAGAAATAAGCAGCGAGAGAATGAATTGATTATTTCTTTTCTGAGA 142 24 BMP2K 639 TTTGGAGAGGATAATTTCTCTAAGTTAACAGAAGAGGGACTATTGGACAGAGAATTTGACCTTCTAAGATCAAGT 143 24 C4orf22 91 CAGCAAAAGACGCTAACAAGTGCTGGTAAAGACCTACCAGATAATTTTCTGACGGCCCTGGCAATGAGAGAAGAA 144 24 C4orf22 117 AATCGCAGTGGAAAACTGAGTTCCGTGATCTTTATTTGTGACAGAAATTCTCATGGGCAAGAGATATCAGGATAC 145 25 AFF1 113 TTAATTCCTGACAAAGGGAGCAGCATTCCATCCAGCTTCTTCCACACTAGTGTCCACCACCAGTCCATTCACACT 146 25 MMRN1 819 GCAGGTATTCCCAGAGATGAGAAACTAAATCAGTCCAGCTTCCAAAAGATGTATCAAATGTTCAATGAAACCACT 147 25 CFI 115 AACGGAACATGCACAGCCGAAGGAAAGTTTAGTGTTTTCTTGAAGCATGGAAATACAGATTCAGAGGGAATAGTT 148 25 ENPEP 466 TTGATGTCTTCGCATCCAATTATTGTGACTGTGACAATCCCTGATGAAATAACATCTGTTTTTGATGGAATATCC 149 25 ALPK1 456 CTTATCTTGCATGGGCAAGGGGATTTCCAAAAAATCTTTGACACCTATTCACAGCACCATACTTCGGTGTGTGAA 150 25 DNAH5 1756 AATGTGTTTGACAACATTAAATCTGTCAAGTTCCACAAAAAGATCTATGATCGAATTCTGTCAATTTCCTCTCAA 151 26 ADAMTS6 116 ACAGATTTTGTGTCCAAACATTTTACAGTAGAATATTTGGGGAAAGATGGACCCCAGTGGAAACATGATTTTTTA 152 26 KIAA0686 1671 GTTTATTGGAAAGCATCACCAGACAGTGCTGGCCTGAAAGACTTTAAACCATCTCATGGGATTCTTGAATTTGCA 153 26 CDC23 363 GCCTTATATTTCCAGAGAGCCCTGAAATTAAATCCTTGGTATCTTGGTGCCTGGACACTAATGGGACATGAGTAC 154 26 PCDHB7 240 GTGCGCATTCTGGTTCTAGACGTAAATGACAACGCCCTTGATTTTGTGCGGTCGCTCTACAAGGTGCAGGTGCCC 155 26 GABRB2 377 AACATCTTACTGAGCACTCTCGAGATAAAAAATGAAATAGCCACATCTGAGGCTGTGATGGGACTTGGAGACCCC 156 26 KIF13A 309 GGTAAAAGCAAATTTGTGCCTTATCGAGATTCAGTCTTCACTTGGCTGCTTAAGGACAACTTGGGGGGCAACAGC 157 27 MDC1 1776 GCTGAATCCCTTACAGCCATTCCTGAGCCTGCCTCTCTCCAGCTTCTTGAGACACCAATTCATGCCTCCCAGATC 158 27 ZNF318 2245 CTGAATTTGGTTAAAGCTCCAGTGTCAAGGTCCCCTTCAAGGGAGCAGGTAATTGAAGACAATATGGTCCCTCAG 159 27 GPR115 66 CTTCTGAATTATCTTGTCATCCCATCAAAGGCCCAAGGTAACAAACCAGGCTACATCCCTAACCTAGGAGAATGT 160 27 PRIM2 227 GTGGCAATCATCCTGAATGAATTTAGAGCCAAACTGTTCAAGGCTTTGGCAGTGAGTATTTTACTTGATTTCTGT 161 27 DDX43 50 ACAGGTCCTGAGGGATATAGTGTCGGCAGAGGTGGTTGCTGGAGAGGCACCTCTAGGCCCCCGGAGGCCGTGGCC 162 27 COL12A1 1082 AAGATGGGAGAAGGAAAGCTTAGGCAAGGATCAGGAGCAACAGCTTCTCGGTTTAAGTCTCCTAGAAACCTCAAA 163 28 PRIM2 227 GTGGCAATCATCCTGAATGAATTTAGAGCCAAACTGTTCAAGGCTTTGGCATTAACAGCCAGGTCCTTGCCTGCT 164 28 MCHR2 266 CTGGTGCTGGTGGTAGTCTTTATCCTGAGTGCTGCCCTTTATCATGTGATACAACTGGTGAACTTACAGATGGAA 165 28 RTN4IP1 149 TTCAAGCCTGGAGATGAGGTCTGGGCTGCAGTTCCTTCTTGGAAACAAGGCACTCTTTCAGAGTTTGTTGTAGTC 166 28 AK124171 245 AGACTCGAAGAAGAAAATCGAAGGCTACTGGAACTTATAAAAGTGAAGGCAAAAGAAGCTGAAGAGACTGATAAT 167 28 ROS1 960 CCAGATTCTGTTCAAGAGTCTTCATTTAGGATTGAAAGAAATGCTTCAAGTTTTCAAATCCTGTGGAATGGTCCC 168 28 TRDN 705 TACTTGGATGGGTACAATGGCTATGGATTTCAGTTTTCTTTCACTCCTGCAGACCGCCCTGGAGAGAGCTCTGGT 10 169 29 LRP11 369 ACGGCAGCTAGTCCTGCCCTGCCAAGAACCACAGGGTCGAGTGAAGATGCAGGGGGTGACTCCTTGGTGGAAAAG 170 29 MAS1 294 TTTGTGGGAAGCAGTAAGAAGAAGAGATTCAAGGAGTTCTTAAAAGTTGTTCTGACCAGGGCTTTCAAAGATGAA 171 29 SLC22A2 199 AATGCTGCAGCTGGAGTTCTCATGGCCATTTCCCCAGCCTATACGTGGATGTTAATTTTTCGCTTAATCCAAGGA 172 29 CARD11 756 AGCGGCCCCGTCACGCTGCACTACAAGGTCAACCACAAAGGGTACCGGAAGCTGGTGAAGGACATGGAGGACGGC 173 29 SEMA3D 209 GATGAGTACCTCTACTCTGGAACAGCTTCTGATTTCTTTGGCAAAGATACTGCATTCACTCGATCCCTTGGGCCT 174 29 TRRAP 122 ACAAAAAATGTTTTGTCTGTGATGTTTCGCTTTTTAAAGACGGAAAATGAAGAAAATGTTCTTATTTGTCTAAGA 175 30 ORC5L 57 TATGTAACACAAACGTTGTTGAAAACTTTAGAGCTCCAACATGTGTTTGTGAATTGTGTTGAATGCTTTACATTG 176 30 DOCK4 506 ACAAAGGAGAAAGGAGAGAAGAAGTTGTTTGGGTTTTTTTTTGTCCCTCTGATGCAAGAAGATGGTAGGACTCTT 177 30 GRM8 649 TTTTTAATGATTGCAGCACCAGATACAATCATATGCTTCTTCCGACGGGTCTTCCTAGGACTTGGCATGTGTTTC 178 30 IRF5 416 AACACCCCACCACCCTTCGAGATCTTCTTCTGCTTTGAGGAAGAATGGCCTGACCGCAAACCCCGAGAGAAGAAG 179 30 ATP6V0A4 181 AAGTTGGGGTTCATAGCCGGTGTGATCAACAGGGAGACGATGGCTTCCTTTGAGCGGTTACTGTGGCGAATCTGC 180 30 DENND2A 852 GACCTCGTCAACAGCCGGTTCCTCAGACAGATGGACAATGAGGACTCCATCCTGCCCCGGAAGCTTCAGGTGGCC 181 31 BRAF 600 CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAG 182 31 TCRBV6S1A1N1 18 CTCTGCTGGGCAGCCCTGTGCCTCCTGGGGGCAGATTACACAGGTGCTGGAGTCTCCCAGACCCCCAGTAACAAG 183 31 SGCZ 131 CAGTCTGACAGGAATGTCACAGTGAATGCAAGAAATTACATGGGGCAGTTAACCGGACAGCTGACCATAGGAGCT 184 31 PRKDC 70 GTTTTTTCCAGAGATTTCGGTTTGCTTGTATTTGTCTGGAAGTCACTCAACAGTATTGAATTTCGTGAATGTAGA 185 31 ZFHX4 2527 CACTTCCTTGCTGCTCAAAACCAATTCCTTCACTCTCTGTTCTTGGAAAGGCCCATGGACATGCCCTACATGATA 186 31 CALB1 199 TGTGGGAAAGAGTTCAATAAGGCTTTTGAGCTGTATAATCAGGACGGCAATGGATACATAGATGAAAATGAACTG 187 32 EIF3E 170 TCTCTTCAGCAGAGAACATGGCTCATTCACTGGTCTCGGTTTGTTTTCTTCAATCACCCCAAAGGTCGCGATAAT 188 32 CDKN2A 135 CGCGGAGCCCAACTGCGCCGACCCCGCCACTCTCACCTGACCCGTGCACGACGCTGCCCGGGAGGGCTTCCTGGA 189 32 SLC28A3 22 AGAGCTGAGGGCTACAGCAACGTGGGCTTCCAGAATAAAGAAAACTTTCTTGAGAACGAGAACACATCAGGAAAC 190 32 FAM120A 764 GATCAGCTCCAGGAGCTCAAGATTGAGAACCTAGATCTCCGAGGAATTCAGCTATCAGCTCTCTTCATGAGTGGA 191 32 GRIN3A 434 GTGGAAACTACAAATCTCACTTCAGGACAATATTTATTAAGGTTTCTAGCCAATACCACTTTCAGAGGCCTCAGT 192 32 SEC16A 604 TATGATGGTGCTGCGTCTGCTTACGCCCAGAACTACAGCTATCCCGAGCCCGAGCGGCCCAGCTCCCGAGCCAGC 193 33 EIF1AX 7 ATGCCCAAGAATAAAGGTGAAGGAGGTAAAAACAGACGCAGGGGTAAGAATGAGAAT 194 33 PHF8 114 GTGATTCTGAAGCCCACTGGAAATCAACTGACCGTGCAATTCCTGGAAGAAAATAGCTTCAGTGTGCCCATCCTG 195 33 CYLC1 378 CCAGAGTCTACTGATACTGAATCAGGAGATGCAAAGAATGCAAGAAATGATTCAAGAAATTTGAAGAAAGCTTCA 196 33 SERPINA7 122 CATGGCTTCCAGCATCTGATCTGTTCACTGAATTTTTCAAAGAAGGAACTGGAATTGCAGATAGGAAATGCCCTC 197 33 GABRE 115 TTTTCCTATCCTGAGAATGAGATGATCTACAAGTGGAAAAATTTCAAGCTTGAAATCAATGAGAAGAACTCCTGG 198 33 SLC2A7 484 AAAACATTTGTGGAGATAAACCGCATTTTTGCCAAGAAAAACAGGGTGAAGCTTCCAGAGGAGAAAGAAGAAACC 199 34 TAF3 579 ACTGGCAGGGAAACAAAGTATCCCTGGAAGGAATTTTTTAAAGAGGAAGAGGCAGATCCCTACAAGTTTAAAATC 200 34 RNF17 525 CTTTCAAAGAGACAGGAGTTACCTAAAAATCCATGGAAGAAATTGTCTATTCACCTCTATTTTGATGGAATGTCA 201 34 DACH1 439 TCCAGAGTTGAGACATCAGTTATTAAGGAGCGTGTTTCTGATAGCCCCTCACCTGCCCCCTCTCTGGAGGAGGGG 202 34 MAP2K1 176 CAAATTTTAGGAAAAGTTAGCATTGCTGTAATAAAAAGCCTGACATATCTGAGGGAGAAGCACAAGATCATGCAC 203 34 ABCA10 427 TTTGACATATATGAAGGACAGATCACTGCAATACTTGAGCATAATGGAGCTGGTAAATCAACACTGCTAAACATT 204 35 LOC391343 183 CACCAGACGCAGTGCAGGCGAGGAGGCCGGGAGGAACTGAAAGACCAGACCCAGTGCAGGCGAGGAGGCTGGGAG 205 35 BIRC6 3210 TCCTACATCTTTCTTCCAGAGGAGGCTTGGTGTGACTTTACCATTCACCTTCCTGCAGCAGTGCTGCTTAAGGAG 206 35 BIRC6 3744 GGTGCACAACAGACCAGTGCAAGATCAGCTTCTCTTTTTTCAGCTGCTACAACAGGACTGACTACTCAACAGCGC 207 35 KIAA1697 712 CTTCTTTCTGTGTCAAAGACATTTTTCTCACAAGTCAATGCTGGAAATGAAGAACTGAAAGAAAAGCTTCCCTTG 208 35 KIAA1697 2208 CACTGCCCACTTTATAAAACAGGAGCCCGGGCAGGAGCACTCTCAACCACAGGACATTCAACCAATTTTGTGGTA 209 36 MUC20 375 TCAGCCCTCTCTGTTGAGACACCAAGTTACGTCAAAATCTCAGGAGCAGCTCCGGTCTCCATAGAGGCTGGGTCA 210 36 C4orf22 134 ATCTTGAAGAAGCAACTGGCATCCGTGATCTTTATTTGTGACAGAAATTCTCATGGGCAAGAGATATCAGGATAC 211 36 TXLNB 2 ATGAAGGCTAATCACTCTGAACAGCTCTCAGCGGAACGACAG 212 36 PRUNE2 972 CCTTCCCCCTTAGATACCAATTATTCCACCTCAGACACTTACACATCACCAACATTTGCTGGAGACGAAAAGGAA 213 36 TTN 30410 GATTATTATGCTCTGCACATCAGGGACACTTTGCCTAAAGACACGGGTTATTATAGAGTCACAGCCACTAACACA 214 37 TTN 10455 AAGCATTCAATGGTGATCAAGTCAGCTGCTTTTGAAAATGAAGCAAAATACATGTTTGAAGCTGAAGATAAGCAC 215 37 TTN 1445 TCCCCTGCAAGAATGTCCCCTGGACGTAGGCTGGAGAAGACAGATGAGTCACAACTTGAGAGACTATATAAACCA 216 37 MUC16 8613 ACCACAGACATGTTGCGCACAAGCTCAGAACCTGAAATCAGTTCACCTCCAAATTTGAGCAGCACCTCAGCTGAA 217 37 MUC16 3435 TATTCATCAACTAGTTCTTGGTCAGATCAGACATCTTGGAGTGACATCACCCTTGGTGCATCTCCTGATGTCACA 11 11 Supplementary Table S4. Authors’Affiliations: Candidate binding peptides for mutated POLA2. Amino acid position Mutated Peptide Predicted HLA-C0701 binding affinity (nM) Co-culture result [IFN- (pg/mL)] 413-422 TRSSGSHFVF 147.35 1106 413-423 TRSSGSHFVFV 280.38 50 413-421 TRSSGSHFV 285.90 60 413-420 TRSSGSHF 518.82 48 420-429 FVFVPSLRDV 599.44 39 Supplementary Table S4. Candidate binding peptides for mutated POLA2. 12
https://openalex.org/W3160349507	https://www.research-collection.ethz.ch/bitstream/20.500.11850/511954/2/MolecularEcologyResources-2021-Bohmann-StrategiesforsamplelabellingandlibrarypreparationinDNAmetabarcoding.pdf	English	null	Strategies for sample labelling and library preparation in DNA metabarcoding studies	Molecular ecology resources	2,021	cc-by	16,089	ETH Library ETH Library Author(s): Author(s): Bohmann, Kristine; Elbrecht, Vasco; Carøe, Christian; Bista, Iliana; Leese, Florian; Bunce, Michael; Yu, Douglas W.; Seymour, Mathew; Dumbrell, Alex J.; Creer, Simon Publication date: 2022-05 Rights / license: g Creative Commons Attribution 4.0 International Originally published in: Molecular Ecology Resources 22(4), https://doi.org/10.1111/1755-0998.13512 Review Article Author(s): Bohmann, Kristine; Elbrecht, Vasco; Carøe, Christian; Bista, Iliana; Leese, Florian; Bunce, Michael; Yu, Douglas W.; Seymour, Mathew; Dumbrell, Alex J.; Creer, Simon Author(s): Bohmann, Kristine; Elbrecht, Vasco; Carøe, Christian; Bista, Iliana; Leese, Florian; Bunce, Michael; Yu, Douglas W.; Seymour, Mathew; Dumbrell, Alex J.; Creer, Simon Received: 28 April 2021 \| Revised: 7 September 2021 \| Accepted: 14 September 2021 DOI: 10.1111/1755-0998.13512 I N V I T E D T E C H N I C A L R E V I E W Strategies for sample labelling and library preparation in DNA metabarcoding studies Kristine Bohmann1 \| Vasco Elbrecht2 \| Christian Carøe1 \| Iliana Bista3,4 \| Florian Leese5 \| Michael Bunce6 \| Douglas W. Yu7,8,9 \| Mathew Seymour10 \| Alex J. Dumbrell11 \| Simon Creer12 1Faculty of Health and Medical Sciences, Section for Evolutionary Genomics, Globe Institute, University of Copenhagen, Copenhagen, Denmark 2Department of Environmental Systems Science, ETH Zurich, Zürich, Switzerland 3Department of Genetics, University of Cambridge, Cambridge, UK 4Tree of Life, Wellcome Sanger Institute, Hinxton, UK 5Aquatic Ecosystem Research, Faculty of Biology, University of Duisburg-Essen, Essen, Germany 6Trace and Environmental DNA (TrEnD) Laboratory, School of Molecular and Life Sciences, Curtin University, Perth, WA, Australia 7State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China 8School of Biological Sciences, Norwich Research Park, University of East Anglia, Norwich, UK 9Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming Yunnan, China 10Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden 11School of Life Sciences, University of Essex, Colchester, UK 12Molecular Ecology and Evolution Group, School of Natural Sciences, Bangor University, Gwynedd, UK Received: 28 April 2021 \| Revised: 7 September 2021 \| Accepted: 14 September 2021 DOI: 10.1111/1755-0998.13512 Received: 28 April 2021 \| Revised: 7 September 2021 \| Accepted: 14 September 2021 DOI: 10.1111/1755-0998.13512 I N V I T E D T E C H N I C A L R E V I E W I N V I T E D T E C H N I C A L R E V I E W 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. © 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample- specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during “library preparation”, that is, when amplicons are prepared for sequencing. Different strate- gies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using me- tabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodi- versity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample- specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Mol Ecol Resour. 2022;22:1231–1246. y cited. ogy Resources published by John Wiley & Sons Ltd. he terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, y cited Funding information NERC Biomolecular Analysis Facility, Grant/Award Number: NE/M02086X/1, NE/N003756/1, NE/N006216/1, NE/ S000291/1, NE/S005560/1 and NE/ S006958/1; Carlsbergfondet, Grant/ Award Number: CF18-1110; H2020 European Research Council, Grant/ Award Number: 856488; Det Frie Forskningsråd, Grant/Award Number: 5051-00140; Wellcome, Grant/Award Number: WT207492, 104640/Z/14/Z and 092096/Z/10/Z Strategies for sample labelling and library preparation in DNA metabarcoding studies Kristine Bohmann1 \| Vasco Elbrecht2 \| Christian Carøe1 \| Iliana Bista3,4 \| Florian Leese5 \| Michael Bunce6 \| Douglas W. Yu7,8,9 \| Mathew Seymour10 \| Alex J. Dumbrell11 \| Simon Creer12 Kristine Bohmann1 \| Vasco Elbrecht2 \| Christian Carøe1 \| Iliana Bista3,4 \| Florian Leese5 \| Michael Bunce6 \| Douglas W. Yu7,8,9 \| Mathew Seymour10 \| Alex J. Dumbrell11 \| Simon Creer12 12Molecular Ecology and Evolution Group, School of Natural Sciences, Bangor University, Gwynedd, UK Originally published in: Molecular Ecology Resources 22(4), https://doi.org/10.1111/1755-0998.13512 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. 1 \| INTRODUCTION main approaches; one-step PCR, two-step PCR, and tagged PCR (Figure 2). All three approaches have advantages, challenges, and limitations which, if not considered, can result in misleading data in- terpretation, and in the very worst case can lead to unusable data and considerable wasted time and money, as for instance in the case of the so-called “tag-jumps” (Carøe & Bohmann, 2020; Esling et al., 2015; Schnell et al., 2015). Despite this, in contrast to discussions on metabarcoding substrate selection, DNA extraction, and data pro- cessing, the strategies for amplicon labelling and library preparation workflows have received little systematic attention in the metabar- coding literature (although see Murray et al., 2015). 1 In recent years, the analysis of environmental DNA (eDNA) and DNA extracted from bulk specimen samples has experienced an enor- mous surge in popularity in basic and applied biodiversity studies seeking to detect e.g., animal, plant, algae, fungi, and bacteria (Bálint et al., 2016; Compson et al., 2020; Creer et al., 2016; Jarman et al., 2018; Lindahl et al., 2013; Taberlet et al., 2012). Within the field of genetic biodiversity assessment, DNA metabarcoding is currently the most widely used approach, as it allows targeted, parallel, and as such relatively cost-effective, identification of multiple taxa from environmental samples, such as soil, water, and faeces, as well as from bulk samples of organisms (Taberlet, Coissac, Pompanon, et al., 2012). Here, the applications of metabarcoding range widely; for example, detection of invasive species (e.g., Pochon et al., 2013); assessment of water quality via identification of freshwater inver- tebrates in bulk specimen samples (e.g., Elbrecht et al., 2017) and environmental samples (e.g., Seymour et al., 2020); identification of plant-pollinator interactions (e.g, Gous et al., 2019; Lucas et al., 2018); detection of vertebrate wildlife via invertebrate “samplers” of vertebrate blood or faeces (e.g., Calvignac-Spencer et al., 2013), and assessment of for example, niche partitioning (e.g., Razgour et al., 2011) and ecosystem services (e.g., Aizpurua et al., 2017) through detection of diet items. Furthermore, metabarcoding is explored for implementation in routine biomonitoring around the world (Aylagas et al., 2018; Li et al., 2018, 2019; Pont et al., 2018, 2021; Zizka et al., 2020; www.danubesurvey.org; www.syke.fi), and is an integral com- ponent of the proposals for the Next Generation of Biomonitoring programmes (Bohan et al., 2017). 1 \| INTRODUCTION Here, we present an overview of the three most commonly used workflows with which to achieve sample-specific labelling and li- brary preparation in metabarcoding studies, and how they can po- tentially influence the resulting data. For the sake of simplicity, we mainly focus on metabarcoding of plants and animals in basic and applied biodiversity studies with sequencing on arguably the most used high-throughput sequencing platform series today, the Illumina sequencing platforms. Note that points raised will be relevant for metabarcoding of other organisms and to high-throughput sequenc- ing platforms with similar labelling structures to Illumina platforms, such as Ion Torrent (Thermo Fischer Scientific), BGI platforms (BGI Genomics), Oxford Nanopore Technologies MinION, and PacBio (Pacific Biosciences). In the present article, we provide critical considerations for researchers to choose the optimal metabarcod- ing strategy for generating reliable data tailored to their individual study; for example, regarding sample type and number, research question, speed of laboratory processing, contamination risk, bud- get, and whether similar studies are to be carried out in the labo- ratory. Ultimately, by gaining detailed and critical insights into the consequences of choosing different metabarcoding workflows, we hope to further increase the potential of metabarcoding as a reliable tool for use across a wide range of applications. Metabarcoding relies on PCR amplification of extracted DNA with primers designed to target a taxonomically informative marker for a selected taxonomic group (Taberlet, Coissac, Pompanon, et al., 2012) (Figure 1). The backbone of metabarcoding analyses is the addition of sample-specific nucleotide identifiers to amplicons and the use of these to assign metabarcoding sequences back to the samples they originated from (“demultiplexing”). This allows pooling of hundreds to thousands of samples for sequencing and utilisation of the capacity of high-throughput sequencing platforms (Figure 1). Amplicon labelling can be achieved at two stages during a metabar- coding workflow: prior to library build, as 5′ nucleotide “tags” on metabarcoding primers, and during library build as library indices. The strategies to achieve this labelling can be categorised into three K E Y W O R D S K E Y W O R D S amplicon sequencing, biodiversity assessment, eDNA, environmental DNA, high-throughput sequencing, Illumina sequencing, library preparation \| 1232 BOHMANN et al. distill the key considerations for researchers seeking to select an appropriate meta- barcoding strategy for their specific study. Ultimately, by gaining insights into the con- sequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications. Funding information Funding information NERC Biomolecular Analysis Facility, Grant/Award Number: NE/M02086X/1, NE/N003756/1, NE/N006216/1, NE/ S000291/1, NE/S005560/1 and NE/ S006958/1; Carlsbergfondet, Grant/ Award Number: CF18-1110; H2020 European Research Council, Grant/ Award Number: 856488; Det Frie Forskningsråd, Grant/Award Number: 5051-00140; Wellcome, Grant/Award Number: WT207492, 104640/Z/14/Z and 092096/Z/10/Z 1231 wileyonlinelibrary.com/journal/men Mol Ecol Resour. 2022;22:1231–1246. \| 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES Today, the most commonly used high-throughput sequencing platform for metabarcoding studies is the Illumina series, where for example the MiSeq, iSeq, HiSeq, NextSeq, and NovaSeq have \| BOHMANN et al. 1233 \| been employed (Jarman et al., 2018). These platforms offer high throughput relatively low error rates and relatively long paired-end 2017; Hope et al., 2014; Quéméré et al., 2013; Shehzad, Riaz, et al., 2012; Singer et al 2019; Stoeck et al 2018) FI G U R E 1 Simplified overview of a metabarcoding workflow. (a–b) DNA extracted from environmental samples such as soil, water, and faeces or from bulk specimen samples. The DNA extracts are typically a complex mix of DNA from target and nontarget organisms. (c) DNA extracts are PCR-amplified with metabarcoding primers that target a taxonomically informative marker for a taxonomic group. Importantly, identifiers unique to each PCR product are added in the form of 5ʹ nucleotide tags on primers and/or as indices added to sequence libraries during library build. (d) The taxonomic markers of hundreds to thousands of samples are sequenced in parallel on a high- throughput sequencing platform producing millions of sequence reads. (e) The sequences can be traced back to the samples they originated from through the nucleotide tags and/or library indices, and (f) can be further analysed. Images courtesy of the Integration and Application Network, University of Maryland Centre for Environmental Science (ian.umces.edu/symbols/) and Illumina.com (c) (d) (e) (f) (a) Environmental or bulk specimen samples DNA from target taxa, e.g. DNA from non-target taxa, e.g. bacteria, fungi PCR amplification with primers for a taxonomically informative marker for a taxonomic group, e.g. generic vertebrate, plant, and insect primers Sample n Unique identifiers attached to each PCR product - 5’ nucleotide tags and/or library indices Demultiplexing by informatically tracing sequences back to samples through the nucleotide tags and/or library indices 1 2 3 Sample 1 Sample 2 Sample n Sample 2 Sample 1 Sample n Sample 1 Sample 2 Sample n 3 3 2 2 1 2 1 3 2 2 3 3 3 3 3 DNA extract Metabarcoding Tracing sequences back to samples High-throughput sequencing Further sequence processing and taxonomic assignment (b) (a) Environmental or bulk specimen samples DNA from target taxa, e.g. DNA from non-target taxa, e.g. bacteria, fungi Sample n DNA extract (b) Sample n (c) PCR amplification with primers for a taxonomically informative marker for a taxonomic group, e.g. 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES index PCR Library sequence F R i5 i7 t t OR Adapter ligation Adapter Index primers PCR i5 i7 t t Two-step PCR - tagged R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs and tags Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Primers with sequence overhangs and tags PCR 1 DNA extract F Sample 1 t t t t t t DNA extract Primers with sequence overhangs F PCR 1 R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs Two-step PCR - untagged Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Sample 1 DNA extract Library sequence Sample 1 F R Sample 1 F PCR R Fusion primers One-step PCR with fusion primers Library sample 1 Library sample 2 Pooling t t } } } Insert nucleotide tagged PCR product Sequence read 1 (r1) Sequence read 2 (r2) 5’ 3’ 5’ 3’ Metabarcoding primer Adapters and indices added during library build 5’ nucleotide tag Sequencing adapter Library index Flow cell bind sequence Metabarcoding marker i7 Forward Reverse i5 t Metabarcoding marker i7 Forward Reverse i5 Adapters and indices added during library build t t t Index read 2 Index read 1 Structure of dual-tagged and dual-indexed Illumina metabarcoding library sequence (a) Strategies for addition of sample-specific identifiers and library build in metabarcoding studies } } } Insert nucleotide tagged PCR product Sequence read 1 (r1) Sequence read 2 (r2) 5’ 3’ 5’ 3’ Metabarcoding primer Adapters and indices added during library build 5’ nucleotide tag Sequencing adapter Library index Flow cell bind sequence Metabarcoding marker i7 Forward Reverse i5 t Metabarcoding marker i7 Forward Reverse i5 Adapters and indices added during library build t t t Index read 2 Index read 1 Structure of dual-tagged and dual-indexed Illumina metabarcoding library sequence (a) Strategies for addition of sample-specific identifiers and library build in metabarcoding studies } } } Insert nucleotide tagged PCR product Sequence read 1 (r1) Sequence read 2 (r2) 5’ 3’ 5’ 3’ Metabarcoding primer Adapters and indices added during library build 5’ nucleotide tag Sequencing adapter Library index Flow cell bind sequence Metabarcoding marker i7 Forward Reverse i5 t Metabarcoding marker i7 Forward Reverse i5 Adapters and indices added during library build t t t Index read 2 Index read 1 tructure of dual-tagged and dual-indexed Illumina metabarcoding library sequence Structure of dual-tagged and dual-indexed Illumina metabarcoding library sequence (a) Metabarcoding primer 5’ nucleotide tag Sequencing adapter Library index Flow cell bind sequence Sequence read 2 (r2) Sequence read 1 Index read 2 Index read 1 Strategies for addition of sample-specific identifiers and library build in metabarcoding studies Strategies for addition of sample-specific identifiers and library build in metabarcoding studies (b) (c) (e) (d) Sample 1 Tagged PCR and library build on amplicon pool DNA extract Primers with tags F PCR R t t F R Tagged PCR amplicon Tagged PCR product sample 1 Tagged PCR product sample 2 Pooling Library build on amplicon pool Incl. 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES index PCR Library sequence F R i5 i7 t t OR Adapter ligation Adapter and index ligation Index primers PCR i5 i7 Library sequence F R i5 i7 t t t t Two-step PCR - tagged R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs and tags Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Primers with sequence overhangs and tags PCR 1 DNA extract F Sample 1 t t t t t t DNA extract Primers with sequence overhangs F PCR 1 R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs Two-step PCR - untagged Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Sample 1 DNA extract Library sequence Sample 1 F R Sample 1 F PCR R Fusion primers One-step PCR with fusion primers Library sample 1 Library sample 2 Pooling t t Strategies for addition of sample-specific identifiers and library build in metabarcoding studies (b) (c) (d) Two-step PCR - tagged R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs and tags Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Primers with sequence overhangs and tags PCR 1 DNA extract F Sample 1 t t t t t t DNA extract Primers with sequence overhangs F PCR 1 R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs Two-step PCR - untagged Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Sample 1 DNA extract Library sequence Sample 1 F R Sample 1 F PCR R Fusion primers One-step PCR with fusion primers Library sample 1 Library sample 2 Pooling t t Strategies for addition of sample-specific identifiers and library build in metabarcoding studies (b) (c) DNA extract Primers with sequence overhangs F PCR 1 R PCR 2 i5 i7 Library sequence Sample 1 F R F R i5 i7 PCR amplicon with sequence overhangs Two-step PCR - untagged Library sample 1 Library sample 2 Pooling Fusion primers with sequence complementary to overhang, adapters and indexes Sample 1 DNA extract Library sequence Sample 1 F R Sample 1 F PCR R Fusion primers One-step PCR with fusion primers Library sample 1 Library sample 2 Pooling t t One-step PCR with fusion primers Library sample 2 Library sample 1 Fusion primers Pooling PCR Sample 1 c) P Two-step PCR - untagged Fusion primers with sequence complementary to overhang, adapters and indexes Primers with sequence overhangs Library sample 2 Library sample 1 PCR 1 PCR 2 Pooling PCR amplicon with sequence overhangs Library sequence Sample 1 Sample 1 (d) Two-step PCR - tagged R PCR 2 i5 i7 Library Sa F R F i5 PCR amplicon with sequence overhangs and tags Fusion primers with sequence complementary to overhang, adapters and indexes Primers with sequence overhangs and tags PCR 1 DNA extract F Sample 1 t t t t t ) Two-step PCR - tagged R Primers with sequence overhangs and tags t Library sample 2 Library sample 1 PCR 2 PCR 1 Pooling PCR amplicon with sequence overhangs and tags Library sequence Sample 1 Sample 1 Sample 1 (e) Sample 1 Tagged PCR and library build on amplicon pool DNA extract Primers with tags F PCR R t t F R Tagged PCR amplicon Tagged PCR product sample 1 Tagged PCR product sample 2 Pooling Library build on amplicon pool Incl. 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES 1234 Structure of dual-tagged and dual-indexed Illumina metabarcoding library sequence (a) Strategies for addition of sample-specific identifiers and library build in metabarcoding studies (b) (c) (e) (d) Sample 1 Tagged PCR and library build on amplicon pool DNA extract Primers with tags F PCR R t t F R Tagged PCR amplicon Tagged PCR product sample 1 Tagged PCR product sample 2 Pooling Library build on amplicon pool Incl. 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES index PCR Excl. 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES generic vertebrate, plant, and insect primers Metabarcoding Unique identifiers attached to each PCR product - 5’ nucleotide tags and/or library indices Sample 2 Sample 1 Demultiplexing by informatically tracing sequences back to samples through the nucleotide tags and/or library indices Demultiplexing by informatically tracing sequences back to samples through the nucleotide tags and/or library indices Sample n FI G U R E 1 Simplified overview of a metabarcoding workflow. (a–b) DNA extracted from environmental samples such as soil, water, and faeces or from bulk specimen samples. The DNA extracts are typically a complex mix of DNA from target and nontarget organisms. (c) DNA extracts are PCR-amplified with metabarcoding primers that target a taxonomically informative marker for a taxonomic group. Importantly, identifiers unique to each PCR product are added in the form of 5ʹ nucleotide tags on primers and/or as indices added to sequence libraries during library build. (d) The taxonomic markers of hundreds to thousands of samples are sequenced in parallel on a high- throughput sequencing platform producing millions of sequence reads. (e) The sequences can be traced back to the samples they originated from through the nucleotide tags and/or library indices, and (f) can be further analysed. Images courtesy of the Integration and Application Network, University of Maryland Centre for Environmental Science (ian.umces.edu/symbols/) and Illumina.com 2017; Hope et al., 2014; Quéméré et al., 2013; Shehzad, Riaz, et al., 2012; Singer et al., 2019; Stoeck et al., 2018). been employed (Jarman et al., 2018). These platforms offer high throughput, relatively low error rates, and relatively long paired-end reads, typically up to 150 bp of each paired read on the iSeq100, NextSeq550/1000/2000, HiSeq 3000/4000, and NovaSeq (up to 250 bp on SP flow cell), and 300 bp of each paired read on the MiSeq platform (www.illumina.com, applied in e.g., Elbrecht et al., The sequencing depth required per sample is commonly much lower in metabarcoding studies than in shotgun sequencing studies (e.g., Srivathsan et al., 2015; Stat et al., 2017), and in metabarcoding studies it is (economically) feasible to sequence tens, hundreds, or BOHMANN et al. \| (2017), Hardy et al. (2017), Elbrecht and Steinke (2018), Seersholm et al. (2018), and Bessey et al. (2020). If indices are used, then each PCR replicate or sample is a sequencing library and as such is returned as a separate fastq file fol- lowing sequencing. It should be noted that most studies add nucleotide tags next to the primers thereby eliminating the need for i5 and i7 “indexing”. 1. The “one-step PCR” approach in which sample DNA extracts are amplified and built into sequence libraries in one reac- tion. Here, metabarcoding primers carry sequencing adapters, nucleotide tags, and/or library indices, referred to as “fusion primers” (Figure 2b). This approach is used in for example, Kozich et al. (2013), Elbrecht and Leese (2015), Sickel et al. (2015), Grealy et al. (2016), Berry et al. (2017), Elbrecht et al. (2017), Hardy et al. (2017), Elbrecht and Steinke (2018), Seersholm et al. (2018), and Bessey et al. (2020). If indices are used, then each PCR replicate or sample is a sequencing library and as such is returned as a separate fastq file fol- lowing sequencing. It should be noted that most studies add nucleotide tags next to the primers thereby eliminating the need for i5 and i7 “indexing”. For all three strategies, it is important to carefully design tags and indices to ensure that oligonucleotide synthesis, PCR, and se- quencing error will not cause them to be unidentifiable or confused (Coissac, 2012; Faircloth & Glenn, 2012). Further, all three strategies offer the option to add extra nucleotides to shift PCR amplicons in relation to each other and thereby to increase sequence complexity on the flow cell (“heterogeneity spacers”, see for example, Bohmann et al., 2018; De Barba et al., 2014; Elbrecht & Leese, 2015). 2. The two-step PCR approach in which sample DNA extracts are PCR-amplified with two primer sets. In the primary reaction the metabarcoding primers carry 5′ sequence overhangs of c. 33– 34 nucleotides in length. These can be with (Clarke et al., 2017; Griffiths et al., 2020; Kitson et al., 2018; Li et al., 2019; Vesterinen et al., 2018) or without (Bista et al., 2017; de Vere et al., 2017; Galan et al., 2017; Miya et al., 2015; Swift et al., 2018; Vesterinen et al., 2018) nucleotide tags (Figure 2c,d). \| Most commonly, two consecutive PCRs are carried out, such as in Miya et al. (2015), de Vere et al. (2017), Galan et al. (2017), Kaunisto et al. (2017), Swift et al. (2018), and Vesterinen et al. (2018). However, a few studies carry out only one reaction with the two primer sets, such as Clarke, Czechowski, et al. (2014). The two-step PCR approach is based on Illumina's 16S rRNA system originally developed for microbiome studies (www.illumina.com). If unique ndexing is used on PCR replicates in the two-step ap- proach, each PCR replicate is an individual sequencing library and as such is returned as a separate fastq file following sequencing. 3. The “tagged PCR” approach, in which sample DNA extracts are PCR amplified with metabarcoding primers that carry 5′ nucleo- tide tags. Following PCR amplification, the individually tagged PCR products are pooled, and ligation-based library preparation is carried out on pools of 5′ tagged amplicons. The ligated adapt- ers can themselves contain indices, which eliminates the need for a second PCR step (e.g., Carøe & Bohmann, 2020; Thomsen et al., 2016), or the adapter ligation can be followed by a PCR step with indexed primers (e.g., Bohmann et al., 2018; Hope et al., 2014). This approach was first demonstrated by Binladen et al. (2007) on the 454 FLX platform and has since been used in for example, Shehzad, McCarthy, et al. (2012), Hibert et al. (2013), Hope et al. (2014), Thomsen et al. (2016), Apothéloz-Perret-Gentil et al. (2017), Sigsgaard et al. (2017), Bakker et al. (2017), Kocher et al. (2017), Thomsen and Sigsgaard (2019), and Lynggaard et al. (2020) (Figure 2e). In this approach, each library pool of PCR rep- licates is a sequencing library and is returned as a separate fastq file, each of which can contain data from a large number of tagged PCR replicates. 3. Metabarcoding approaches can be divided into three overall strategies for adding nucleotide tags and library indices (Taberlet et al., 2018) (Figure 2): 1. The “one-step PCR” approach in which sample DNA extracts are amplified and built into sequence libraries in one reac- tion. Here, metabarcoding primers carry sequencing adapters, nucleotide tags, and/or library indices, referred to as “fusion primers” (Figure 2b). This approach is used in for example, Kozich et al. (2013), Elbrecht and Leese (2015), Sickel et al. (2015), Grealy et al. (2016), Berry et al. (2017), Elbrecht et al. \| FI G U R E 2 Metabarcoding approaches can be divided into three overall strategies for adding nucleotide tags and library indices. (a) The composition of a dual-tagged and dual-indexed metabarcoding Illumina library sequence. Note that the metabarcoding marker, primers, and tags are sequenced as Illumina read 1 and read 2, while index reads are sequenced separately as i7 and i5 reads and used to multiplex sequencing libraries. (b–e) Strategies for adding nucleotide tags and indices to metabarcoding markers. The one-step PCR (b) is depicted with the use of nucleotide tags, which eliminates the need for indices FI G U R E 2 Metabarcoding approaches can be divided into three overall strategies for adding nucleotide tags and library indices. (a) The composition of a dual-tagged and dual-indexed metabarcoding Illumina library sequence. Note that the metabarcoding marker, primers, and tags are sequenced as Illumina read 1 and read 2, while index reads are sequenced separately as i7 and i5 reads and used to multiplex sequencing libraries. (b–e) Strategies for adding nucleotide tags and indices to metabarcoding markers. The one-step PCR (b) is depicted with the use of nucleotide tags, which eliminates the need for indices even thousands of samples per sequencing run. To allow pooling and parallel sequencing of this magnitude, different molecular labelling systems have been developed. For metabarcoding studies, the addi- tion of sample-specific identifiers to PCR amplicons can be achieved either as nucleotide tags during the metabarcoding PCR, or as library indices when converting amplicons into sequencing libraries, that is, as part of the workflow of adding sequencing adapters to amplicons. A metabarcoding sequencing library consists of amplicons carrying sequencing adapters and indices and can consist of one or more PCR products from one or more samples as outlined below. Note that given the inconsistent use of terminology in the metabarcoding lit- erature, for clarity, we use the original term for nucleotide tags in amplicon sequencing as used by Binladen et al., (2007), and Illumina's terminology to describe the nucleotide reads that are used to de- multiplex sequencing libraries, the i5 and i7 index reads. That is, 5′ nucleotide tags are sequenced with the metabarcoding marker and primers in the Illumina sequencing read 1 (and read 2 for paired-end sequencing), while library indices are sequenced as separate index reads, i.e., if dual-indexing is performed as i5 and i7 reads (Figure 2a) (https://support.illumina.com). 2 \| TAGGING AND INDEXING APPROACHES IN METABARCODING STUDIES index PCR Excl. index PCR Library sequence F R i5 i7 t t OR Adapter ligation Adapter and index ligation Index primers PCR i5 i7 Library sequence F R i5 i7 t t t t Index primers Tagged PCR product sample 2 Tagged PCR product sample 1 Incl. index PCR PCR Adapter ligation Library sequence Library build on amplicon pool Pooling PCR Tagged PCR amplicon Sample 1 Adapter and index ligation Excl. index PCR Library sequence BOHMANN et al. 1235 to low reported use of this method, its high cost and workload and thereby limited throughput (Zizka et al., 2019). In the two-step approach, sample-specific labelling is not neces- sarily carried out during the metabarcoding PCR (Figure 2c,d). If not labelled, there is a risk of cross-contamination between unlabelled PCR products when handling them prior to the second PCR (Zizka et al., 2019). Therefore, this metabarcoding approach has the great- est theoretical risk of cross-contamination between PCR products (Figure 2c, Table 1). The risk of this kind of cross-contamination is eliminated if tagging is carried out in the first PCR, see for example Kitson et al. (2018). If untagged metabarcoding primers are used in the two-step PCR approach (Figure 2c), then cross-contamination can be eliminated if the two PCRs are carried out in the same re- action, that is, both two primer sets are included, see for example Clarke, Czechowski, et al. (2014). 3 \| PROS AND CONS OF METABARCODING APPROACHES The ability to tag and index amplicons to fully harvest the power of high-throughput sequencing comes at a price as the labelling and pooling of hundreds of PCR replicates is highly complex and entails costs associated with preventing, detecting, and eliminating errors and biases. None of the metabarcoding approaches presented here is perfect; rather each of them has pros and cons. Below, we outline the advantages and disadvantages, specifically addressing issues related to cross-contamination risk, PCR amplification efficiency, chimera formation, tag-jumping, index-misassignment, cost, and workload. The issues associated with each metabarcoding strat- egy are important to keep in mind for choosing a metabarcoding strategy and for designing laboratory workflows and interpreting results. Irrespective of the chosen approach, cross-contamination can be detected and filtered out by including sample replicates, PCR repli- cates, and positive and negative controls. Thus, these should be in- cluded in the laboratory workflow and sequencing (e.g., Bista et al., 2017). An important measure that enables one to filter out potential contamination during data processing is to use different nucleotide tag or library index combinations on each sample's individual PCR replicates. This will allow for stringent sequence processing across each sample's PCR replicates, that is, a restrictive approach in which only sequences that are shared by a number of a sample's PCR rep- licates are retained (see Alberdi et al., 2018, applied in, for example, Giguet-Covex et al., 2014; De Barba et al., 2014; Hope et al., 2014; Cohen et al., 2020; Lynggaard et al., 2021; Yang et al., 2021). 3.1 \| Cross-contamination risk During the metabarcoding PCR, here specified as the PCR in which the metabarcoding marker is targeted, relatively short DNA se- quences (typically <350 bp) are enriched through amplification. Especially when targeting trace amounts of DNA, PCR amplifica- tion can be highly susceptible to contamination and thereby to false positives. The risk of contamination when preparing metabarcod- ing PCRs, that is from the surroundings or laboratory reagents, is the same no matter which of the three overall metabarcoding ap- proaches is used. Moreover, regardless of the metabarcoding strat- egy employed, cross-contamination can happen between nucleotide tagged and indexed primer stocks (which are delivered at high mo- larity). The risk of this happening will be similar between the strate- gies and will depend on the number of samples and the chosen setup within the employed strategy. In the following, we will therefore focus on how the three main metabarcoding approaches differ in their ability to allow detection of cross-contamination between PCR products after the metabarcoding PCR. \| The sequence over- hangs allow the resulting amplicons to be targeted by the second round of primers, which carry sequencing adapters and indices. In this article, we discuss the three main metabarcoding strat- egies. One approach not mentioned here is library preparation on individual unlabelled PCR products through a ligation-based library preparation protocol with or without an index PCR step. However, such ligation based protocol would entail several protocol steps to be carried out on each PCR product, such as end-repair and ligation of adapters (e.g., carrying indices such as in Illumina's TruSeq Nano DNA Library Prep kit, see Zizka et al., 2019). The reason that we do not consider this approach a main metabarcoding strategy is due 2. The two-step PCR approach in which sample DNA extracts are PCR-amplified with two primer sets. In the primary reaction the metabarcoding primers carry 5′ sequence overhangs of c. 33– 34 nucleotides in length. These can be with (Clarke et al., 2017; Griffiths et al., 2020; Kitson et al., 2018; Li et al., 2019; Vesterinen et al., 2018) or without (Bista et al., 2017; de Vere et al., 2017; Galan et al., 2017; Miya et al., 2015; Swift et al., 2018; Vesterinen et al., 2018) nucleotide tags (Figure 2c,d). The sequence over- hangs allow the resulting amplicons to be targeted by the second round of primers, which carry sequencing adapters and indices. 1236 BOHMANN et al. 3.2 The longest 5ʹ-nucleotide additions are found in the one-step PCR ap- proach where up to 60 nucleotides (sequence adapters and tags) are added to one or both of the primers, making the complete primer often over 80 bp long (e.g., Elbrecht & Leese, 2015). In the two-step PCR approach (Figure 2c,d), the sequence overhangs on the me- tabarcoding primers used in the first PCR are approximately half the length of the fusion primers, for example, 33–34 nucleotides if using Illumina Nextera Indices. The tagged PCR approach has the shortest nucleotide additions to the metabarcoding primers (Figure 2e) with tags of typically 5–10 nucleotides in length (e.g. Alberdi et al., 2018; Coissac, 2012; De Barba et al., 2014). The long additions to the me- tabarcoding primers in the one-step PCR approach cause a decrease in PCR efficiency, as witnessed by an increase in CT values (Murray et al., 2015). A comparison of PCR efficiency to other metabarcoding strategies has not, to our knowledge, been formally assessed for the two-step PCR approach, but the two-step PCR approach has been shown to have higher consistency as compared to the one-step fu- sion primer approach (Zizka et al., 2019). Even the short nucleotide additions in the tagged PCR approach have been shown to decrease PCR efficiency, as witnessed by a significant increase in CT values (Schnell et al., 2015). Thus, no method is free of decreased PCR effi- ciency caused by the nucleotide additions to 5′-end of metabarcod- ing primers. However, it has to our knowledge, not been formally tested whether - and to what extent - the shorter nucleotide tag additions in the tagged PCR approach offers greater PCR efficiency and taxonomic detection than the two other approaches, and thereby it can only be speculated that it is the most sensitive when it comes to detection of taxa in low abundance. It should be noted that increasing the cycle number in the PCR amplifications is not an ac- ceptable solution to increase sensitivity, as increased cycle number will reduce taxonomic diversity (Kelly et al., 2019; Piñol et al., 2015). Regardless of metabarcoding strategy, we stress the importance of optimising PCR amplifications (usually by qPCR) to detect PCR inhi- bition, identify samples with low template quantity, and track PCR efficiency issues (Murray et al., 2015; Yang et al., 2021). 3.2 1237 g g re Metabarcoding strategy One-step PCR Two-step PCR Tagged PCR With 5′ nucleotide tags, without i5 and i7 indices Without 5′ nucleotide tags on metabarcoding primers With 5′ nucleotide tags on metabarcoding primers Library preparation with T4 DNA polymerase blunt- ending and post-ligation PCR Library preparation without T4 DNA polymerase blunt-ending and post-ligation PCR ing and workload ↓ ↑ ↑ ↑ ↑ f cross-contamination between PCR products ↓ ↑ ↓ ↓ ↓ mps No No No Yes No tial for index misassignment/library bleeding on e flow cell No (only if indices are used) Yes Yes (if indices are used) Yes Yes ase in PCR efficiency due to nucleotide additions metabarcoding primers High Potentially high Potentially high Low Low of metabarcoding primers ↑ ↓ ↑ ↓ ↓ er PCR steps prior to sequencing 1 2 2 2 1 TA B LE 1 Features of the three main metabarcoding strategies Feature Metabarcoding strategy One-step PCR Two-step PCR Tagged PCR With 5′ nucleotide tags, without i5 and i7 indices Without 5′ nucleotide tags on metabarcoding primers With 5′ nucleotide tags on metabarcoding primers Library preparation with T4 DNA polymerase blunt- ending and post-ligation PCR Library preparation without T4 DNA polymerase blunt-ending and post-ligation PCR Handling and workload ↓ ↑ ↑ ↑ ↑ Risk of cross-contamination between PCR products ↓ ↑ ↓ ↓ ↓ Tag-jumps No No No Yes No Potential for index misassignment/library bleeding on the flow cell No (only if indices are used) Yes Yes (if indices are used) Yes Yes Decrease in PCR efficiency due to nucleotide additions to metabarcoding primers High Potentially high Potentially high Low Low Cost of metabarcoding primers ↑ ↓ ↑ ↓ ↓ Number PCR steps prior to sequencing 1 2 2 2 1 of amplifying (trace copy number) target DNA from different taxa (Taberlet, Coissac, Pompanon, et al., 2012) potentially distorted by primer biases, inhibitors, and potentially abundant predator or host DNA (e.g., Clarke, Soubrier, et al., 2014; Deagle et al., 2014; Murray et al., 2015). Therefore, it is important to take the effect of 5′ nucle- otide additions to metabarcoding primers into account. The three main metabarcoding strategies have different lengths of nucleotide additions on the 5′-end of metabarcoding primers. 3.2 PCR amplification introduces biases, such as primer biases, and errors, such as nucleotide substitutions and chimeras (Haas et al., 2011; Murray et al., 2015; Piñol et al., 2015; Polz & Cavanaugh, 1998). Two of the three main metabarcoding strategies allow prac- titioners to carry out only a single PCR step before sequencing, namely the one-step PCR approach and the tagged PCR approach in which PCR-free library building is carried out (Figure 2b,e, Table 1). Because an extra PCR step adds an additional risk of introducing errors, these two approaches offer an advantage over the two-step PCR method (Figure 2c,d) and the tagged PCR approach in which the workflow includes an index PCR step (Figure 2e). It should be noted that the number of cycles in the indexing PCR is typically kept low to minimize PCR errors (e.g., eight cycles: Bohmann et al., 2018). Throughout any of these workflows there is a need to keep PCR cy- cles to a minimum, which might be especially true of metabarcoding workflows with two PCR steps. PCR products are labelled during the metabarcoding PCR ampli- fication in the one-step PCR approach (Figure 2b), the two-step PCR approach where tagging is carried out in the first PCR (Figure 2d), and the tagged PCR approach (Figure 2e). If the resulting PCR products carry different tag combinations then cross-contamination between them is obviously not of concern. However, if the same tag com- binations occur across multiple samples, then cross-contamination between them can be an issue. A solution is to process them in sepa- rate batches to avoid cross-contamination. Some laboratories do not reuse tag-primer combinations to eliminate cross-contamination risk (see Murray et al., 2015). Aside from minimizing the number of PCR steps, the effect of 5′ nucleotide additions to metabarcoding primers should be con- sidered as they are likely to decrease PCR efficiency (Murray et al., 2015; Schnell et al., 2015). Bulk sample and eDNA extracts consist of complex mixtures of DNA from a large number of organisms, which especially in the case of eDNA can be degraded (Taberlet et al., 2012). With DNA extracts, the primers are faced with the task BOHMANN et al. 3.3 \| Chimeras and tag-jumps Chimeras can be formed during all PCR steps in any metabarcod- ing workflow (Figure 2b–e). Chimeras are amplicons which com- bine sequences from two or more different template molecules, and the majority are thought to result from incomplete primer ex- tension during the elongation phase of the PCR cycle (Judo et al., 1998; Meyerhans et al., 1990; Shin et al., 2014; Wang & Wang, 1997). The probability of chimera formation increases when simi- lar template sequences are amplified in the same PCR reaction ( Judo et al., 1998, Smyth et al., 2010, but see also Fonseca et al., 2012), such as during the metabarcoding PCR or during the index PCR amplification of pools of tagged amplicons (Figure 2e). There are different consequences of chimeric sequences depending on where they arise. If they are created during a PCR amplification of a single sample's DNA extract, the chimeras will be intrasam- ple chimeras, which can be falsely interpreted as novel taxa and erroneously inflate measures of diversity. On the other hand, if chimeras are created during a PCR amplification of pooled tagged amplicons, such as in the tagged PCR approach (Figure 2e), the chimeras may be intersample chimeras. Such intersample chime- ras can result in tag-jumps and false attribution of amplicon se- quences to samples, which can lead to false positives and inflation of diversity (Schnell et al., 2015). To avoid tag-jumps in the tagged PCR approach, and thereby prevent false assignment of sequences to samples, it is important to refine index PCR parameters to decrease the likelihood of chi- mera formation, or better yet, to omit the index PCR step (Figure 2e). Furthermore, blunt-ending using T4 DNA polymerase should be cir- cumvented during library preparation (Carøe & Bohmann, 2020; Palkopoulou et al., 2016; Schnell et al., 2015). If both T4 DNA poly- merase blunt-ending and index PCR are eliminated during library preparation of pools of tagged amplicons, tag-jumps can practically be eliminated (Carøe & Bohmann, 2020). If the library preparation protocol contains a T4 DNA blunt- ending step and/or an index PCR step, and thereby can be assumed to generate tag-jumps, they can be detected and removed by using “twin-tags” during the original PCRs (e.g., F1-R1, F2-R2, etc.), be- cause tag-jumped sequences would then produce nontwinned tag combinations not used in the set-up (e.g., F1-R2, F2-R3, etc.) (e.g. Schnell et al., 2015; Yang et al., 2021). 3.2 TA B LE 1 Features of the three main metabarcoding strategies Theoretically, the reduced PCR efficiency in the one-step and two-step PCR approaches caused by the long overhangs on prim- ers might be counteracted by spiking the PCRs with metabarcod- ing primers without any 5ʹ attachments (e.g., Murray et al., 2015). However, this has been shown to have modest PCR efficiency im- provements for the one-step approach (e.g., Murray et al., 2015). Alternatively, a pre-enrichment can be carried out before the me- tabarcoding PCR. That is, running a PCR with metabarcoding prim- ers with no nucleotide additions prior to the metabarcoding PCR, as done in Zizka et al. (2019) and Elbrecht and Steinke (2018) for 1238 BOHMANN et al. programmes such as UCHIME (Edgar et al., 2011) can be used for further clean-up. the one-step PCR approach. However, this not only introduces another PCR amplification step, but can increase the risk of cross- contamination between PCR products due to the initial unlabelled PCR amplification step (e.g., Murray et al., 2015). Note that adding such a pre-enrichment step to the one-step approach can cause it to be mistaken for a two-step PCR approach. the one-step PCR approach. However, this not only introduces another PCR amplification step, but can increase the risk of cross- contamination between PCR products due to the initial unlabelled PCR amplification step (e.g., Murray et al., 2015). Note that adding such a pre-enrichment step to the one-step approach can cause it to be mistaken for a two-step PCR approach. Inter-sample chimeras can cause havoc in metabarcoding stud- ies. They can only occur in the tagged PCR approach where library build is carried out on pooled tagged amplicons from different sam- ples (Figure 2e, Table 1). Here, tag-jumps can create sequences with new combinations of the nucleotide tags used in the amplicon pool (Schnell et al., 2015). If the new combinations of tags are already used in the amplicon pool, it will cause false assignment of sequences to samples, which should be avoided at all cost (Esling et al., 2015; Schnell et al., 2015). Such tag-jumps can cause negative controls to accumulate a number of sequences following bioinformatic sorting of sequences to samples, which makes sequencing of negative con- trols a valuable tool to detect tag-jumps. 3.2 Apart from the length of the nucleotide additions, it has been investigated whether differences in nucleotide tag sequences can result in biases in the tagged PCR approach. Although some studies show that such tag bias is an issue (Berry et al., 2011; O’Donnell et al., 2016), other studies show that it is not (Leray & Knowlton, 2017; Yang et al., 2021). If tag bias does exist, it should theoreti- cally be minimised if different tags are used on each sample's PCR replicates. The rate of tag-jumping has been estimated from ca. 2% to up to 49% of total sequences (Carøe & Bohmann, 2020; Esling et al., 2015; Schnell et al., 2015). This broad range can be caused by fac- tors affecting intersample chimera formation during the index PCR. For example, DNA template and primer concentration, PCR cycle number, and sequence similarity (e.g., Carøe & Bohmann, 2020; Judo et al., 1998; Smyth et al., 2010). The range of tag-jump pro- portions highlights the unreliability of including an index PCR step in the tagged PCR approach. It should be noted that tag-jumps can also occur due to T4 DNA polymerase activity in the blunt-ending step during library preparation, as demonstrated in library building for the Roche/454 sequencing platform (van Orsouw et al., 2007; Palkopoulou et al., 2016) and for the Illumina sequencing platform (Carøe & Bohmann, 2020). 3.4 \| Misassignment of library indices Incorrect assignment of indices between pooled libraries can cause sequence reads to be incorrectly assigned to libraries. Misassigned indices have been attributed to the formation of mixed clusters on the sequencing flow cell, that is, clusters originating from two dif- ferent template molecules or clusters growing into each other, to low levels of free index primers present in the sequence library and to bulk amplification of pooled libraries (Costello et al., 2018; Nelson et al., 2014; Sinha et al., 2017; Valk et al., 2019; Vodak et al., 2018). Regardless of how index misassignment occurs, if it occurs in metabarcoding studies it can cause incorrect assignment of am- plicon sequences to libraries, which can cause incorrect assignment of sequences to samples and false positives. This phenomenon can affect metabarcoding approaches that include indexing of libraries (Figure 2, Table 1). To avoid index misassignment it is recommended to dual-index libraries with unique library index combinations (Kircher et al., 2012; Sinha et al., 2017), www.illumina.com). Further, stringent bead purification (or size selection) can remove free adapt- ers/primers from the libraries (Owens et al., 2018). The labelling in the different metabarcoding approaches further allows for account- ing for potential incorrect assignment of sequences to libraries. In the tagged PCR approach, unique tagging of PCR replicates across all pooled libraries can be used to account for (and detect) index mis- assignment. However, this can be costly. In the one-step PCR ap- proach, it is common to eliminate the use of i7 and i5 library indices, instead relying on 5′ nucleotide tags, which creates a single library that is free of index misassignment (Table 1). As with tag-jumping, the extent of incorrect assignment of indices and spillover of taxa between samples can be detected through inclusion of positive con- trols consisting of taxa not expected to occur in the data set and by comparing all observed to all used combinations of used indices when demultiplexing libraries. The fusion primers for the one-step PCR approach are the most expensive metabarcoding primers amongst the three approaches. This is because differently labelled versions are purchased for each metabarcoding primer set and because the increased oligo length results in lower yield of the full length product. If indexing is used instead of tagging and unique matching indices are used to account for index misassignment, one-step PCR can become increasingly expensive for larger scale studies. 3.4 \| Misassignment of library indices However all of this needs to be factored against the potential cost of repeating runs due to arte- facts and contamination, and the fact that only a single PCR step is needed to go from sample extract to library. The tagged two-step PCR primers will be the second-most expensive (Figure 2d) due to their length and individual labelling. In the tagged PCR approach (Figure 2e), the metabarcoding prim- ers are relatively inexpensive as they only add 5′ tags of 5–10 nu- cleotides in length. However, these need to be purchased in many tagged versions for each metabarcoding primer set. Furthermore, if tag-jumping is to be taken into account by only using each tag once in a library amplicon pool, for example, by only amplifying with twin forward and reverse tags, then metabarcoding primer sets have to be ordered in many differently labelled versions (Schnell et al., 2015). To keep costs down, this twin-tagging needs to be balanced by pool- ing fewer PCR products into each library and thereby creating more sequence libraries, but this then increases expenses to library prepa- ration (Figure 2e). However, if a library preparation protocol is used that does not create tag-jumps, tags can be freely combined, which lowers the number of tagged primers that must be purchased (Carøe & Bohmann, 2020; Schnell et al., 2015). In contrast to the other two metabarcoding approaches, the tagged PCR approach includes ligation-based library preparation of pools of amplicons, and the cost of this therefore has to be taken into account. The cost can be kept low if a protocol that does not generate tag-jumps is used and only a few libraries have to be made. It is important not to mistake tag-jumping, index misas- signment, or cross-contamination between PCR products with cross-contamination of the primers themselves. Due to the high concentration of primers upon synthesis, cross-contamination (e.g., by aerosols) can manifest itself as low numbers of sequence reads and could be misinterpreted as tag-jumps or index-bleeding. Due to the risk of primer cross-contamination, some laboratories avoid ordering primers in 96-well plates. As mentioned, the risk of cross-contamination between nucleotide tagged primer stocks and indexed primer stocks, which could for example occur during resus- pension of primers, will generally be the same no matter which of the three overall metabarcoding approaches is used. 3.5 \| Cost Metabarcoding primers in the tagged and one-step PCR approaches are labelled, whereas the metabarcoding primers in the two-step ap- proach can be either labelled or not (Figure 2). Due to the different labelling systems in the three primary metabarcoding approaches, there are different costs associated with them. Metabarcoding primers in the tagged and one-step PCR approaches are labelled, whereas the metabarcoding primers in the two-step ap- proach can be either labelled or not (Figure 2). Due to the different labelling systems in the three primary metabarcoding approaches, there are different costs associated with them. set. However, note that such controls do not enable confident elimination of false positives caused by tag-jumps. The extent of tag-jumping can also be assessed by comparing all observed combi- nations of used tags to all originally used tag combinations (Schnell et al., 2015; Zepeda Mendoza et al., 2016). the primers are unlabelled and any cross-contamination between the primers will not have consequences. 3.3 \| Chimeras and tag-jumps However, using twin tags comes at the price of buying many more versions of tagged prim- ers and building more libraries (Schnell et al., 2015). If twin tags are not used, chimera removal software can remove some chimeric sequences carrying false combinations of used tags (Schnell et al., 2015). All metabarcoding approaches are prone to intra-sample chi- meras. However, as chimera formation increases when similar se- quences are amplified in the same PCR reaction (e.g. Judo et al., 1998; Smyth et al., 2010), the use of metabarcoding primers with long 5′ overhangs, as in the one-step and two-step approaches, might be more prone to chimera formation since they carry long and similar sequences at the 5ʹ end of the primers. However, this hy- pothesis requires testing. Intrasample chimeras can be reduced by limiting the number of PCR cycles and extending elongation time (Haas et al., 2011; Qiu et al., 2001). Also, if samples are subjected to multiple, independent PCRs, chimeras can be filtered out by keeping only sequences that occur in multiple PCR replicates, the “restric- tive approach” described in Alberdi et al., (2018). Chimera detection The extent of tag-jumping and spillover of taxa between sam- ples can be detected through inclusion of positive controls con- sisting of synthetic oligos or taxa not expected to occur in the data BOHMANN et al. 1239 3.6 \| Laboratory workload The one-step PCR approach is without doubt the quickest method for generating sequence-ready libraries, as it only requires a single PCR-step to achieve both amplification and library preparation of the metabarcoding amplicons (Figure 2b). Researchers have used this method in research and commercial scenarios to turn around sequence data in 12–24 h in the field on the iSeq platform (Bunce, unpublished data). In some applications, especially requiring timely interventions, the rapid turnaround time of the one-step PCR ap- proach may be a consideration. The workload for the two-step PCR approach and the tagged PCR approach depends, to some extent, on how many sample extracts and PCR replicates are to be pro- cessed. If it is a relatively high number, the tagged PCR approach is the quickest due to the library build being performed on pooled amplicons rather than through a PCR step on individual PCR prod- ucts. However, as with all molecular biological workflows, carefully organised liquid handling and automation provide solutions to high- throughput studies. When choosing a metabarcoding approach, the need for future multiplexing of the metabarcoding primers should be considered. That is, to use several metabarcoding primer sets that target differ- ent markers and taxonomic groups within the same PCR reaction to simultaneously screen for many taxonomic groups and thereby keep costs and work load at a minimum (e.g., De Barba et al., 2014). For this, the nucleotide tagged primers in the tagged PCR approach should theoretically be the most applicable, whereas the long addi- tions to the metabarcoding primers in the one-step and two-step PCR approaches might be less conducive to multiplexing due to the extensive sequence homology. Lastly, it should be noted that whatever metabarcoding strategy is chosen, it should be clear from the present article that one should not change workflows within an experiment. Moreover, there is some justified concern within the metabarcoding community that the nuances in metabarcoding workflows makes interlaboratory comparison difficult (Blackman et al., 2019; Murray et al., 2015; Zizka et al., 2019). 4 \| CHOOSING A METABARCODING APPROACH It is clear that there is no such thing as a perfect metabarcoding sample-labelling approach, and that choosing which one is right for a given study or laboratory should be an informed trade-off of pros and cons balanced to the needs (Table 1). Within metabarcoding studies, those needs can range widely. 3.4 \| Misassignment of library indices The two primer sets in the untagged two-step PCR approach (Figure 2c) have good potential for being used up, as the first unlabelled metabar- coding primer set can be used across many samples and the second primer set can be used across different metabarcoding primer sets. A multitude of combinations of the above metabarcoding study parameters exist, and as demonstrated by this article, the signifi- cance of the pros and cons of the metabarcoding approaches will differ with them. For example, while the tagged PCR approach (Figure 2e) may excel in amplifying low abundance templates given the shorter nucleotide additions to the metabarcoding primers than the one-step PCR primers (Murray et al., 2015; Zizka et al., 2019), the one-step PCR offers a quicker turnaround (Figure 2b). However, the one-step PCR strategy comes at the cost of buying long fusion primers, and is only worthwhile if the metabarcoding primers are to be used again. 3.4 \| Misassignment of library indices If the first PCR step in the two-step PCR approach is carried out without tags (Figure 2c), If a large number of metabarcoding primer sets are used, the two-step approach, where primers in the first PCR do not carry tags (Figure 2c), offers a relatively inexpensive solution. This means that the same primer set can be used across multiple samples and projects. This has the benefit that trying out new metabarcoding primer sets does not entail buying many labelled versions of the metabarcoding primer sets, as it does in the other metabarcod- ing approaches (Figure 2b,d,e). However, the second primer set in the two-step PCR approach is costly as it has to include both the 1240 BOHMANN et al. from a number of samples within, for example, a geographic location is the goal (Grealy et al., 2016; Schnell et al., 2018). Sample types can range from bulk specimen samples consisting of high quality DNA from pools of entire organisms (Tang et al., 2015) to environmental samples in which DNA from target organisms can be fragmented and scarce (Stat et al., 2017). Furthermore, studies differ in how many metabarcoding primer sets are used - from only one ( Bohmann et al., 2011; Drinkwater et al., 2018) to several (De Barba et al., 2014; Drummond et al., 2015; Zhang et al., 2018). Furthermore, the bud- get for a metabarcoding project will differ between studies, as will whether the metabarcoding primers are to be used in future studies. Lastly, some applications of metabarcoding, such as biosecurity or forensics, will necessitate a “high bar” for data fidelity and controls. sequence complementary to the sequence overhang, the sequence adapters, and the library indices (Figure 2c). It is worth noting that many labelled index primers will have to be purchased if twin dual- indices are used to account for incorrect assignment of indices to libraries. This second primer set is, however, applicable across differ- ent metabarcoding primer sets and can thereby be used across many metabarcoding studies. For all three approaches, cost-effectiveness will be increased if the purchased primers are depleted effectively, that is, if they are not only to be used in one small study. 5 \| APPLICATIONS ON OTHER SEQUENCING PLATFORMS We thus stress the importance of being informed about the pros and cons of the chosen metabarcoding approach with regards to cross-contamination risk, PCR amplification efficiency, chimera for- mation, tag-jumping, index-misassignment, cost, and workload and to include appropriate quality assurance and quality control mea- sures. This will help ensure that the generated data will facilitate informed data analysis and interpretation. We advocate that me- tabarcoding publications should include detailed information about the metabarcoding strategy and how its challenges have been taken into account in the laboratory, data processing, and interpretation of results. Furthermore, it may be appropriate to eventually develop a set of metabarcoding guidelines similar to the MIQE guidelines for qPCR (Bustin et al., 2009) to establish standard reporting practises, which would ultimately further increase the power and reliability of metabarcoding. 5 \| APPLICATIONS ON OTHER SEQUENCING PLATFORMS Metabarcoding studies range from those that look for one or a few taxa within sample units ( Bohmann et al., 2018) to studies that look for many taxa within sample units (Seersholm et al., 2018), and sample numbers can range from tens (Elbrecht et al., 2017), to hun- dreds (Galan et al., 2017; Rodgers et al., 2017) or even thousands (Ji et al., 2021; Schnell et al., 2018). The research question and ex- perimental set-up can require taxonomic identifications to be made within individual samples (Coghlan et al., 2012), while in other stud- ies, taxonomic identifications from pools of individual samples or Although to a more limited extent, other second generation se- quencing technologies than Illumina are used in metabarcoding. For example, Ion Torrent (Thermo Fischer Scientific) and BGI platforms (BGI Genomics) (Braukmann et al., 2019; Forin-Wiart et al., 2018; Schnell et al., 2018; Yang et al., 2020). These technologies require the addition of sequencing adapters similar to Illumina platforms BOHMANN et al. 1241 and have similar labelling structure. Therefore, discussions regard- ing labelling strategies in the present article are largely applicable to metabarcoding on these other platforms. For example, the one-step (Schnell et al., 2018) and the two-step PCR approach (Braukmann et al., 2019; Nota et al., 2019) have been used on the Ion Torrent plat- form, and the tagged PCR approach has been used on BGI's MGISEQ platform (Yang et al., 2020). Further, third generation technologies yielding long reads have been employed in metabarcoding; Pacific Biosciences (PacBio) (James et al., 2016; Tedersoo et al., 2018) and the portable Oxford Nanopore Technologies MinION sequencer (Karst et al., 2021). These platforms also rely on the addition of se- quencing adapters. The high error rate of these platforms (Dohm et al., 2020) compared to Illumina platforms (Stoler & Nekrutenko, 2021) makes correct taxa identification and sample specific label- ling difficult. However, solutions to this are being developed (Karst et al., 2021). It is likely that metabarcoding applications will probably follow the platform with the highest sequencing fidelity although in some applications speed and portability may also increasingly be- come factors in platform choice. of uptake by stakeholders who will employ metabarcoding for en- vironmental management. Reputational setbacks as the result of practitioners not executing their metabarcoding workflows well will probably resonate across a variety of biomonitoring, forensic, and bioseurity applications. CONFLICT OF INTEREST Metabarcoding of environmental DNA has some commonali- ties with the field of ancient DNA in which low quality and quantity of target DNA is also targeted amongst nontarget, and potentially more abundant, templates. In the early days of ancient DNA studies, PCR-based techniques, including amplifying already amplified DNA to enhance signals, were used, which caused authentication issues, as amplification of modern templates was mistaken for true ancient signals. This was followed by urgent calls for precautions to ensure reliability and authenticity of ancient DNA sequences (Cooper & Poinar, 2000; Pääbo et al., 2004). Also similarly to the field of an- cient DNA, the take-home message should be that metabarcoding is becoming a self-critical and self-correcting field in which techni- cal reliability is promoted and rewarded, with the long-term benefit The authors declare no conflict of interest. The authors declare no conflict of interest. ACKNOWLEDGEMENTS KB was supported by Independent Research Fund Denmark, DFF grant 5051-00140 and the European Research Council (ERC) under the European Union's Horizon 2020 research and innova- tion programme (grant agreement No 856488). AJD was sup- ported by the U.K. Natural Environment Research Council (NERC) grants NE/S006958/1, NE/M02086X/1, NE/S005560/1, and NE/ S000291/1. SC benefitted from NERC Highlight Topic funding grant NE/N006216/1 and SC acknowledges support from NERC grant NE/N003756/1 and NE/M02086X/1. CC was supported by the Carlsberg Foundation (CF18-1110, ”Archives”). IB was supported by Wellcome grants WT207492 and 104640/Z/14/Z, 092096/Z/10/Z. MS was supported by European Research Council grant 856506. FL, VE and SC are members of EU COST Action DNAqua-Net (CA15219). Moreover, the authors would like to thank their collective research groups and collaborators whose laboratory and bioinformatic ex- periences over many years contributed to the ideas, initiation, and completion of this manuscript. All metabarcoding strategies can generate robust data. However, like all laboratory workflows if they are not executed well or are in- appropriate for the application, they may lead to flawed data. We advocate that just because PCR is a relatively simple method it does not mean that metabarcoding is simple, and there are many traps in metabarcoding workflows that can trip-up new users. Here, we have presented an overview of the three main metabarcoding strategies for assessment of biodiversity on Illumina sequencing platforms, and the downstream consequences for the resulting data with regards to cross-contamination risk, PCR amplification efficiency, chimera formation, tag-jumping, index-misassignment, as well as cost and workload. In doing so we wish to enable researchers and practition- ers to make an informed choice of which metabarcoding strategy is best suited for their specific study. Ultimately, this is to avoid the worst case scenario: generation of unusable data and wasting a con- siderable amount of time and money, or even worse making wrong conclusions due to flawed data. 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https://openalex.org/W3201311380	https://www.mdpi.com/2076-2615/11/9/2727/pdf?version=1632279217	English	null	Rumen (Calicophoron/Paramphistomum spp.) and Liver Flukes (Fasciola hepatica) in Cattle—Prevalence, Distribution, and Impact of Management Factors in Germany	Animals	2,021	cc-by	7,758	Citation: Forstmaier, T.; Knubben-Schweizer, G.; Strube, C.; Zablotski, Y.; Wenzel, C. Rumen (Calicophoron/Paramphistomum spp.) and Liver Flukes (Fasciola hepatica) in Cattle—Prevalence, Distribution, and Impact of Management Factors in Germany. Animals 2021, 11, 2727. https://doi.org/10.3390/ani11092727 Abstract: This study was carried out to determine the prevalence of rumen ﬂukes on German cattle farms via the sedimentation technique, and to identify the rumen ﬂuke species occurring in Germany. Additionally, the prevalence of patent Fasciola hepatica infections was determined. Furthermore, a short questionnaire was answered by the farmers. A prevalence of 5.5% and 9.5% was detected for rumen ﬂukes and liver ﬂukes, respectively. Coinfections occurred on 2.1% of farms. In northern Germany, the rumen ﬂuke prevalence was higher than in southern Germany, while for liver ﬂuke the distribution was reversed. Rumen ﬂukes were mostly identiﬁed as Calicophoron daubneyi, but in four cases, sequencing revealed Paramphistomum leydeni for the ﬁrst time in Germany. Grazing and feeding of fresh grass, as well as organic farming, were signiﬁcantly associated with rumen and liver ﬂuke occurrence. In contrast, suckler cow husbandry only had an inﬂuence on the occurrence of rumen ﬂukes, but not liver ﬂukes. Trematode eggs could be detected in both, farms with and without deworming. Since there were only a few studies about Paramphistomidosis in Germany, more attention should be paid to these parasitic diseases for animal welfare and animal health reasons. Academic Editors: Maria Teresa Manfredi, Alessia L. Gazzonis, Michele Mortarino, Luca Villa and Sergio Zanzani Received: 28 August 2021 Accepted: 16 September 2021 Published: 18 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: paramphistomidosis; rumen ﬂukes; Calicophoron daubneyi; Paramphistomum leydeni; coinfection; faecal examination; modelling; risk factors; organic farming; ruminants Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Tanja Forstmaier 1, Gabriela Knubben-Schweizer 1, Christina Strube 2 , Yury Zablotski 1 and Christoph Wenzel 1,* Tanja Forstmaier 1, Gabriela Knubben-Schweizer 1, Christina Strube 2 , Yury Zablotski 1 and Christoph Wenzel 1,* 1 Clinic for Ruminants with Ambulatory and Herd Health Services, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, 85764 Oberschleissheim, Germany; T.Forstmaier@med.vetmed.uni-muenchen.de (T.F.); gknubben@med.vetmed.uni-muenchen.de (G.K.-S.); Y.Zablotski@med.vetmed.uni-muenchen.de (Y.Z.) 1 Clinic for Ruminants with Ambulatory and Herd Health Services, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, 85764 Oberschleissheim, Germany; T.Forstmaier@med.vetmed.uni-muenchen.de (T.F.); gknubben@med.vetmed.uni-muenchen.de (G.K.-S.); Y.Zablotski@med.vetmed.uni-muenchen.de (Y.Z.) 2 Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30559 Hanover, Germany; Christina.Strube@tiho-hannover.de 2 Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30559 Hanover, Germany; Christina.Strube@tiho-hannover.de y * Correspondence: c.wenzel@lmu.de Simple Summary: Paramphistomidosis is a parasitic disease of ruminants caused by so-called rumen ﬂukes. To determine the current prevalence in German cattle, faecal samples from 614 herds were examined for parasite eggs. In addition, the occurring rumen ﬂuke species were determined, resulting in Calicophoron daubneyi and Paramphistomum leydeni. In the course of the work, the occurrence of the common liver ﬂuke, causing fasciolosis, was also documented. Rumen ﬂuke eggs could be detected in 5.5% of German cattle farms, liver ﬂukes in 9.5%. In 2.1% of the farms, both ﬂuke types occurred. Regional differences between northern and southern Germany were detected. Cattle grazing and fed with fresh grass were more likely to be infected with rumen and liver ﬂukes than cattle without such access. Cattle from organic farms were also more likely to have ﬂuke infections than those from conventional farms, in suckler cows, however, this association only applied to rumen ﬂukes. animals animals 1. Introduction Trematodes of the family Paramphistomidae, so-called rumen ﬂukes, e.g., Paramphisto- mum cervi, Paramphistomum leydeni, or Calicophoron daubneyi, cause paramphistomidosis in ruminants [1]. Rumen ﬂukes mainly infect domestic and wild ruminants [2], but also new world camelids may be affected [3]. Parasite development includes an intermediate host, https://www.mdpi.com/journal/animals Animals 2021, 11, 2727. https://doi.org/10.3390/ani11092727 2 of 11 Animals 2021, 11, 2727 represented by different genera of air-breathing freshwater snails [4]. Calicophoron daubneyi, nowadays the most frequently recorded rumen ﬂuke in northern, southern, and western Europe [5], and the liver ﬂuke Fasciola hepatica share the same intermediate host, the dwarf pond snail Galba truncatula [6]. Therefore, coinfections are possible in both the intermediate, as well as the ﬁnal host [7,8]. Infection of the ﬁnal host with rumen ﬂuke metacercariae is followed by two phases. During the intestinal phase, juvenile ﬂukes migrate within the mucosa of the small intestine and abomasum towards the rumen. This may result in diarrhoea and weight loss up to cachexia, and, in case of massive infection, even death of the animals [9–11]. In the ruminal phase, ﬂukes attach to the mucosa and mature into adult egg-laying worms. Ruminal paramphistomidosis is usually clinically inapparent, despite the occurrence of pathological lesions caused by the parasites [12]. p g y p Currently, coproscopy by sedimentation is the only practicable method for diagnosing rumen ﬂuke infections in live animals, although it can only detect patent infections [13]. During prepatency, faeces can be sieved, and the material retained examined for juvenile stages with a stereomicroscope; however, the method is only meaningful in positive cases. Post mortem detection includes the examination of the upper digestive tract at the abattoir or during necropsy. Serological methods are not commercially available yet. Recently, however, a coproantigen-based ELISA with high analytical sensitivity and speciﬁcity has been developed [14], which is a promising future tool to detect current infections in routine diagnosis or epidemiological studies. A few years ago, paramphistomidosis has been reported as an emerging parasitic disease in ruminants in Europe [15]. 1. Introduction For cattle, prevalences of up to 45% positive herds in central France [16], up to 32% in the retrospective analysis of laboratory records [17] and 52% at abattoirs in Ireland [18], 59% positive herds in Wales [19], 61% positive herds [20], and 6% and 19% at abattoirs in north-western Spain [21,22], 22% positive herds and 28% at abattoirs in Belgium [23], and 16% positive herds in the Netherlands [24] have been determined. So far, no comparable ﬁgures are available for cattle in Germany. From 1950 to 2000, only a few case reports have been published [25]. Based on recent ﬁndings of C. daubneyi in German cattle herds, it can be assumed that the parasitosis is also spreading in this country [26]. For the liver ﬂuke Fasciola hepatica, a seroprevalence of 15% was most recently reported for Germany, and the risk of infection was associated, among other factors, with grazing and feeding fresh grass [27,28]. Due to the same intermediate host, on all farms with liver ﬂuke infection, cattle can potentially also get infected with C. daubneyi. For example, on 46% of Welsh farms coinfections of these two trematodes were observed [19]. p Therefore, one aim of this study was to determine the prevalence of paramphistomi- dosis in German cattle herds and to identify the infecting rumen ﬂuke species. In order to present comprehensive data on ﬂuke infections and to take into account the shared intermediate host G. truncatula, the study also aimed to investigate the prevalence of patent F. hepatica infections, and to determine risk factors for both trematodes. 2.2. Questionnaire Survey The questionnaire provided to the participating farmers included the following questions: federal state, cattle breed, production type (dairy cattle/fattening beef cat- tle/suckler beef cattle farming [hereafter referred to as suckler cow]), agricultural system (organic/conventional), grazing patterns or feeding of fresh grass, number of young and adult animals, deworming scheme and known problems with rumen or liver ﬂukes. Faecal sampling was carried out according to a provided manual, either during defecation or by collecting freshly excreted faeces. The sampling scheme included eight young (six months to 2.5 years or before ﬁrst calving) and eight adult cattle (older than 2.5 years or after ﬁrst calving) at each farm, and samples of four animals each were pooled for coproscopical analyses. Overall, 90% of farms complied with this pattern (n = 555), but the other 59 farms (less samples collected than speciﬁed and only from young or adult cattle) were also included in the ﬁnal analysis. 2.1. Sample Size Calculation and Sampled Farms At the beginning of the study, the sample size was calculated to collect enough samples for reliable statistical analysis. A rather low prevalence of 5.0% positive herds was assumed, as there are only few reports for Germany so far [25,26]. Additionally, the seroprevalence of 17.7% for F. hepatica in the federal state of Bavaria [27] was included. Faecal samples were collected from October 2018 to December 2020. Sampling was carried out as part of the German research project “PraeRi” [29] in dairy farms (n = 285), and via media acquisition (calls in different journals and social media) as well as direct contact to veterinary practices, animal health funds and animal health services (n = 329). In total, 614 German farms were sampled. To take account of regional differences, Germany was divided into four regions. In the region North, 179 farms were sampled, in East 76 farms, in South 277 farms and in West 82 farms (Table 1). Resulting prevalences were calculated for Germany as a whole, each of the four regions and, additionally, the federal states of Bavaria (n = 205) and 3 of 11 Animals 2021, 11, 2727 Lower Saxony (n = 92), because these federal states include the highest number of cattle in Germany and the required sample size has been achieved. Farms with co-infections were included in the calculations for both rumen ﬂukes and common liver ﬂuke, respectively. Table 1. Number of sampled farms per German region and federal state. Region Number of Farms per Region Federal State Number of Farms per Federal State North 179 Schleswig-Holstein 51 Hamburg 1 Lower Saxony 92 Bremen 3 Mecklenburg-Western Pomerania 32 East 76 Berlin 0 Brandenburg 16 Saxony 9 Saxony-Anhalt 15 Thuringia 36 South 277 Baden-Wurttemberg 72 Bavaria 205 West 82 North Rhine-Westphalia 45 Hesse 20 Rhineland-Palatinate 16 Saarland 1 Germany 614 Table 1. Number of sampled farms per German region and federal state. 3.1. Study Population Of the 614 participating farms, 571 were dairy farms (region North: n = 173, East: n = 57, South: n = 270, West: n = 71) and 43 suckler cow farms (North: n = 6, East: n = 19, South: n = 7, West: n = 11). No fattening beef farms participated in the study. In total, 506 conventional and 106 organic farms took part in the study, two farms did not provide information. On 457 farms, cattle where grazed or fed with fresh grass, while on 156 farms this was not the case. One farmer did not provide any information on grazing and feeding fresh grass at all. 2.4. Molecular Identiﬁcation of Rumen Fluke Species 2.4. Molecular Identiﬁcation of Rumen Fluke Species From samples positive for rumen flukes, usually 10–20 eggs were isolated and subjected to species identification by amplifying and sequencing the ITS-2 region, as described previ- ously [26]. In brief, genomic DNA was extracted from the eggs with the DirectPCR® Lysis Reagent (Cell) (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and amplified in a 50 µL reaction volume containing 1 µL DreamTaq DNA Polymerase (5 U/µL) (ThermoFisher Scientific, Schwerte, Germany), 5 µL 10x DreamTaq buffer, 1 µL dNTP mix (10 mM each), 2 µL of each primer (ITS-2For and ITS-2Rev [30], 10 µM each), and 10 µL DNA template. Thermocycling was conducted at 95 ◦C for 3 min, 40 cycles of 95 ◦C for 30 s, 53 ◦C for 1 min, 72 ◦C for 45 s, and 72 ◦C for 10 min. The amplification products were custom-sequenced (Seq-lab Sequence Laboratories, Göttingen, Germany) and compared with available sequences in NCBI (National Center for Biotechnology Information) GenBank. 2.5. Statistical Analysis The statistical analyses were carried out using R version 4.0.3. (The R Foundation for Statistical Computing, Vienna, Austria) [31] and Microsoft Excel 2019 (Microsoft Corpo- ration, Redmond, WA, USA). The 95% conﬁdence interval (CI) for rumen and liver ﬂuke prevalences in Germany were calculated using Wald approximation. Logistic regression was carried out to estimate prevalences in the four study regions and two federal states (Bavaria and Lower Saxony), as well as for the production type (dairy/suckler cows) and the agricultural system (organic/conventional). All contrasts (differences) were assessed af- ter model-ﬁtting by the estimated least-squares marginal means (emmeans) with the Tukey p-value correction for multiple comparisons [32]. The ﬁrst level of any variable was used as an intercept. Associations of the variable “coproscopic result” with “agricultural system”, “breed”, and “grazing/feeding fresh grass” were tested via a Pearson’s Chi-Square Test. p-values ≤0.05 were considered statistically signiﬁcant in all analyses. 2.3. Coproscopical Examination Samples received were refrigerated for one day up to eight weeks prior to examination with the sedimentation technique, which was carried out as follows: First, the pooled sample was thoroughly mixed and approximately 10 g faeces were homogenised with tap water. The obtained homogenous suspension was washed through a sieve (mesh size–1500 µm) into a 600 mL beaker by rinsing the sieve with a strong water jet until the beaker was ﬁlled to the 500 mL mark. Each sample was allowed to sediment for at least 15 min before the supernatant was decanted and the beaker reﬁlled with tap water. This process was repeated until the supernatant became clear. The received sediment was transferred into a petri dish through a ﬁne-meshed sieve (mesh size 300 µm). Approximately 8 mL of the sediment were pipetted in an examining chamber, where the sample was stained with three drops 1% methylene blue and examined microscopically for ﬂuke eggs, which were differentiated by their colour. 4 of 11 Animals 2021, 11, 2727 3.2. Prevalence of Rumen and Liver Flukes in Germany The rumen ﬂuke prevalence in Germany amounted to 5.5% (95% CI 3.7–7.4%, 34/614). The highest prevalence was observed in the region North (8.4%), the lowest in South (3.6%). For F. hepatica, a prevalence of 9.5% (95% CI 7.1–11.8%, 58/614) was determined for Germany. The region South revealed the highest prevalence with 14.8%, whereas in West no farm was tested positive. Detailed regional results are provided in Figure 1. Coinfections were identiﬁed on 13 German farms (2.1%, 95% CI 1.0–3.3%). The highest regional coinfection rate was determined in East with coinfections on four farms (5.3%, 95% CI 1.9–12.4%), while logically no coinfections occurred in West (0.0%, 95% CI n. a.). In North, coinfections were detected on three farms (1.7%, 95% CI 0.6–4.9%) and in South on six farms (2.2%, 95% CI 0.1–4.5%). 5 of 11 r farms 95% CI Animals 2021, 11, 2727 South on six farms (2.2%, 95% CI 0.1–4.5%). Figure 1. Prevalence and 95% confidence interval for rumen fluke and F. hepatica prevalence in German regions (coinfections included). Abbreviations: CI, confidence interval; n. a., not applicable. Figure 1. Prevalence and 95% conﬁdence interval for rumen ﬂuke and F. hepatica prevalence in German regions (coinfections included). Abbreviations: CI, conﬁdence interval; n. a., not applicable. Figure 1. Prevalence and 95% confidence interval for rumen fluke and F. hepatica prevalence in German regions (coinfections included). Abbreviations: CI, confidence interval; n. a., not applicable. Figure 1. Prevalence and 95% conﬁdence interval for rumen ﬂuke and F. hepatica prevalence in German regions (coinfections included). Abbreviations: CI, conﬁdence interval; n. a., not applicable. In the statistical comparison of the regions, only the rumen fluke prevalence difference between the regions North and South was statistically significant. Farms in North have a 2.3 time higher odds of their cattle being infected than farms in South (Table 2). These regional differences were also reflected when comparing the two federal states with the highest number of cattle in Germany: In the federal state Lower Saxony (North), rumen fluke prevalence was 10.9% (10/92, 95% CI: 5.8%–18.6%) compared to 4.4% in Bavaria (South; 9/205, 95% CI 2.4%–8.3%), decreasing the odds for a positive result in Bavaria by 0 4 (p = 0 047) In the statistical comparison of the regions, only the rumen ﬂuke prevalence difference between the regions North and South was statistically signiﬁcant. 3.2. Prevalence of Rumen and Liver Flukes in Germany Farms in North have a 2.3 time higher odds of their cattle being infected than farms in South (Table 2). These regional differences were also reﬂected when comparing the two federal states with the highest number of cattle in Germany: In the federal state Lower Saxony (North), rumen ﬂuke prevalence was 10.9% (10/92, 95% CI: 5.8%–18.6%) compared to 4.4% in Bavaria (South; 9/205, 95% CI 2.4%–8.3%), decreasing the odds for a positive result in Bavaria by 0.4 (p = 0.047). y (p ) The comparison between North and South was also statistically significant for F. hepatica. Cattle on a farm in North have 0.4 times lower odds of being infected with liver flukes than those in South (Table 2). Again, comparison of the two federal states mirrored this picture: Liver fluke prevalence in Lower Saxony was 6.5% (6/92, 95% CI: 3.2%–13.9%) and in Bavaria 16.1% (33/205, 95%CI: 11.6%–21.6%), where a farm has 2.6 times higher odds of its cattle being infected than a farm in Lower Saxony (p = 0.031). Table 2. Odds ratio, 95% conﬁdence interval and p-value for rumen ﬂuke and F. hepatica prevalence per German region. Note that in the region West no F. hepatica positive samples were observed. Predictor OR 95% CI p-Value Rumen Flukes South (Intercept) 0.04 0.02–0.07 <0.001 North 2.29 1.08–5.76 0.038 East 2.10 0.75–6.41 0.145 West 0.97 0.22–3.43 0.965 F. hepatica South (Intercept) 0.17 0.12–0.24 <0.001 North 0.39 0.18–0.73 0.007 East 0.52 0.18–1.13 0.134 Abbreviations: OR, odds ratio; CI, conﬁdence interval. The comparison between North and South was also statistically significant for F. hepatica. Cattle on a farm in North have 0.4 times lower odds of being infected with liver fl k h h i S h (T bl 2) A i i f h f d l i d Table 2. Odds ratio, 95% conﬁdence interval and p-value for rumen ﬂuke and F. hepatica prevalence per German region. Note that in the region West no F. hepatica positive samples were observed. The comparison between North and South was also statistically signiﬁcant for F. hepat- ica. Cattle on a farm in North have 0.4 times lower odds of being infected with liver ﬂukes than those in South (Table 2). 3.2. Prevalence of Rumen and Liver Flukes in Germany Again, comparison of the two federal states mirrored this picture: Liver ﬂuke prevalence in Lower Saxony was 6.5% (6/92, 95% CI: 3.2%–13.9%) and in Bavaria 16.1% (33/205, 95%CI: 11.6%–21.6%), where a farm has 2.6 times higher odds of its cattle being infected than a farm in Lower Saxony (p = 0.031). 3.3. Rumen Fluke Species Identiﬁcation Molecular identiﬁcation of infecting rumen ﬂuke species was successful in 24 of the 34 affected farms. Calicophoron daubneyi was identiﬁed on 20 farms, seven of which were located in the region North, ﬁve each in East and South, and three in West. Paramphistomum Animals 2021, 11, 2727 6 of 11 leydeni was detected in the four remaining farms, three of them in North and one in East. Five samples each from the regions North and South could not be identiﬁed. leydeni was detected in the four remaining farms, three of them in North and one in East. Five samples each from the regions North and South could not be identiﬁed. 3.4. Impact of Management Factors on Rumen and Liver Fluke Occurrence and Awareness of Farmers 3.4. Impact of Management Factors on Rumen and Liver Fluke Occurrence and Awareness of Farmers The frequency of patent rumen and liver ﬂuke infections in relation to the management factors production type and agricultural system is shown in Table 3. Due to a rather inhomogeneous and partly incomplete data, the management factors grazing/feeding fresh grass and anthelmintic treatment were only evaluated descriptively (Table 4). Table 3. Prevalence of patent rumen ﬂuke, F. hepatica and co-infections in German cattle farms (n = 614) related to the management factors production type and agricultural system. Note that percent calculation was omitted if less than ﬁve farms (total values) were included. Table 3. Prevalence of patent rumen ﬂuke, F. hepatica and co-infections in German cattle farms (n = 614) related to the management factors production type and agricultural system. Note that percent calculation was omitted if less than ﬁve farms (total values) were included. Total Rumen Flukes a 95% CI F. hepatica a 95% CI Co-Infection 95% CI n n % % n % % n % % Production type Dairy cows 571 23 4.0 2.8–6.1 54 9.5 7.3–12.1 9 1.6 0.9–3.1 Suckler cows 43 11 25.6 14.2–39.4 4 9.3 3.7–21.6 4 9.3 3.2–20.8 Agricultural system Organic 106 11 10.4 5.7–17.4 29 27.4 19.5–36.2 3 2.8 0.9–7.9 Conventional 506 22 4.3 3.0–6.6 29 5.7 4.1–8.2 10 2.0 1.1–3.6 No information 2 1 n. a. n. a. 0 n. a. n. a. 0 n. a. n. a. a. Including coinfections. Abbreviations: n. a., not applicable; CI, conﬁdence interval. Table 4. Descriptive data on the occurrence of patent rumen ﬂuke, F. 4. Discussion The expected rumen ﬂuke prevalence of 5.0% in Germany is in line with the prevalence of 5.5% determined by this study. This is a low ﬁgure compared to other European studies reporting prevalences between 6% and 61% positive herds in the studied region or country [16–24]. However, the comparability with other studies is not always reliable due to different approaches. In some studies, samples were taken only at one abattoir [23], or data from veterinary laboratories were evaluated retrospectively [16–18]. Furthermore, prevalences differ between individual animal or herd level [20,24]. In the present study, representativity is probably given by reaching the predeﬁned number of samples in every region. However, only the samples collected via the PraeRi project [29] were randomly selected, while the other farms were based on the farmers’ willingness to participate in the call, a compromise that had to be made to ensure high numbers of farms within the four regions. The questionnaire was designed to provide the most objective answers possible. On 285 farms, trained and coordinated interviewers using a standard procedure ﬁlled the questionnaire, in the other cases the farmers did it themselves. Paramphistomidosis was detected throughout Germany, as rumen ﬂuke eggs were found in farms of all four regions. This ﬁnding is in line with Huson et al. [15], who identiﬁed paramphistomidosis as an emerging parasitic disease in Europe. Interestingly, prevalences were signiﬁcantly higher in the north than the south of Germany, while the opposite was true for F. hepatica infections. This was unexpected since both trematodes share the intermediate host G. truncatula and, thus, have a similar epidemiology [33,34]. One reason for the contrariness between north and south might be a competition of rumen and liver ﬂuke stages within the intermediate host. This is supported by Jones et al. [19], who found a signiﬁcant negative correlation between F. hepatica and C. daubneyi infection intensities in farms in the UK. Another possible reason is, that Bavaria due to the typical local breed (German Simmental) has probably less international animal trade than other regions with worldwide more “common” breeds such as Holstein Friesian. If this is true, then we expect a slow increase in rumen ﬂukes in south Germany going more in line with liver ﬂuke prevalence in the future. Nevertheless, this result of our study requires further research. 3.3. Rumen Fluke Species Identiﬁcation In contrast, a signiﬁcant correlation Animals 2021, 11, 2727 7 of 11 was found between the breed and F. hepatica infections, since this ﬂuke species was detected more often on farms with German Simmental herds compared to farms with Holstein Friesian herds (p < 0.001). Overall, on the farms tested positive for ﬂukes, mainly adult animals were affected. Both rumen and liver ﬂuke infections showed a statistically signiﬁcant relationship with organic agriculture, where cattle were more often infected than on conventional farms (p = 0.01 and p < 0.001, respectively). Regarding diet, grazing was evaluated together with feeding of fresh grass. Cattle grazing or fed with fresh grass were more likely to be infected with rumen and liver ﬂuke than cattle without such access (p < 0.001). Notably, three positive dairy farms (rumen ﬂukes once, liver ﬂuke twice) categorised as “not grazing” did not provide information about feeding of fresh grass. Deworming was carried out on 274 farms, not on 338 farms, and two farms did not provide any information. Of the farms that treat with anthelmintics, 29 use fasciolicides (10 use triclabendazole, 7 oxyclozanide, 6 closantel, 4 albendazole, 1 oxyclozanide + lev- amisole + triclabendazole, 1 oxyclozanide + closantel). Of these, 23 farms had dairy cows and six had suckler cows. In relation to the total number of these production types, more suckler cow herds are treated with fasciolicides than dairy cow herds. None of the farms without access to grazing or fresh grass reported to deworm with faciolicides. A number of 101 farms dewormed with other drugs, mostly those against gastrointestinal nematodes. No information on the drug used was provided by 144 farms. 3.3. Rumen Fluke Species Identiﬁcation hepatica and co-infections in German cattle farms (n = 614) related to the management factors grazing/feeding fresh grass and anthelminthic treatment. Note that percent calculation was omitted if less than ﬁve farms (total values) were included. Table 4. Descriptive data on the occurrence of patent rumen ﬂuke, F. hepatica and co-infections in German cattle farms (n = 614) related to the management factors grazing/feeding fresh grass and anthelminthic treatment. Note that percent calculation was omitted if less than ﬁve farms (total values) were included. Total Negative Rumen Flukes a F. hepatica a Co-Infection n n % n % n % n % Grazing/feeding fresh grass All herds with access 457 381 83.4 33 7.2 56 12.3 13 2.8 All herds without access 156 153 98.1 1 0.6 2 1.3 0 0.0 Dairy cow herds with access 415 350 84.3 22 5.3 52 12.5 9 2.2 Dairy cow herds without access 155 152 98.1 1 0.6 2 1.3 0 0.0 Suckler cow herds with access 42 31 73.8 11 26.2 4 9.5 4 9.5 Suckler cow herds without access 1 1 n. a. 0 n. a. 0 n. a. 0 n. a. No information 1 1 n. a. 0 n. a. 0 n. a. 0 n. a. Anthelminthic treatment None 338 311 92.0 8 2.4 20 5.9 1 0.3 Fasciolicides 29 20 69.0 5 17.2 5 17.2 1 3.4 Others than fasciolicides 101 85 84.2 11 10.9 11 10.9 6 5.9 Not speciﬁed 144 118 81.9 10 6.9 21 14.6 5 3.5 No information 2 1 n. a. 0 n. a. 1 n. a. 0 n. a. a. Including coinfections. Abbreviations: n. a., not applicable. Rumen ﬂuke prevalence in German dairy herds was 4.0% (95% CI: 2.8%–6.1%) and in suckler cow herds 25.6% (95% CI: 14.2%–39.4%). According to these results, suckler cow herds have 7.7 times higher odds of being infected with rumen ﬂukes than dairy herds (p < 0.001). Fasciola hepatica prevalence in dairy herds amounted to 9.5% (95% CI: 7.3%–12.1%) and in suckler cow herds to 9.3% (95% CI: 3.7%–21.6%) with no statistically signiﬁcant difference between these production types (p = 0.974). Cattle breeds on dairy farms were predominantly Holstein Friesian in the North, East, and West, and German Simmental or German Brown Swiss in the South. There was no statistical correlation between the breed and rumen ﬂuke infections (p = 0.07). 4. Discussion In eastern Germany, the same prevalence values were determined for rumen and liver ﬂukes, with the latter occurring signiﬁcantly less frequently than in the south. Surprisingly, no liver ﬂuke eggs were detected in samples from the west (exclusively based on the farmers’ Animals 2021, 11, 2727 8 of 11 willingness to participate), although patent infections in cattle were recently reported [26]. One possible explanation is that the sample size (86 samples) from this region was too small to detect a possibly very low F. hepatica infection extensity, or that farmers may have chosen not to participate if they had known problems with fasciolosis. Unfortunately, no other recent data on the occurrence on liver ﬂukes in western Germany are available. In a bulk tank milk (BTM) study conducted in 2008, seroprevalences amounted to 9–19% in the German federal states included in the region “West” [27], but this region was not included in a recent BTM study on fasciolosis in Germany examining samples from 2017 to 2019 [28]. Although seroprevalence values are difﬁcult to compare with the prevalence determined in our investigation, as the sensitivity differs and only patent infections can be detected by coproscopy [13,35], the most recent seroprevalence study conﬁrms higher exposure to liver ﬂukes in the south than in the north of Germany [28]. However, F. hepatica BTM seroprevalence in the region “East” was only 1% [28] compared to nearly 8% patent infections in our study. This may be explained by different sample sets (BTM samples were collected in 2017–2019, faecal samples in 2018–2020, and participating farms overlapped only partially), but needs to be clariﬁed in future studies. Overall, the reason for the observed regional differences in patent rumen and liver ﬂuke infections cannot be reliably explained by this study. Other authors assume climatic and environmental factors, as well as the import of animals as possible reasons for re- gional differences in prevalence [19,36,37]. Again, further research is required to elucidate underlying epidemiological factors. The detection of C. daubneyi in all four regions is in line with the ﬁndings at European level, as this species has been mainly detected in recent decades [16,17,19,22,24]. In contrast, P. leydeni, which occurred on four farms, was identiﬁed for the ﬁrst time in Germany. In Europe, P. leydeni has so far been found rarely overall [24,36,38], but in Argentina it appears to be the most ubiquitous species [39]. In contrast to C. 4. Discussion Animals 2021, 11, 2727 9 of 11 This has already been considered by other authors as a cause for the detection of trematode infections in farms without grazing [17,21]. This has already been considered by other authors as a cause for the detection of trematode infections in farms without grazing [17,21]. The evaluation of the susceptibility of different cattle breeds to rumen and liver ﬂukes cannot be conclusively assessed due to insufﬁcient representativity of the samples. In dairy farms, it was not possible to establish a link between rumen ﬂuke detection and the cattle breed tested. This is in line with other published results [21,37,42]. The breed distribution in suckler cow farms was too various for analysis. Although German Simmental cattle were more frequently infected with F. hepatica than Holstein Friesians, this is most likely due to the generally higher liver ﬂuke prevalence in the south of Germany, where mainly German Simmental cattle are kept. For this reason, a predisposition of a speciﬁc cattle breed is rather unlikely. In most of the farms studied, rumen and liver ﬂuke infections were detected only in adult animals (after ﬁrst calving or older than 2.5 years). However, some farms had not sampled enough young animals. Most authors also found that the prevalence of rumen ﬂukes was higher in animals older than 2.5 years than in younger animals [20,21,37], while others did not show any association between age and rumen ﬂuke infection [42]. Since the drug used for deworming was not speciﬁed for 144 farms, the effect of deworming on rumen and liver ﬂuke prevalence could not be conclusively determined. 4. Discussion daubneyi, whose intermediate host is the lymnaeid snail G. truncatula, P. leydeni has planorbid snails as intermediate hosts. However, in order to be able to analyse the distribution pattern of the two rumen ﬂuke species more precisely, the epidemiology of the various Paramphistomidae must be further investigated. g Cattle grazing or fed with fresh grass were more likely to be infected with rumen or liver ﬂuke than cattle without such access. This result was expected since the development of the parasites requires freshwater snails as intermediate host and infective metacercariae encyst on plants. The variable “grazing/ feeding fresh grass” is very likely the reason for the higher prevalence of both ﬂuke types on organic farms, because due to the EU legislation cattle must have permanent access to open space, preferably to pastures. Additionally, grazing and feeding of fresh grass seems to be the reason for the higher risk of suckler cows of being infected with rumen ﬂukes compared to dairy cows. This result is consistent with studies from Spain and the Netherlands [22,24]. In our study, a very similar prevalence of F. hepatica in dairy and suckler cow farms was found. The reason for this could be that suckler cow herds are more likely to be dewormed with fasciolicides, as they are known to have a higher risk of infection compared to dairy herds. Since treatment with triclabendazole and closantel against liver ﬂukes is usually not effective against rumen ﬂuke infection [5], the prevalence of rumen ﬂukes remains high. In addition, when ruminants are treated against F. hepatica, egg excretion on pastures is reduced. As a result, rumen ﬂukes have less competition at the intermediate host level and are, therefore, able to infect more snails [19]. An unexpected result is the detection of trematode infections in three farms without grazing (rumen ﬂukes once, liver ﬂuke twice) in this study. As there was no information on the feeding regime available, it can only be assumed that the feeding of fresh grass or ﬁeld dried hay is the cause of the infection. The possibility of infection by feeding fresh grass or ﬁeld dried hay contaminated with metacercariae has already been proved for F. hepatica [40,41] and this could also apply to an infection with rumen ﬂukes [21]. It would also be possible that cattle already infected elsewhere entered the farm via livestock trades. References Paramphistomum daubneyi sp. nov. from cattle and its snail host in the Kenya Highlands. Para [CrossRef] 7. Jones, R.A.; Williams, H.W.; Dalesman, S.; Brophy, P.M. Conﬁrmation of Galba truncatula as an intermediate host snail for Calicophoron daubneyi in Great Britain, with evidence of alternative snail species hosting Fasciola hepatica. Parasites Vectors 2015, 8, 656. [CrossRef] [PubMed] 8. Szmidt-Adjide, V.; Abrous, M.; Adjide, C.C.; Dreyfuss, G.; Lecompte, A.; Cabaret, J.; Rondelaud, D. Prevalence of Paramphistomum daubneyi infection in cattle in central France. Vet. Parasitol. 2000, 87, 133–138. [CrossRef] 9. Mason, C.; Stevenson, H.; Cox, A.; Dick, I.; Rodger, C. Disease associated with immature paramphistom Rec. 2012, 170, 343–344. [CrossRef] nson, H.; Cox, A.; Dick, I.; Rodger, C. Disease associated with immature paramphistome infection in sheep. 43–344. [CrossRef] 10. Millar, M.; Colloff, A.; Scholes, S. Disease associated with immature paramphistome infection. Vet. Rec. 2012, 171, 509–510. [CrossRef] 11. O’Shaughnessy, J.; Garcia-Campos, A.; McAloon, C.G.; Fagan, S.; De Waal, T.; McElroy, M.; Casey, M.; Good, B.; Mulcahy, G.; Fagan, J.; et al. Epidemiological investigation of a severe rumen ﬂuke outbreak on an Irish dairy farm. Parasitology 2017, 145, 948–952. [CrossRef] [PubMed] 12. Fuertes, M.; Perez, V.; Benavides, J.; Gonzalez-Lanza, M.C.; Mezo, M.; Gonzalez-Warleta, M.; Giraldez, F.J.; Fernandez, M.; Manga-Gonzalez, M.Y.; Ferreras, M.C. Pathological changes in cattle naturally infected by Calicophoron daubneyi adult ﬂukes. Vet. Parasitol. 2015, 209, 188–196. [CrossRef] [PubMed] 13. Sargison, N.; Francis, E.; Davison, C.; Bronsvoort, B.M.; Handel, I.; Mazeri, S. Observations on the biology, epidemiology and economic relevance of rumen ﬂukes (Paramphistomidae) in cattle kept in a temperate environment. Vet. Parasitol. 2016, 219, 7–16. [CrossRef] 14. Huson, K.M.; Atcheson, E.; Oliver, N.A.M.; Best, P.; Barley, J.P.; Hanna, R.E.B.; McNeilly, T.N.; Fang, Y.; Haldenby, S.; Paterson, S.; et al. Transcriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, Calicophoron daubneyi reveals adaptation to a unique host environment. Mol. Cell Proteom. 2021, 20, 100055. [CrossRef] [PubMed] y p q 15. Huson, K.M.; Oliver, N.A.M.; Robinson, M.W. Paramphistomosis of Ruminants: An Emerging Parasitic Disease in Europe. Trends Parasitol. 2017, 33, 836–844. [CrossRef] 16. Mage, C.; Bourgne, H.; Toullieu, J.M.; Rondelaud, D.; Dreyfuss, G. Fasciola hepatica and Paramphistomum daubneyi: Changes in prevalences of natural infections in cattle and in Lymnaea truncatula from central France over the past 12 years. Vet. Res. 2002, 33, 439–447. [CrossRef] [PubMed] 17. 5. Conclusions Our study revealed that the prevalence of rumen ﬂukes is generally rather low in Germany and distributes unevenly. Comparing rumen and liver ﬂuke occurrence, the distribution between the regions North and South is counterbalanced. C. daubneyi was the most frequently identiﬁed species, while P. leydeni was ﬁrst recorded in Germany. A statistically signiﬁcant association was observed between the prevalence of ﬂukes and grazing/feeding fresh grass and, therefore, also between prevalence and organic farming and additionally, in case of rumen ﬂukes, with suckler cow husbandry. For reasons of animal welfare, animal health and economic viability, the ﬂuke prevalence in Germany should continue to be monitored or repeated in a few years. Perhaps a serological method for rumen ﬂukes can than already be used [14] in addition to serological liver ﬂuke detec- tion. Finally, paramphistomidosis should be given more attention by veterinarians and farmers [43] and many (epidemiological) research questions remain to be addressed. Author Contributions: Conceptualisation, G.K.-S., C.S. and C.W.; formal analysis, T.F. and Y.Z.; investigation, T.F. and C.S.; supervision, G.K.-S., C.S. and C.W.; visualisation, T.F.; resources, G.K.-S. and C.S.; Writing—Original draft preparation, T.F.; Writing—Review and editing, G.K.-S., C.S., Y.Z. and C.W.; project administration, G.K.-S., C.S. and C.W.; funding acquisition, G.K.-S. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by Boehringer Ingelheim Vetmedica GmbH (research cooperation number 402039). The PraeRi-Project, with which 285 farms were sampled, was funded by the Federal Ministry of Food and Agriculture and Federal Ofﬁce for Agriculture and Food, grant numbers 2814HS006 (University of Veterinary Medicine Hannover), 2814HS007 (Freie Universität Berlin), and 2814HS008 (Ludwig-Maximilians-Universität München). Institutional Review Board Statement: Ethical review and approval was not required because all animals were sampled by non-invasive procedures (faecal sampling). Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. Data Availability Statement: Data supporting reported results is contained within the article. Acknowledgments: The authors wish to thank all veterinarians and farmers who participated in sample collection. Furthermore, we are grateful to Ingrid Hartmann, Sandra Kirsch, Sue Jauernig, Lily Schöffmann, Sabrina Würﬂ, and Bettina Rinjes for assistance in faecal examinations and Ulla Küttler for excellent technical assistance in molecular analyses. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 10 of 11 Animals 2021, 11, 2727 References 1. Eduardo, S.L. The taxonomy of the family Paramphistomidae Fischoeder, 1901 with special reference to the morphology of species occurring in ruminants. I. General considerations. Syst. Parasitol. 1982, 4, 7–57. [CrossRef] p g y , , [ ] 2. Sey, O. Revision of the amphistomes of European ruminants. Parasitol. Hung. 1980, 13, 13–25. y p p g 3. Millar, M.; Foster, A.; Mitchell, G.; Skuce, P.; Wessels, J.; Velo-Rego, E.; Collins, R.; Stevenson, H. Rumen ﬂuke in South American camelids in Great Britain. Vet. Rec. 2017, 181, 123–124. 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https://openalex.org/W4366278215	https://acp.copernicus.org/preprints/acp-2022-858/acp-2022-858.pdf	English	null	Comment on acp-2022-858	null	2,023	cc-by	13,558	Atlantic Multi-decadal Oscillation Modulates the Relationship Between El Niño-Southern Oscillation and Fire Weather in Australia Guanyu Liu1, Jing Li1, Tong Ying1 1 Department of Atmospheric and Oceanic Sciences, School of Physics, Peking University, Beijing, China, 100871 1 Department of Atmospheric and Oceanic Sciences, School of Physics, Peking University, Be Abstract El Niño–Southern Oscillation (ENSO) is an important driver of fire weather in Australia. The correlation between ENSO and Australian fire weather has strengthened over the recent two decades. However, the causes for this change have not been well investigated. Here, using reanalysis datasets and numerical model simulations, we show that the Atlantic not been well investigated. Here, using reanalysis datasets and numerical model simulations, we show that the Atlantic Multi-decadal Oscillation (AMO) could potentially modulate the ENSO-Australian fire weather relationship. The correlation 10 between ENSO and Australia fire weather index (FWI) increases from 0.17 to 0.70 when AMO shifts from its negative phase to its positive phase. This strengthening effect can be explained by the atmospheric teleconnection mechanisms. Specifically, the positive AMO phase, manifested as a warming North and Tropical Atlantic, generates Rossby wave trains and results in high pressures over Australia, which increases local temperature and wind speed but decreases precipitation. This signal superimposes ENSO and thus serves to enhance the ENSO effect on Australian fire weather. 15 Multi-decadal Oscillation (AMO) could potentially modulate the ENSO-Australian fire weather relationship. The correlation 10 between ENSO and Australia fire weather index (FWI) increases from 0.17 to 0.70 when AMO shifts from its negative phase to its positive phase. This strengthening effect can be explained by the atmospheric teleconnection mechanisms. Specifically, the positive AMO phase, manifested as a warming North and Tropical Atlantic, generates Rossby wave trains and results in high pressures over Australia, which increases local temperature and wind speed but decreases precipitation. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. 1 Introduction Australia is the main fire-burning area in the world, as it has more fire-prone lands than the other continents due to the highly flammable biota (Duc et al., 2018). This region frequently suffers from wildfires in austral spring and summer each year (Cai et al., 2009; Dowdy, 2018), especially in the catastrophic 2019/20 wildfire season (Abram et al., 2021; Arriagada et al., 2020). Wildfires in Australia also have a significant impact on the environment (Damany-Pearce et al., 2022; Nguyen et al., 20 2021), human health (Nguyen et al., 2021; Yu et al., 2020) and ecological systems (Liu et al., 2022). Moreover, as the frequency and intensity of heat waves and droughts in the region increase due to climate warming, wildfires in the region are expected to become more intense, and the related impacts might be more far-reaching (Clarke and Evans, 2018; Jain et al., 2021). Australia is the main fire-burning area in the world, as it has more fire-prone lands than the other continents due to the highly flammable biota (Duc et al., 2018). This region frequently suffers from wildfires in austral spring and summer each year (Cai et al., 2009; Dowdy, 2018), especially in the catastrophic 2019/20 wildfire season (Abram et al., 2021; Arriagada et al., 2020). Wildfires in Australia also have a significant impact on the environment (Damany-Pearce et al., 2022; Nguyen et al., 20 2021) h h lth (N t l 2021 Y t l 2020) d l i l t (Li t l 2022) M th Australia is the main fire-burning area in the world, as it has more fire-prone lands than the other continents due to the highly flammable biota (Duc et al., 2018). This region frequently suffers from wildfires in austral spring and summer each year (Cai et al., 2009; Dowdy, 2018), especially in the catastrophic 2019/20 wildfire season (Abram et al., 2021; Arriagada et al., et al., 2009; Dowdy, 2018), especially in the catastrophic 2019/20 wildfire season (Abram et al., 2021; Arriagada et al., 2020). Wildfires in Australia also have a significant impact on the environment (Damany-Pearce et al., 2022; Nguyen et al., 20 2021), human health (Nguyen et al., 2021; Yu et al., 2020) and ecological systems (Liu et al., 2022). 1 Introduction We hope the results can provide insights into the causes of the variability of fire weather in Australia and improve wildfire modeling and forecast in this area. 1 Introduction Moreover, as the frequency and intensity of heat waves and droughts in the region increase due to climate warming, wildfires in the region are expected to become more intense, and the related impacts might be more far-reaching (Clarke and Evans, 2018; Jain et al., 2021). 2020). Wildfires in Australia also have a significant impact on the environment (Damany-Pearce et al., 2022; Nguyen et al., 20 2021), human health (Nguyen et al., 2021; Yu et al., 2020) and ecological systems (Liu et al., 2022). Moreover, as the frequency and intensity of heat waves and droughts in the region increase due to climate warming, wildfires in the region are expected to become more intense, and the related impacts might be more far-reaching (Clarke and Evans, 2018; Jain et al., 2021). 25 As an area with great productivity, wildfire occurrence and spread in Australia are primarily determined by fire weather, so the enhancement of fire weather indicates the increase of fire (Bradstock, 2010; Clarke et al., 2019). Australian fire weather is closely related to the El Niño-Southern Oscillation (ENSO), the leading climate mode controlling key determinants of fire (moisture and temperature variability) (Risbey et al., 2009). In general, El Niño events (the warm phase of ENSO) starve As an area with great productivity, wildfire occurrence and spread in Australia are primarily determined by fire weather, so the enhancement of fire weather indicates the increase of fire (Bradstock, 2010; Clarke et al., 2019). Australian fire weather is closely related to the El Niño-Southern Oscillation (ENSO), the leading climate mode controlling key determinants of fire (moisture and temperature variability) (Risbey et al., 2009). In general, El Niño events (the warm phase of ENSO) starve 1 1 Australia of precipitation and promote long-term droughts and fire weather (Chen et al., 2017; M. Mariani et al., 2016). 30 Moreover, the relationship between ENSO and Australian fire weather has strengthened (Michela Mariani et al., 2018) in the 21st century, but the causes for this strengthening remain unclear. Additionally, the underlying mechanism accounting for this change is poorly understood. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Australia of precipitation and promote long-term droughts and fire weather (Chen et al., 2017; M. Mariani et al., 2016). 1 Introduction 30 Moreover, the relationship between ENSO and Australian fire weather has strengthened (Michela Mariani et al., 2018) in the 21st century, but the causes for this strengthening remain unclear. Additionally, the underlying mechanism accounting for this change is poorly understood. Australia of precipitation and promote long-term droughts and fire weather (Chen et al., 2017; M. Mariani et al., 2016). 30 Moreover, the relationship between ENSO and Australian fire weather has strengthened (Michela Mariani et al., 2018) in the 21st century, but the causes for this strengthening remain unclear. Additionally, the underlying mechanism accounting for this change is poorly understood. Decadal climate variability may also impact fire weather and impose interannual variability. For example, the Atlantic- 35 induced anomalies have a significant impact on Pacific SST and circulation changes (Chikamoto et al., 2016; Chikamoto et al., 2020; Lv et al., 2022), contributing nearly 75% of the tropical SST changes during the satellite era (Li et al., 2016). Markedly, Atlantic decadal variability, i.e., Atlantic Multi-decadal Oscillation (AMO), shifts its phase from negative to positive in the late 1990s. This shift may potentially influence ENSO as well as its teleconnection patterns, including the meteorological conditions in Australia that are closely related to fire weather. In this study, we investigate the Atlantic 40 impact on Australian fire weather and its potential contribution to the ENSO-Australian fire weather relationship. We also attempt to clarify the possible mechanism accounting for this impact. We hope the results can provide insights into the causes of the variability of fire weather in Australia and improve wildfire modeling and forecast in this area. meteorological conditions in Australia that are closely related to fire weather. In this study, we investigate the Atlantic 40 impact on Australian fire weather and its potential contribution to the ENSO-Australian fire weather relationship. We also attempt to clarify the possible mechanism accounting for this impact. We hope the results can provide insights into the causes of the variability of fire weather in Australia and improve wildfire modeling and forecast in this area. meteorological conditions in Australia that are closely related to fire weather. In this study, we investigate the Atlantic 40 impact on Australian fire weather and its potential contribution to the ENSO-Australian fire weather relationship. We also attempt to clarify the possible mechanism accounting for this impact. 2.1 Fire weather index, meteorological data, and climate variability index 80 80 In addition, simple stochastic variability can drive perceived decadal changes and needs to be considered a potential driver of variable ENSO behaviors. In order to obtain a more robust conclusion, we estimated all p-values by considering the autocorrelation using the method by Storch and Zwiers (2000). 2.1 Fire weather index, meteorological data, and climate variability index 60 Finally, climate variability indices, such as monthly AMO and Niño 3.4 indices from 1981 to 2019 from the NCAR climate data guide (Trenberth, 1997; Trenberth and Stepaniak, 2001; Trenberth and Shea, 2006) are used to represent the variability Finally, climate variability indices, such as monthly AMO and Niño 3.4 indices from 1981 to 2019 from the NCAR climate data guide (Trenberth, 1997; Trenberth and Stepaniak, 2001; Trenberth and Shea, 2006) are used to represent the variability of AMO and ENSO respectively. Moreover, we have explored dataset dependence of the main results by using various SST 70 datasets, including COBE-SST 2 (COBE), the Met Office Hadley Centre’s SST (Hadley), and Kaplan Extended SST v2 (Kaplan), NOAA Extended Reconstruction SST V4 (NOAA V4) and NOAA Extended Reconstruction SST V5 (NOAA V5) (Huang et al., 2015; Huang et al., 2017; Kaplan et al., 1998; Liu et al., 2015; Rayner et al., 2003). Both the general trends and variability of AMO and the ENSO-Australian FWI relationship under different AMO phases, as revealed by these datasets, are highly consistent (Figure S1-S2). 75 Finally, climate variability indices, such as monthly AMO and Niño 3.4 indices from 1981 to 2019 from the NCAR climate data guide (Trenberth, 1997; Trenberth and Stepaniak, 2001; Trenberth and Shea, 2006) are used to represent the variability of AMO and ENSO respectively. Moreover, we have explored dataset dependence of the main results by using various SST 70 datasets, including COBE-SST 2 (COBE), the Met Office Hadley Centre’s SST (Hadley), and Kaplan Extended SST v2 (Kaplan), NOAA Extended Reconstruction SST V4 (NOAA V4) and NOAA Extended Reconstruction SST V5 (NOAA V5) (Huang et al., 2015; Huang et al., 2017; Kaplan et al., 1998; Liu et al., 2015; Rayner et al., 2003). Both the general trends and variability of AMO and the ENSO-Australian FWI relationship under different AMO phases, as revealed by these datasets, are highly consistent (Figure S1-S2). 75 Subsequently, we calculated the meteorological variable composite maps of ENSO (AMO). These composite maps are defined as the meteorological variables of Niño 3.4 (AMO) positive phase years minus those in Niño 3.4 (AMO) negative phase years. The positive phase year of Niño 3.4 is defined when the absolute value of the moving average of the Niño 3.4 index in three months exceeds 0.5 ℃for at least five months, and vice versa (Trenberth, 1997). 2.1 Fire weather index, meteorological data, and climate variability index Fire occurrence and spread are determined by the confluence of sufficient fuel, an ignition source, and suitable weather, i.e., the fire triangle including heat, fuel, and oxygen (Krawchuk et al., 2009). In areas of high biomass (abundant fuel), such as Australia, fire occurrence over time is primarily modulated by fuel moisture (i.e., weather) and ignitions (lightning and humans) (Bradstock, 2010). The Fire Weather Index (FWI) is a numeric rating of fire intensity that depends on weather 50 conditions and has been shown as an effective indicator of fire danger because it contains both a component of fuel availability (drought conditions) and a measure of the ease of spread (Simpson et al., 2014). Daily FWI (0.25° x 0.25°, 1981- 2019) is obtained from the historical data of fire danger indices from the Copernicus Emergency Management Service (Copernicus Contractor, 2021). This dataset provides a complete historical reconstruction of meteorological conditions favorable to the start, spread and sustainability of fires. The daily FWI is then converted to monthly FWI for later analysis. 55 Meteorological variables, such as surface temperature, precipitation, and wind speed, are critical factors in calculating FWI (Lawson, 2008). Therefore, we primarily use monthly meteorological variables from the re-gridded and interpolated ERA5 2 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. reanalysis datasets (Hersbach,2019) from climate data store (CDS) disks to examine responses of meteorological variables to remote SST forcing. According to Lawson (2008), we select 2m temperature (T2M), total precipitation (TP), sea level 60 pressure (SLP) and 10m wind speed (WND10) from 1981 to 2019, with a resolution of 0.25 ° × 0.25 ° . WND10 is calculated by the UV component of 10m wind (U10, V10) to represent the intensity of the 10m wind. In order to verify the robustness of our results, we also used the same variables from the NCEP-NCAR Reanalysis 1 datasets and MERRA-2 datasets (Global Modeling and Assimilation Office, 2015a; Global Modeling and Assimilation Office, 2015b; Kalnay et al., 1996), and re-gridded and interpolated ERA5 reanalysis datasets (Hersbach,2019) from 1959 to 2019. All meteorological 65 variables were linearly detrended to minimize the impact of global warming on our analysis. 2.2 Ocean basin experiments 85 To examine the meteorological responses and physical mechanism of the Atlantic impacts on Australia, we performed a series of ocean basin experiments using the Community Earth System Model-Community Atmosphere Model version 4 (CESM-CAM4) (Gent et al., 2011). The North and Tropical Atlantic Sea Surface Temperature (SST) variabilities were 3 3 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. respectively added to the model to examine the response of meteorological variables in Australia to these remote forcings. Specifically, the monthly SST variability from 1979 to 2015 is added in the North Atlantic region of 25 ° N- 90 75 ° N and the tropical Pacific region of 20 ° N-20 ° S, with a buffer zone of 10 ° added to the north and south of each region. SST forcings over the other regions remain seasonal varying climatological SST. An ensemble simulation with eight members is performed, and the ensemble means are considered the atmospheric response to SST forcing in the target ocean basin. We call this experiment the Ocean Basin Experiments (OBE). These experiments are similar to previous studies (Liu et al., 2022; Wang et al., 2018) 95 forcings. Specifically, the monthly SST variability from 1979 to 2015 is added in the North Atlantic region of 25 ° N- 90 75 ° N and the tropical Pacific region of 20 ° N-20 ° S, with a buffer zone of 10 ° added to the north and south of each region. SST forcings over the other regions remain seasonal varying climatological SST. An ensemble simulation with eight members is performed, and the ensemble means are considered the atmospheric response to SST forcing in the target ocean basin. We call this experiment the Ocean Basin Experiments (OBE). These experiments are similar to previous studies (Liu et al., 2022; Wang et al., 2018) 95 Since the peak season for fire weather in Australia is mainly the local spring (SON, Earl, and Simmonds, 2017), we selected the model responses to SON North Atlantic SST (25°N-65°N, 10°W-60°W) and Tropical Atlantic SST (0-20° N, 10°W-60°W) in the OBE for further analysis. 3.1 Influence of AMO on the relationship between ENSO and Australian FWI As shown in previous studies, Australian fire weather is closely correlated with ENSO (Harris et al., 2014; Mariani et al., 2016). Here, we also found that Australian FWI is significantly positively correlated at R≈0.53 (p<0.01) with Niño 3.4 index. El Niño events (anomalously positive Niño 3.4 index) induce warmer-than-average temperatures and reduced precipitation across most of Australia in SON. These meteorological anomalies create favorable conditions for igniting and 105 spreading wildfires (Keeley et al., 2022; Littell et al., 2016). In addition to temperature and precipitation, Australia is dominated by a high-pressure center and enhanced northwest winds, which also contribute to wildfires by expanding the burnt area (Clements et al., 2008; Koo et al., 2010). However, the correlation between ENSO and Australian fire weather is not stable but changes over time. The previous study 110 indicates that the impact of ENSO on Southern Hemisphere fire weather, including in Australia, has become stronger since the start of this century (Mariani et al., 2018). Our analysis also found that the ENSO-Australian FWI correlation increased from 0.34 during 1981-1999 to 0.66 during 2000-2019 (Figure S3 a). However, the other two major southern hemisphere wildfire regions, South Africa and South America, do not show obvious trends (Figure S3 b, c). Mariani et al. (2018) However, the correlation between ENSO and Australian fire weather is not stable but changes over time. The previous study 110 indicates that the impact of ENSO on Southern Hemisphere fire weather, including in Australia, has become stronger since the start of this century (Mariani et al., 2018). Our analysis also found that the ENSO-Australian FWI correlation increased from 0.34 during 1981-1999 to 0.66 during 2000-2019 (Figure S3 a). However, the other two major southern hemisphere wildfire regions, South Africa and South America, do not show obvious trends (Figure S3 b, c). Mariani et al. (2018) hypothesized that this correlation transition is likely due to global warming. Given that the global warming trend slowed 115 down or even paused between ~2000 and 2015, this hypothesis seems unjustified, motivating us to find other potential causes. hypothesized that this correlation transition is likely due to global warming. Given that the global warming trend slowed 115 down or even paused between ~2000 and 2015, this hypothesis seems unjustified, motivating us to find other potential causes. hypothesized that this correlation transition is likely due to global warming. 3.1 Influence of AMO on the relationship between ENSO and Australian FWI Given that the global warming trend slowed 115 down or even paused between ~2000 and 2015, this hypothesis seems unjustified, motivating us to find other potential causes. 4 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Around 2000, a significant global decadal climate variability, the AMO, also transitioned from its negative to its positive phase. Atlantic climate variability has been shown to have a broad impact on the global climate system, including the Pacific 120 Ocean (Chikamoto et al., 2016), the Indian Ocean (Xue et al., 2018), and even the Antarctic (Ren et al., 2022). Indeed, the ENSO-Australian FWI relationship dramatically changed between different AMO phases, with their correlation coefficients increasing from 0.17 to 0.70 between negative and positive AMO phases (Figure 1a), similar to the correlation increase before and after 2000 (Figure S1a) but with a greater contrast. We further calculate a running correlation between ENSO and Australian FWI and compare its time series with that of the AMO (Figure 1b). It is clearly seen that at the negative AMO 125 phase, the ENSO-Australia FWI correlation is low and mostly below 0.3, whereas it increases to above 0.6 or even 0.8 at positive AMO. The time of the transition of the two time series also agrees well, in the late 1990s, although the correlation time series shows a slight lead mainly due to the smoothing treatment. Australian FWI and compare its time series with that of the AMO (Figure 1b). It is clearly seen that at the negative AMO 125 phase, the ENSO-Australia FWI correlation is low and mostly below 0.3, whereas it increases to above 0.6 or even 0.8 at positive AMO. The time of the transition of the two time series also agrees well, in the late 1990s, although the correlation time series shows a slight lead mainly due to the smoothing treatment. 3.2 Impact of North and Tropical Atlantic on Australian FWI To explain why AMO may reinforce the ENSO-Australian fire weather relationship. It is necessary to examine the impact on 130 Australian meteorological conditions, especially the coherent impact between ENSO and AMO. We approach this question by analyzing reanalysis datasets and OBE simulation results. To explain why AMO may reinforce the ENSO-Australian fire weather relationship. It is necessary to examine the impact on 130 Australian meteorological conditions, especially the coherent impact between ENSO and AMO. We approach this question by analyzing reanalysis datasets and OBE simulation results. As mentioned above, positive ENSO, i.e., El Niño events, corresponds to higher SLP, higher temperature, and lower precipitation, which is conducive to fire generation. For the surface wind, the composite field of the surface wind has the 135 same direction (Southeast wind) as the climatology surface wind, which plays a vital role in strengthening the wind speed and accelerating the expansion of wildfires. This relationship is further corroborated by the composite difference maps of temperature, precipitation, and circulation field between positive and negative Niño 3.4 index conditions We first compare these El Niño-related meteorological responses in Australia during positive (Figure 2d-f) and negative 140 AMO phases (Figure 2g-i) and find that temperature increase and precipitation decrease are both stronger during El Niño when AMO is at its positive phase. The response of SLP and surface winds are even more intense during positive AMO phases compared with the negative phases (Figure 2f & i). Specifically, there might be higher SLP and stronger surface wind during El Niño in the positive phase of AMO. This phenomenon indicates that AMO may potentially enhance the relationship between ENSO and Australian FWI. 145 We further conduct separate composite analysis of the Australian meteorological fields during positive and negative AMO phases (Figure 2j-l), which appears to show similar patterns to that of ENSO (Figure 2a-c). Specifically, the positive AMO 5 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. also corresponds to warmer temperatures and lower precipitation, although the area with significant precipitation changes is mostly concentrated in the southern part. Given that wildfires are also more intense in south Australia (Hennessy et al., 2005), 150 these AMO-associated meteorological anomalies thus also serve to enhance fire weather. Positive AMO also corresponds to the easterly anomaly wind in eastern Australia. 3.2 Impact of North and Tropical Atlantic on Australian FWI Due to the topography of the Great Watershed in eastern Australia, the easterly wind adiabatically sinks, increasing temperature and reducing humidity, resulting in a high temperature and low humidity environment over this biomass-rich area (Kriwoken, 1996). These similarities suggest that positive AMO may reinforce the ENSO effect on Australian FWI and result in more severe and extensive wildfires. We also examined the 155 differences in the ENSO events between the two periods with different AMO phases (1981-1999 and 2000-2019, Figure S4), but found no significant difference in their spatial patterns (Figure S4e & f), indicating that ENSO flavor is an unlikely factor for the shifted ENSO-Australian FWI relationship. also corresponds to warmer temperatures and lower precipitation, although the area with significant precipitation changes is mostly concentrated in the southern part. Given that wildfires are also more intense in south Australia (Hennessy et al., 2005), 150 these AMO-associated meteorological anomalies thus also serve to enhance fire weather. Positive AMO also corresponds to the easterly anomaly wind in eastern Australia. Due to the topography of the Great Watershed in eastern Australia, the easterly wind adiabatically sinks, increasing temperature and reducing humidity, resulting in a high temperature and low humidity environment over this biomass-rich area (Kriwoken, 1996). These similarities suggest that positive AMO may 150 reinforce the ENSO effect on Australian FWI and result in more severe and extensive wildfires. We also examined the 155 differences in the ENSO events between the two periods with different AMO phases (1981-1999 and 2000-2019, Figure S4), but found no significant difference in their spatial patterns (Figure S4e & f), indicating that ENSO flavor is an unlikely factor for the shifted ENSO-Australian FWI relationship. reinforce the ENSO effect on Australian FWI and result in more severe and extensive wildfires. We also examined the 155 differences in the ENSO events between the two periods with different AMO phases (1981-1999 and 2000-2019, Figure S4), but found no significant difference in their spatial patterns (Figure S4e & f), indicating that ENSO flavor is an unlikely factor for the shifted ENSO-Australian FWI relationship. Because reanalysis datasets contain many different physical processes, to isolate the AMO effect, we continue to examine 160 the responses of major meteorological variables (T2M, TP, SLP, U10, V10) to North and Tropical Atlantic in the OBE. 3.2 Impact of North and Tropical Atlantic on Australian FWI The detrended and normalized SON meteorological variables are regressed on the detrended and normalized SON Tropical Atlantic and North Atlantic SST in OBE. The regression coefficients, representing the responses of local meteorological variables to remote SST forcings in the corresponding ocean basin, are shown in Figure 3. Because reanalysis datasets contain many different physical processes, to isolate the AMO effect, we continue to examine 160 the responses of major meteorological variables (T2M, TP, SLP, U10, V10) to North and Tropical Atlantic in the OBE. The detrended and normalized SON meteorological variables are regressed on the detrended and normalized SON Tropical Atlantic and North Atlantic SST in OBE. The regression coefficients, representing the responses of local meteorological variables to remote SST forcings in the corresponding ocean basin, are shown in Figure 3. 165 For the Tropical Atlantic, the anomalously high SST in this region corresponds to higher T2M and lower TP in southern Australia (Figure 3a-b), the primary wildfire regime. This anomaly also corresponds to anomalously easterly and northerly winds in eastern Australia (Figure 3c). These winds will sink due to topographic factors, increasing temperature, and reduced humidity. These meteorological anomalies may reduce moisture in combustible plants and increase surface dryness, creating 165 165 For the Tropical Atlantic, the anomalously high SST in this region corresponds to higher T2M and lower TP in southern Australia (Figure 3a-b), the primary wildfire regime. This anomaly also corresponds to anomalously easterly and northerly winds in eastern Australia (Figure 3c). These winds will sink due to topographic factors, increasing temperature, and reduced humidity. These meteorological anomalies may reduce moisture in combustible plants and increase surface dryness, creating favorable conditions for igniting and spreading wildfires. Similarly, although the responses of these meteorological variables 170 are relatively weaker to the North Atlantic forcing (Figure 3d-f), their change directions agree with those under the Tropical Atlantic forcing, i.e., positive North Atlantic SST anomalies correspond to higher T2M and less TP in southern Australia. Therefore, the impact from tropical Atlantic appears stronger and more significant. Nonetheless, the impact of North Atlantic on precipitation is statistically significant across southern and western Australia and cannot be ignored. favorable conditions for igniting and spreading wildfires. 3.2 Impact of North and Tropical Atlantic on Australian FWI Similarly, although the responses of these meteorological variables 170 are relatively weaker to the North Atlantic forcing (Figure 3d-f), their change directions agree with those under the Tropical Atlantic forcing, i.e., positive North Atlantic SST anomalies correspond to higher T2M and less TP in southern Australia. Therefore, the impact from tropical Atlantic appears stronger and more significant. Nonetheless, the impact of North Atlantic on precipitation is statistically significant across southern and western Australia and cannot be ignored. 175 The consistent results in reanalysis (Figure 2) and OBE (Figure 3) indicate that the positive AMO phase, associated with warm North and Tropical Atlantic SST anomalies, induces warmer and drier weather in Australia, especially in the southern part. This inducing reinforces the positive ENSO signal on Australian fire weather, thus enhancing the ENSO-fire weather relationship there. In addition, the modulation effect of AMO on Australian climate is also significant in different periods 175 175 The consistent results in reanalysis (Figure 2) and OBE (Figure 3) indicate that the positive AMO phase, associated with warm North and Tropical Atlantic SST anomalies, induces warmer and drier weather in Australia, especially in the southern part. This inducing reinforces the positive ENSO signal on Australian fire weather, thus enhancing the ENSO-fire weather relationship there. In addition, the modulation effect of AMO on Australian climate is also significant in different periods 6 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. (1981-2019 and 1959-2019) and different reanalysis data sets (ERA5, NCEP-NCAR, and MERRA-2), which further 180 confirms the robustness of our findings (Figure S5-S7). (1981-2019 and 1959-2019) and different reanalysis data sets (ERA5, NCEP-NCAR, and MERRA-2), which further 180 confirms the robustness of our findings (Figure S5-S7). 180 3.3 Possible mechanism accounting for this modulation To clarify the physical process that the Atlantic impacts on Australia, we examine the responses of the 500 hPa geopotential height (GPH) responses in the North and Tropical Atlantic OBE and the mechanisms by which North Atlantic and the Tropical Atlantic respectively affect Australian FWI. Following Sardeshmukh and Hoskins (1988), the dynamics of Rossby 185 waves can be diagnosed by analyzing the barotropic vorticity equation at 200 hPa. Specifically, we diagnosed the dynamics of Rossby waves by analyzing the barotropic vorticity equation at 200 hPa, i.e., the Rossby wave source (RWS) that quantifies vorticity forcing associated with low-level convergence and upper-level divergence. To clarify the physical process that the Atlantic impacts on Australia, we examine the respon height (GPH) responses in the North and Tropical Atlantic OBE and the mechanisms by Tropical Atlantic respectively affect Australian FWI. Following Sardeshmukh and Hoskins (1988), the dynamics of Rossby 185 waves can be diagnosed by analyzing the barotropic vorticity equation at 200 hPa. Specifically, we diagnosed the dynamics of Rossby waves by analyzing the barotropic vorticity equation at 200 hPa, i.e., the Rossby wave source (RWS) that quantifies vorticity forcing associated with low-level convergence and upper-level divergence. Tropical Atlantic respectively affect Australian FWI. Following Sardeshmukh and Hoskins (1988), the dynamics of Rossby 185 waves can be diagnosed by analyzing the barotropic vorticity equation at 200 hPa. Specifically, we diagnosed the dynamics of Rossby waves by analyzing the barotropic vorticity equation at 200 hPa, i.e., the Rossby wave source (RWS) that quantifies vorticity forcing associated with low-level convergence and upper-level divergence. For Tropical Atlantic, the thermal forcing in the tropical Atlantic drives changes to the zonal Walker circulation. This change 190 may lead to upward vertical motion and local convection over the Atlantic, whereby the corresponding low-level convergence and upper-level divergence subsequently produce an intensification of the local Hadley circulation (Li et al., 2014; Li et al., 2015). In doing so, upper-level convergence is enhanced at the descending branch of the Hadley cell (Simpkins et al., 2014). This enhancement consequently intensifies the local Hadley circulation and becomes a vital source For Tropical Atlantic, the thermal forcing in the tropical Atlantic drives changes to the zonal Walker circulation. For North Atlantic, the warmer Atlantic heats the air above, thus forming a local high-pressure center in the upper troposphere. This signal generates the Rossby wave source over the North Atlantic (Figure 4d). The corresponding Rossby wave train will propagate from the west to the east with alternating high and lower pressure centers, which arrive in Australia as a high-pressure anomaly (Figure 4b and 4d). This high pressure corresponds to descending motions over 205 Australia with drier and hotter air, unfavorable to cloud and rain formation. The southward propagation of Rossby waves originating from the Atlantic is also supported by previous works (Miller et al., 2007; Zhao et al., 2019), which form the basis of the teleconnection between the North Atlantic and Australia. Previous studies also indicate that the AMO can modulate ENSO effects by the similar Rossby wave dynamics (Lin and Li, 2012; Nagaraju et al., 2018). 3.3 Possible mechanism accounting for this modulation This change 190 may lead to upward vertical motion and local convection over the Atlantic, whereby the corresponding low-level convergence and upper-level divergence subsequently produce an intensification of the local Hadley circulation (Li et al., 2014; Li et al., 2015). In doing so, upper-level convergence is enhanced at the descending branch of the Hadley cell (Simpkins et al., 2014). This enhancement consequently intensifies the local Hadley circulation and becomes a vital source of Rossby waves that propagate eastward with the climatological mean flow in the Southern Hemisphere (Figure 4c). This 195 Rossby wave source is clear over the South Atlantic (Figure 4c), and the corresponding Rossby wave will consequently propagate to Australia and intensify the high pressure there (Figure 4a). In summary, anomalous deep convection in response to increased SSTs in Tropical Atlantic drives anomalous divergence of the large-scale flow that extends away from the local heating by modulating the Hadley and Walker circulations. This process has been discussed in detail by Simpkins et al. (2014). 200 of Rossby waves that propagate eastward with the climatological mean flow in the Southern Hemisphere (Figure 4c). This 195 Rossby wave source is clear over the South Atlantic (Figure 4c), and the corresponding Rossby wave will consequently propagate to Australia and intensify the high pressure there (Figure 4a). In summary, anomalous deep convection in response to increased SSTs in Tropical Atlantic drives anomalous divergence of the large-scale flow that extends away from the local heating by modulating the Hadley and Walker circulations. This process has been discussed in detail by Simpkins et al. (2014). 200 For North Atlantic, the warmer Atlantic heats the air above, thus forming a local high-pressure center in the upper troposphere. This signal generates the Rossby wave source over the North Atlantic (Figure 4d). The corresponding Rossby wave train will propagate from the west to the east with alternating high and lower pressure centers, which arrive in Australia as a high-pressure anomaly (Figure 4b and 4d). This high pressure corresponds to descending motions over 205 Australia with drier and hotter air, unfavorable to cloud and rain formation. The southward propagation of Rossby waves originating from the Atlantic is also supported by previous works (Miller et al., 2007; Zhao et al., 2019), which form the basis of the teleconnection between the North Atlantic and Australia. 3.3 Possible mechanism accounting for this modulation Previous studies also indicate that the AMO can modulate ENSO effects by the similar Rossby wave dynamics (Lin and Li, 2012; Nagaraju et al., 2018). For North Atlantic, the warmer Atlantic heats the air above, thus forming a local high-pressure center in the upper troposphere. This signal generates the Rossby wave source over the North Atlantic (Figure 4d). The corresponding Rossby wave train will propagate from the west to the east with alternating high and lower pressure centers, which arrive in Australia as a high-pressure anomaly (Figure 4b and 4d). This high pressure corresponds to descending motions over 205 Australia with drier and hotter air, unfavorable to cloud and rain formation. The southward propagation of Rossby waves originating from the Atlantic is also supported by previous works (Miller et al., 2007; Zhao et al., 2019), which form the basis of the teleconnection between the North Atlantic and Australia. Previous studies also indicate that the AMO can modulate ENSO effects by the similar Rossby wave dynamics (Lin and Li, 2012; Nagaraju et al., 2018). Australia as a high-pressure anomaly (Figure 4b and 4d). This high pressure corresponds to descending motions over 205 Australia with drier and hotter air, unfavorable to cloud and rain formation. The southward propagation of Rossby waves originating from the Atlantic is also supported by previous works (Miller et al., 2007; Zhao et al., 2019), which form the basis of the teleconnection between the North Atlantic and Australia. Previous studies also indicate that the AMO can modulate ENSO effects by the similar Rossby wave dynamics (Lin and Li, 2012; Nagaraju et al., 2018). 7 7 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4 Conclusions 210 This wave train arrives in Australia as a higher-pressure anomaly, inducing descending air and leading to warmer and drier meteorological conditions conducive to wildfire generation. These meteorological anomalies superimpose the positive ENSO-induced meteorological 220 changes and enhance the response of Australian fire weather to ENSO. Previous studies also point out that the positive AMO, characterized by the basin-wide Atlantic warming, induces an Atlantic-Pacific sea surface temperature seesaw, strengthening the Walker circulation over the Pacific (Chafik et al., 2016; McGregor et al., 2014; Wang et al., 2013). This strengthened Walker circulation might also amplify ENSO’s effects on Australian FWI. conducive to wildfire generation. These meteorological anomalies superimpose the positive ENSO-induced meteorological 220 changes and enhance the response of Australian fire weather to ENSO. Previous studies also point out that the positive AMO, characterized by the basin-wide Atlantic warming, induces an Atlantic-Pacific sea surface temperature seesaw, strengthening the Walker circulation over the Pacific (Chafik et al., 2016; McGregor et al., 2014; Wang et al., 2013). This strengthened Walker circulation might also amplify ENSO’s effects on Australian FWI. 225 Compared with previous studies, this work sheds light on the teleconnection between AMO and Australian climate and fire weather on the decadal scale. The Australian mega-fire in the austral spring of 2019 has been attributed to the El Niño event, positive southern annular mode, and the positive Indian Ocean Dipole event of the year (Abram et al., 2021; Nolan et al., 2020; van Oldenborgh et al., 2021). Our study reveals that this year also corresponds to higher than normal Atlantic SST 225 Compared with previous studies, this work sheds light on the teleconnection between AMO and Australian climate and fire weather on the decadal scale. The Australian mega-fire in the austral spring of 2019 has been attributed to the El Niño event, positive southern annular mode, and the positive Indian Ocean Dipole event of the year (Abram et al., 2021; Nolan et al., 2020; van Oldenborgh et al., 2021). Our study reveals that this year also corresponds to higher than normal Atlantic SST (Figure S8), which may strengthen the Australian atmospheric response to the El Niño event, further leading to prolonged 230 dry seasons and increased heat waves. Admittedly, the Pacific decadal variability (such as the Pacific Decadal Oscillation) also plays an essential role in Australia’s climate (Power et al., 1999). 4 Conclusions 210 Fire weather in Australia is closely related to the variability of ENSO. The correlation between them has been strengthened in the recent two decades, yet the cause has not been fully clarified. By analyzing reanalysis datasets and conducting ocean basin experiments using a global climate model, our study offers a plausible explanation that the AMO modulates the ENSO-Australian fire weather relationship. The correlation coefficient between ENSO and the Australian Fire Weather Fire weather in Australia is closely related to the variability of ENSO. The correlation between them has been strengthened in the recent two decades, yet the cause has not been fully clarified. By analyzing reanalysis datasets and conducting ocean basin experiments using a global climate model, our study offers a plausible explanation that the AMO modulates the ENSO-Australian fire weather relationship. The correlation coefficient between ENSO and the Australian Fire Weather Index (FWI) increases from 0.17 to 0.70 when AMO transitions from negative to possible phases. Under positive AMO, El 215 Niño conditions correspond to stronger temperature increase, precipitation decrease, stronger surface winds favorable for wildfire generation, and vice versa. Physically, positive AMO, associated with warmer North and Tropical Atlantic SST, forms a local low-pressure center, which propagates to the southwest as a Rossby wave train. This wave train arrives in Australia as a higher-pressure anomaly, inducing descending air and leading to warmer and drier meteorological conditions Index (FWI) increases from 0.17 to 0.70 when AMO transitions from negative to possible phases. Under positive AMO, El 215 Niño conditions correspond to stronger temperature increase, precipitation decrease, stronger surface winds favorable for wildfire generation, and vice versa. Physically, positive AMO, associated with warmer North and Tropical Atlantic SST, forms a local low-pressure center, which propagates to the southwest as a Rossby wave train. This wave train arrives in Australia as a higher-pressure anomaly, inducing descending air and leading to warmer and drier meteorological conditions conducive to wildfire generation. These meteorological anomalies superimpose the positive ENSO-induced meteorological 220 Index (FWI) increases from 0.17 to 0.70 when AMO transitions from negative to possible phases. Under positive AMO, El 215 Niño conditions correspond to stronger temperature increase, precipitation decrease, stronger surface winds favorable for wildfire generation, and vice versa. Physically, positive AMO, associated with warmer North and Tropical Atlantic SST, forms a local low-pressure center, which propagates to the southwest as a Rossby wave train. Author contributions The paper was written by GY and JL and designed by JL. The data analysis was performed by GY and TY. All the co- authors contributed to the interpretation of results and the improvement of this paper. 4 Conclusions 210 The meteorological reanalysis data is downloaded from ERA5 monthly averaged data in the climate data store (CDS) (https://doi.org/10.24381/cds.adbb2d47). The NCEP-NCAR Reanalysis 1 data is downloaded from Physical Sciences Library (https://psl.noaa.gov/data/gridded/data.ncep.reanalysis.html). The MERRA-2 data is downloaded from MDISC 245 (https://disc.gsfc.nasa.gov/datasets/M2TMNXFLX_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_flx_Nx and https://disc.gsfc.nasa.gov/datasets/M2TMNXSLV_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_slv_Nx). AMO and Niño 3.4 indexes are obtained from the NCAR climate data guide for the AMO index (https://climatedataguide.ucar.edu/climate-data/atlantic-multi-decadal-oscillation-amo) and Niño 3.4 index (https://climatedataguide.ucar.edu/climate-data/nino-sst-indices-nino-12-3-34-4-oni-and-tni), respectively. The sea surface 250 temperature data is obtained from NOAA Extended Reconstructed Sea Surface Temperature (SST) V4 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v4.html) NOAA Extended Reconstructed Sea Surface Temperature (SST) V5 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v5.html) COBE-SST 2 (https://psl.noaa.gov/data/gridded/data.cobe2.html), HadISST (https://www.metoffice.gov.uk/hadobs/hadisst/), and Kaplan Extended SST V2 (https://psl.noaa.gov/data/gridded/data.kaplan_sst.html). 255 The history Fire Weather Index (FWI) data is downloaded from fire danger indices historical data in Copernicus Emergency Service (https://doi.org/10.24381/cds.0e89c522). The meteorological reanalysis data is downloaded from ERA5 monthly averaged data in the climate data store (CDS) (https://doi.org/10.24381/cds.adbb2d47). The NCEP-NCAR Reanalysis 1 data is downloaded from Physical Sciences Library (https://psl.noaa.gov/data/gridded/data.ncep.reanalysis.html). The MERRA-2 data is downloaded from MDISC 245 (https://disc.gsfc.nasa.gov/datasets/M2TMNXFLX_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_flx_Nx and https://disc.gsfc.nasa.gov/datasets/M2TMNXSLV_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_slv_Nx). AMO and Niño 3.4 indexes are obtained from the NCAR climate data guide for the AMO index (https://climatedataguide.ucar.edu/climate-data/atlantic-multi-decadal-oscillation-amo) and Niño 3.4 index (https://climatedataguide.ucar.edu/climate-data/nino-sst-indices-nino-12-3-34-4-oni-and-tni), respectively. The sea surface 250 temperature data is obtained from NOAA Extended Reconstructed Sea Surface Temperature (SST) V4 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v4.html) NOAA Extended Reconstructed Sea Surface Temperature (SST) V5 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v5.html) COBE-SST 2 (https://psl.noaa.gov/data/gridded/data.cobe2.html), HadISST (https://www.metoffice.gov.uk/hadobs/hadisst/), and Kaplan Extended SST V2 (https://psl.noaa.gov/data/gridded/data.kaplan_sst.html). 255 245 (https://climatedataguide.ucar.edu/climate-data/nino-sst-indices-nino-12-3-34-4-oni-and-tni), respectively. The sea surface 250 temperature data is obtained from NOAA Extended Reconstructed Sea Surface Temperature (SST) V4 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v4.html) NOAA Extended Reconstructed Sea Surface Temperature (SST) V5 (https://psl.noaa.gov/data/gridded/data.noaa.ersst.v5.html) COBE-SST 2 (https://psl.noaa.gov/data/gridded/data.cobe2.html), HadISST (https://www.metoffice.gov.uk/hadobs/hadisst/), and Kaplan Extended SST V2 (https://psl.noaa.gov/data/gridded/data.kaplan_sst.html). 255 This study is funded by the National Natural Science Foundation of China (NSFC) Grants No. 41975023. 4 Conclusions 210 However, previous research indicates that the Pacific variability may be at least partly triggered by that of the Atlantic (Li et al., 2016; Ren et al., 2021), highlighting the vital role of the latter in the Earth’s climate system. Note that another major decadal climate variability, the Interdecadal Pacific (Figure S8), which may strengthen the Australian atmospheric response to the El Niño event, further leading to prolonged 230 dry seasons and increased heat waves. Admittedly, the Pacific decadal variability (such as the Pacific Decadal Oscillation) also plays an essential role in Australia’s climate (Power et al., 1999). However, previous research indicates that the Pacific variability may be at least partly triggered by that of the Atlantic (Li et al., 2016; Ren et al., 2021), highlighting the vital role of the latter in the Earth’s climate system. Note that another major decadal climate variability, the Interdecadal Pacific Oscillation (IPO), also changed its phase in the late 1990s. We also investigated its modulation effect on ENSO and 235 Australian FWI but found it not as significant as that of AMO in both observation and simulation (Figures not shown). In the future, we plan to investigate the impact of other ocean basins on Australian fire weather and the ENSO-Australian fire weather relationship in the future. Oscillation (IPO), also changed its phase in the late 1990s. We also investigated its modulation effect on ENSO and 235 Australian FWI but found it not as significant as that of AMO in both observation and simulation (Figures not shown). In the future, we plan to investigate the impact of other ocean basins on Australian fire weather and the ENSO-Australian fire weather relationship in the future. Data availability 240 8 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. The history Fire Weather Index (FWI) data is downloaded from fire danger indices historical data in Copernicus Emergency Service (https://doi.org/10.24381/cds.0e89c522). The meteorological reanalysis data is downloaded from ERA5 monthly averaged data in the climate data store (CDS) (https://doi.org/10.24381/cds.adbb2d47). The NCEP-NCAR Reanalysis 1 data is downloaded from Physical Sciences Library (https://psl.noaa.gov/data/gridded/data.ncep.reanalysis.html). The MERRA-2 data is downloaded from MDISC 245 (https://disc.gsfc.nasa.gov/datasets/M2TMNXFLX_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_flx_Nx and https://disc.gsfc.nasa.gov/datasets/M2TMNXSLV_5.12.4/summary?keywords=MERRA2_100.tavgM_2d_slv_Nx). AMO and Niño 3.4 indexes are obtained from the NCAR climate data guide for the AMO index (https://climatedataguide.ucar.edu/climate-data/atlantic-multi-decadal-oscillation-amo) and Niño 3.4 index The history Fire Weather Index (FWI) data is downloaded from fire danger indices historical data in Copernicus Emergency Service (https://doi.org/10.24381/cds.0e89c522). 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Disclaimer Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps an 265 institutional affiliations. Acknowledgement We thank ECMWF for providing the ERA5 reanalysis data. We also acknowledge the efforts of CAM4 Working Groups an Support Team for developing and maintaining the CAM4 model. 270 Support Team for developing and maintaining the CAM4 model. 270 This study is funded by the National Natural Science Foundation of China (NSFC) Grants No. 41 This study is funded by the National Natural Science Foundation of China (NSFC) Grants No. 41975023. 9 9 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. 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Y.: Interhemispheric influence of Indo-Pacific convection oscillation on Southern Hemisphere precipitation through southward propagation of Rossby waves, Clim. Dyn., 52(5-6), 3203-3221, https://doi.org/10.1007/s00382-018-4324-y, 2019. 13 40 Figure 1. (a) Scatter plots for detrended, standardized SON Niño 3.4 index and the corresponding reanalysis mean Australia FWI from 1981 to 2019. The red upward triangles represent positive AMO indices, while the blue circles represent negative ones. The lines are linear fit lines. The correlation coefficient (R=0.71) corresponding to AMO+ passed the significance test of p-value<0.05, while the other one did not. References 275 (b) The solid black line represents the sliding correlation coefficient between detrended SON FWI in Australia and detrended SON Niño 3.4 index with a sliding window of 10 years. The shaded area 45 represents the annual AMO index. Red is positive, and blue is negative. All the correlation coefficients assume autocorrelation to the time series. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. 440 Figure 1. (a) Scatter plots for detrended, standardized SON Niño 3.4 index and the corresponding reanalysis mean Australia FWI from 1981 to 2019. The red upward triangles represent positive AMO indices, while the blue circles represent negative ones. The lines are linear fit lines. The correlation coefficient (R=0.71) corresponding to AMO+ passed the significance test of p-value<0.05, while the other one did not. (b) The solid black line represents the sliding correlation coefficient between Figure 1. (a) Scatter plots for detrended, standardized SON Niño 3.4 index and the corresponding reanalysis mean Australia FWI from 1981 to 2019. The red upward triangles represent positive AMO indices, while the blue circles represent negative ones. The lines are linear fit lines. The correlation coefficient (R=0.71) corresponding to AMO+ passed the significance test of p-value<0.05, while the other one did not. (b) The solid black line represents the sliding correlation coefficient between Figure 1. (a) Scatter plots for detrended, standardized SON Niño 3.4 index and the corresponding reanalysis mean Australia FWI from 1981 to 2019. The red upward triangles represent positive AMO indices, while the blue circles represent negative ones. The lines are linear fit lines. The correlation coefficient (R=0.71) corresponding to AMO+ passed the significance test of p-value<0.05, while the other one did not. (b) The solid black line represents the sliding correlation coefficient between Figure 1. (a) Scatter plots for detrended, standardized SON Niño 3.4 index and the corresponding reanalysis mean Australia FWI from 1981 to 2019. The red upward triangles represent positive AMO indices, while the blue circles represent negative ones. The lines are linear fit lines. The correlation coefficient (R=0.71) corresponding to AMO+ passed the significance test of p-value<0.05, while the other one did not. (b) The solid black line represents the sliding correlation coefficient between detrended SON FWI in Australia and detrended SON Niño 3.4 index with a sliding window of 10 years. The shaded area 445 represents the annual AMO index. Red is positive, and blue is negative. References 275 All the correlation coefficients assume autocorrelation to the time series. 445 detrended SON FWI in Australia and detrended SON Niño 3.4 index with a sliding window of 10 years. The shaded area 445 represents the annual AMO index. Red is positive, and blue is negative. All the correlation coefficients assume autocorrelation to the time series. detrended SON FWI in Australia and detrended SON Niño 3.4 index with a sliding window of 10 years. The shaded area 445 represents the annual AMO index. Red is positive, and blue is negative. All the correlation coefficients assume autocorrelation to the time series. 14 14 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. The difference maps for the detrended and normalized reanalysis SON (a, d, g, j) 2m temperature (T2M), (b, e, h, 450 k) Total Precipitation (TP), and (c, f, I, l) Sea Level Pressure (SLP)+10m zonal and meridional winds (U10+V10) in conditions with (a-c) ENSO composite (El Niño composite minus La Niña composite), (d-f) ENSO composite with AMO+, (g-i) ENSO composite with AMO-, and (j-l) AMO composite from 1981 to 2019. The composite results are calculated using meteorological variables with positive indices minus those with negative ones. The area with white dots passed the significance test of p-value < 0.05 by Student’s t-test. 455 Figure 2. The difference maps for the detrended and normalized reanalysis SON (a, d, g, j) 2m temperature (T2M), (b, e, h, 450 k) Total Precipitation (TP), and (c, f, I, l) Sea Level Pressure (SLP)+10m zonal and meridional winds (U10+V10) in conditions with (a-c) ENSO composite (El Niño composite minus La Niña composite), (d-f) ENSO composite with AMO+, (g-i) ENSO composite with AMO-, and (j-l) AMO composite from 1981 to 2019. The composite results are calculated using meteorological variables with positive indices minus those with negative ones. The area with white dots passed the significance test of p-value < 0.05 by Student’s t-test. 455 15 https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 3. References 275 Regression coefficients of the ensemble mean detrended and normalized SON (a, d) T2M, (b, e) TP and (c, f) SLP+U10+V10 onto detrended and normalized SON (a-c) Tropical Atlantic (10°-60°W, 0-20°N) SST and (d-f) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The area with white dots passed the significance test of p ≤0.05 by 0 Student’s t-test. Figure 3. Regression coefficients of the ensemble mean detrended and normalized SON (a, d) T2M, (b, e) TP and (c, f) SLP+U10+V10 onto detrended and normalized SON (a-c) Tropical Atlantic (10°-60°W, 0-20°N) SST and (d-f) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The area with white dots passed the significance test of p ≤0.05 by 60 Student’s t-test. Figure 3. Regression coefficients of the ensemble mean detrended and normalized SON (a, d) T2M, (b, e) TP and (c, f) SLP+U10+V10 onto detrended and normalized SON (a-c) Tropical Atlantic (10°-60°W, 0-20°N) SST and (d-f) North Atlantic (10° 60°W 25 45°N) SST in the OBE The area with white dots passed the significance test of p ≤0 05 by 0 Figure 3. Regression coefficients of the ensemble mean detrended and normalized SON (a, d) T2M, (b, e) TP and (c, f) SLP+U10+V10 onto detrended and normalized SON (a-c) Tropical Atlantic (10°-60°W, 0-20°N) SST and (d-f) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The area with white dots passed the significance test of p ≤0.05 by 460 Student’s t-test. ( ) p ( , ) ( ) Atlantic (10°-60°W, 25-45°N) SST in the OBE. The area with white dots passed the significance test of p ≤0.05 by 460 Student’s t-test. 16 Figure 4. (a-b) Regression coefficients of detrended and normalized SON 500 hPa GPH onto detrended and normalized SON (a) Tropical Atlantic (10°-60°W, 0-20°N) and (b) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The red solid lines represent the isolines from 0.2 to 1 with an interval of 0.2, and the blue solid lines represent the isolines from - 0.2 to - 1 with an interval of - 0.2. (c-d) 200hPa Rossby wave source anomaly in (c) Tropical Atlantic OBE, and (d) North Atlantic OBE. The area with white dots passed the significance test of p ≤0.05 by Student’s t-test. https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. References 275 (a-b) Regression coefficients of detrended and normalized SON 500 hPa GPH onto detrended and normalized SON (a) Tropical Atlantic (10°-60°W, 0-20°N) and (b) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The red solid https://doi.org/10.5194/acp-2022-858 Preprint. Discussion started: 29 March 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. (a-b) Regression coefficients of detrended and normalized SON 500 hPa GPH onto detrended and normalized SON (a) Tropical Atlantic (10°-60°W, 0-20°N) and (b) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The red solid lines represent the isolines from 0.2 to 1 with an interval of 0.2, and the blue solid lines represent the isolines from - 0.2 to - 65 1 with an interval of - 0.2. (c-d) 200hPa Rossby wave source anomaly in (c) Tropical Atlantic OBE, and (d) North Atlantic OBE. The area with white dots passed the significance test of p ≤0.05 by Student’s t-test. Figure 4. (a-b) Regression coefficients of detrended and normalized SON 500 hPa GPH onto detrended and normalized SON (a) Tropical Atlantic (10°-60°W, 0-20°N) and (b) North Atlantic (10°-60°W, 25-45°N) SST in the OBE. The red solid lines represent the isolines from 0.2 to 1 with an interval of 0.2, and the blue solid lines represent the isolines from - 0.2 to - 465 1 with an interval of - 0.2. (c-d) 200hPa Rossby wave source anomaly in (c) Tropical Atlantic OBE, and (d) North Atlantic OBE. The area with white dots passed the significance test of p ≤0.05 by Student’s t-test. lines represent the isolines from 0.2 to 1 with an interval of 0.2, and the blue solid lines represent the isolines from - 0.2 to - 465 1 with an interval of - 0.2. (c-d) 200hPa Rossby wave source anomaly in (c) Tropical Atlantic OBE, and (d) North Atlantic OBE. The area with white dots passed the significance test of p ≤0.05 by Student’s t-test. 17
https://openalex.org/W2773339774	https://eprints.qut.edu.au/208498/1/water_09_00949.pdf	English	null	Stormwater Biofilters as Barriers against Campylobacter jejuni, Cryptosporidium Oocysts and Adenoviruses; Results from a Laboratory Trial	Water	2,017	cc-by	9,754	This may be the author’s version of a work that was submitted/accepted for publication in the following source: Chandrasena, Gayani, Deletic, Ana, Lintern, Anna, Henry, Rebekah, & McCarthy, David Chandrasena, Gayani, Deletic, Ana, Lintern, Anna, Henry, Rebekah, & McCarthy, David (2017) Stormwater bioﬁlters as barriers against Campylobacter jejuni, Cryp- tosporidium Oocysts and adenoviruses; results from a laboratory trial. Water (Switzerland), 9(12), Article number: 949. ( ) Stormwater bioﬁlters as barriers against Campylobacter jejuni, Cryp- tosporidium Oocysts and adenoviruses; results from a laboratory trial. Water (Switzerland), 9(12), Article number: 949. This ﬁle was downloaded from: https://eprints.qut.edu.au/208498/ Stormwater Bioﬁlters as Barriers against Campylobacter jejuni, Cryptosporidium Oocysts and Adenoviruses; Results from a Laboratory Trial Gayani Chandrasena 1,2, Ana Deletic 1,2,3, Anna Lintern 4, Rebekah Henry 1,2 and Gayani Chandrasena 1,2, Ana Deletic 1,2,3, Anna Lintern 4, Rebekah Henry 1,2 and David McCarthy 1,2,* Gayani Chandrasena 1,2, Ana Deletic 1,2,3, Anna Lintern 4, Rebekah Henry 1,2 and David McCarthy 1,2,* 1 Environmental and Public Health Microbiology Laboratory (EPHM Lab), Monash Water for Liveability, Department of Civil Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; gayani.chandrasena@gmail.com (G.C.); a.deletic@unsw.edu.au (A.D.); Rebekah.Henry@monash.edu (R.H.) 2 Cooperative Research Centre for Water Sensitive Cities, Melbourne, Victoria 3800, Australia 1 Environmental and Public Health Microbiology Laboratory (EPHM Lab), Monash Water for Liveability, Department of Civil Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; gayani.chandrasena@gmail.com (G.C.); a.deletic@unsw.edu.au (A.D.); Rebekah.Henry@monash.edu (R.H.) 2 Cooperative Research Centre for Water Sensitive Cities, Melbourne, Victoria 3800, Australia 3 School of Civil and Environmental Engineering, University of New South Wales, Sydney NSW 2052, Australia 4 Department of Civil Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; Anna.Lintern@monash.edu 1 Environmental and Public Health Microbiology Laboratory (EPHM Lab), Monash Water for Liveability, Department of Civil Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; gayani.chandrasena@gmail.com (G.C.); a.deletic@unsw.edu.au (A.D.); Rebekah.Henry@monash.edu (R.H.) 2 Cooperative Research Centre for Water Sensitive Cities, Melbourne, Victoria 3800, Australia 3 School of Civil and Environmental Engineering, University of New South Wales, Sydney NSW 2052, Australia 4 Department of Civil Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; Anna.Lintern@monash.edu * Correspondence: David.McCarthy@monash.edu; Tel.: +61-3-9905-4944 Correspondence: David.McCarthy@monash.edu; Tel.: +61-3-9905-4944 Received: 24 September 2017; Accepted: 1 December 2017; Published: 6 December 2017 Received: 24 September 2017; Accepted: 1 December 2017; Published: 6 December 2017 Abstract: Bioﬁlters are a widely used stormwater treatment technology. However; other than some evidence regarding non-pathogenic indicator microorganisms; there are signiﬁcant knowledge gaps in the capacity of stormwater bioﬁlters to remove actual pathogens and how this removal is impacted by bioﬁlter design elements and operational conditions. In this study; we explored the capacity of stormwater bioﬁlters to remove three reference pathogens (Campylobacter spp.; adenovirus and Cryptosporidium oocysts) and compared these to commonly used indicator microorganisms (E. coli; FRNA coliphages and Clostridium perfringens). Two different bioﬁlter designs; each having a submerged zone (SZ); were tested under extended dry weather periods (up to 4 weeks) and different event volumes (the equivalent of 1–2 pore volumes) in a laboratory trial. These systems were able to consistently reduce the concentrations of all tested reference pathogens (average log reduction in Campylobacter spp. Stormwater Bioﬁlters as Barriers against Campylobacter jejuni, Cryptosporidium Oocysts and Adenoviruses; Results from a Laboratory Trial Gayani Chandrasena 1,2, Ana Deletic 1,2,3, Anna Lintern 4, Rebekah Henry 1,2 and = 0.7; adenovirus = 1.0 and Cryptosporidium oocysts = 1.7) and two of the indicators (average log reduction in E. coli = 1.2 and C. perfringens = 2.1). However; none of the tested indicators consistently mimicked the removal performance of their corresponding reference pathogens after extended dry weather periods and during larger simulated storm events. This indicates that the behaviour of these pathogens in stormwater bioﬁlters are not adequately represented by their corresponding indicator microorganisms and that to optimise bioﬁlter designs for pathogen removal it is critical to further study pathogen removal processes in these systems. Keywords: adenoviruses; Campylobacter; Cryptosporidium oocysts; rain garden; stormwater harvesting; stormwater management © © 2017 by the authors. This work is covered by copyright. Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. If the docu- ment is available under a Creative Commons License (or other speciﬁed license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recog- nise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to qut.copyright@qut.edu.au License: Creative Commons: Attribution 4.0 Notice: Please note that this document may not be the Version of Record (i.e. published version) of the work. Author manuscript versions (as Sub- mitted for peer review or as Accepted for publication after peer review) can be identiﬁed by an absence of publisher branding and/or typeset appear- ance. If there is any doubt, please refer to the published source. https://doi.org/10.3390/w9120949 https://doi.org/10.3390/w9120949 water water Keywords: adenoviruses; Campylobacter; Cryptosporidium oocysts; rain garden; stormwater harvesting; stormwater management 1. Introduction Stormwater harvesting has great potential to reduce the pressure on existing water resources while protecting receiving waters from degradation [1,2]. However, stormwater contains pathogens, which can cause human health risks when stormwater is harvested for re-use or is discharged into recreational water bodies without treatment [3]. Bioﬁlters, also known as bioretention systems or rain gardens, are a common stormwater treatment technology. Stormwater bioﬁlters consist of planted soil-based ﬁlter media with a high sand content, a sand transition layer and a drainage layer. These systems, which could be lined, are sometimes installed with a raised outlet to create a submerged zone (SZ). Water 2017, 9, 949; doi:10.3390/w9120949 www.mdpi.com/journal/water Water 2017, 9, 949 2 of 12 This SZ has been shown to improve the treatment performance of biofilters by promoting denitrification [4] d i i i l lif d ﬁl di i d i d d d h i d [5] This SZ has been shown to improve the treatment performance of biofilters by promoting denitrification [4] and maintaining plant life and ﬁlter media consistency during extended dry weather periods [5]. maintaining plant life and ﬁlter media consistency during extended dry weather periods [5]. Almost all of the literature on microbial removal in bioﬁlters is limited to indicator microorganisms (e.g., [6–10]), even though it is well established that indicators and pathogens respond differently to various stresses found in the natural environment [2,11]. Obtaining data on the capacity of bioﬁlters to remove other faecal microorganisms (including ‘reference’ pathogens, as suggested by various health organisations [3]) is crucial to validate the use of bioﬁlters to treat water for stormwater harvesting and also to protect recreational waters that receive a stormwater discharge. Previous work on indicator microorganism removal by stormwater bioﬁlters has demonstrated that some bioﬁlter design elements and operational conditions greatly inﬂuence microorganism removal mechanisms [12]. For instance, vegetation with extensive root structures was shown to improve E. coli retention in stormwater bioﬁlters [13]. Filter media modiﬁed with either antimicrobial compounds [14,15] or adsorption enhancements [16,17] were found to improve faecal indicator microorganism attenuation rates. Intermittent wetting/drying operation is generally associated with poor removal performances due to decreased microbial adsorption and increased microbial desorption [7,8,17]. Adsorption kinetics suggest that overall removal rates of microorganisms from stormwater will vary greatly with event volume [18]. 1. Introduction However, almost all previous laboratory studies have tested microbial removal in bioﬁlters using a single event size (i.e., a single dosing volume), even though storm sizes vary greatly in practice. Further, it is not yet known whether the pathogens and faecal indicator microorganisms have the same attenuation processes and whether the bioﬁlter design and operational conditions affect pathogens and faecal indicator microorganism attenuation mechanisms in the same way. As such, it must be determined whether pathogen removal can be represented by the behaviour of its indicator microorganisms, or whether speciﬁc testing of pathogen removal by stormwater bioﬁlters is necessary. The primary objective of this laboratory study was to understand whether stormwater bioﬁlters are an effective barrier against three of the most commonly used reference pathogens: Campylobacter jejuni, Cryptosporidium oocysts and adenoviruses. Further, the study investigates how these removal rates are inﬂuenced by the design (presence of vegetation) and operational characteristics of these systems, such as extended dry weather periods and different sized rainfall events. Finally, this study explored the potential for commonly used faecal indicator microorganisms (E. coli, FRNA coliphages, Clostridium perfringens) can be used to represent reference pathogens. 2.2.1. Semi-Natural Stormwater Preparation Semi-natural stormwater was used to dose the columns similar to previous laboratory-scale experiments [21]. This was to ensure consistency of the inﬂow and to avoid the logistical constraints of collecting large volumes of natural stormwater. Stormwater was prepared by mixing dechlorinated tap water with sediment collected from a local stormwater wetland inlet and sieved through a 1 mm sieve. This mixture was then supplemented with laboratory grade chemicals to obtain target pollutant concentrations consistent with Australian urban stormwater quality ([3,22,23]; Table 1). Table 1. Target and measured semi-natural stormwater pollutant concentrations. Table 1. Target and measured semi-natural stormwater pollutant concentrations. Stormwater Pollutant Unit Inﬂow Concentration Main Source Target Measured in the Inﬂow during This Study 1 E. coli MPN/100 mL 5.9 × 104 2.8 × 104 (4.16) raw sewage FRNA coliphages pfu/100 mL 5.5 26.7 (20.83) laboratory culture C. perfringens orgs/100 mL 925 1.4 × 103 (1.73) laboratory culture Campylobacter spp. MPNIU/L 33.1 65 (19.49) laboratory culture Adenoviruses MPNIU/L 1 11 (6.50) laboratory culture Cryptosporidium oocysts oocysts/L 17.6 9 (2.53) laboratory culture Total suspended solids mg/L 100 86 (1.44) sediment Total phosphorus mg/L 0.35 0.42 (1.18) sediment, KH2PO4 Total nitrogen mg/L 2.2 2.92 (1.17) sediment, KNO3, NH4Cl, C6H5O2N Cadmium mg/L 4.5 × 10−3 8.1 × 10−3 (1.40) Cd(NO3)2 in HNO3 Chromium mg/L 2.5 × 10−2 5.4 × 10−2 (1.58) Cr(NO3)3 Copper mg/L 5.0 × 10−2 8.0 × 10−2 (1.36) CuSO4 Lead mg/L 1.4 × 10−1 2.7 × 10−1 (1.47) Pb(NO3)2 Manganese mg/L 2.3 × 10−1 1.9 × 10−1 (1.11) Mn(NO3)2 Nickel mg/L 3.1 × 10−2 5.0 × 10−2 (1.30) Ni(NO3)2 Zinc mg/L 2.5 × 10−1 2.6 × 10−1 (1.18) ZnCl2 Note: 1 measured concentrations are presented as Geometric mean (Geometric standard deviation). MPN—Most Probable Number; MPNIU: Most Probable Number of Infectious Units; pfu: plaque forming units; orgs: organisms. Note: 1 measured concentrations are presented as Geometric mean (Geometric standard deviation). MPN—Most Probable Number; MPNIU: Most Probable Number of Infectious Units; pfu: plaque forming units; orgs: organisms. Raw sewage sampled from the inlet channel at the sewage treatment plant in Pakenham, Australia, was used as the source of faecal microorganisms and was mixed into the synthetic stormwater (a 1:500 ratio of sewage: stormwater mixture by volume). Use of raw sewage allowed a better representation of the natural variability of faecal microorganism concentrations and composition in natural stormwater than using a monoculture of laboratory microbial strains to spike the semi-natural stormwater. 2.1. Experimental Conﬁgurations This laboratory-scale study involved six bioﬁlter columns that were part of thirty-bioﬁlter columns originally constructed for another parallel study [19] in a greenhouse. Details of the design of the columns can be found in [19]. Brieﬂy, each bioﬁlter column consisted of a 1140 mm tall, 240 mm diameter PVC column. A 280 mm tall clear Perspex pipe was plastic welded to the top of each column, to serve as a ponding zone [4]. All columns had 400 mm deep ﬁlter media. The top 100 mm of the ﬁlter media in each conﬁguration was mixed with an ameliorant to support plant growth (see [20] for more information). A perforated plastic collection pipe was installed at the bottom of each column to collect the treated water. An SZ was created in all columns by conveying the treated water through a raised outﬂow pipe which created a 440 mm deep SZ below the ﬁlter media layer. The SZs consisted of a gravel drainage layer (70 mm deep), a coarse sand layer (70 mm deep) and a washed sand layer (300 mm deep) mixed with 5% sugarcane mulch and 5% pinewood chips without bark by volume. Bioﬁlter layer depths and compositions were based on the current best practice guidelines in Australia [5]. Three bioﬁlter columns were planted with Leptospermum continentale (small shrub) (hereinafter LC) while the remaining three columns were left 3 of 12 Water 2017, 9, 949 unplanted as an un-vegetated control (hereinafter WS). L. continentale was selected for the current study as it showed good faecal microorganism removal in the authors’ previous work [13]. unplanted as an un-vegetated control (hereinafter WS). L. continentale was selected for the current study as it showed good faecal microorganism removal in the authors’ previous work [13]. 2.2. Dosing and Sampling 2.2.2. Dosing Frequency and Volumes 2.2.2. Dosing Frequency and Volumes The dosing frequency and volumes are outlined in Figure 1. Since, the current study was conducted in parallel to another study [19], the dosing frequency was designed to accommodate the scopes of both studies. Each biofilter column was dosed with 13 L of semi-natural stormwater twice a week (using at least three 3–5 L passes/rounds to ensure consistent inflow into all of the columns). This weekly dosing frequency and volume reflect Melbourne’s historical climate patterns [21]. The 13 L dosing volume was estimated to be equivalent to a rainfall event of 5.75 mm and at the same time equal to 0.7 pore volumes (PV) of the biofilter columns. Pore volume estimated from a KCl tracer test conducted in un-vegetated pilot columns prior to week 10 (See Supplementary Material for more details on tracer test). The dosing frequency and volumes are outlined in Figure 1. Since, the current study was conducted in parallel to another study [19], the dosing frequency was designed to accommodate the scopes of both studies. Each bioﬁlter column was dosed with 13 L of semi-natural stormwater twice a week (using at least three 3–5 L passes/rounds to ensure consistent inﬂow into all of the columns). This weekly dosing frequency and volume reﬂect Melbourne’s historical climate patterns [21]. The 13 L dosing volume was estimated to be equivalent to a rainfall event of 5.75 mm and at the same time equal to 0.7 pore volumes (PV) of the bioﬁlter columns. Pore volume estimated from a KCl tracer test conducted in un-vegetated pilot columns prior to week 10 (See Supplementary Materials for more details on tracer test). ) Dosing volumes were increased on the days where the performance of the systems was evaluated (i.e., when inflow and outflow water samples were collected). The volumes used were: (1) 20 L, which is the 1 in 1-month average recurrence interval (ARI) event and equivalent to 1 PV (equivalent loading of 8.84 mm per event) or (2) 40 L which is equal to a 1 in 3-month ARI event and 2 PVs (equivalent loading of 17.68 mm per event). In total, samples were collected from three 40 L dosing events and two 20 L dosing events for the current study. Dosing volumes were increased on the days where the performance of the systems was evaluated (i.e., when inﬂow and outﬂow water samples were collected). 2.2.1. Semi-Natural Stormwater Preparation This process of mixing semi-natural stormwater with raw sewage at a 1:500 dilution ratio resulted in E. coli levels which reﬂected natural stormwater variability (Table 1 and [3]), but undetectable levels of other fecal microorganisms of interest in this study: pathogen indicators (FRNA coliphages, C. perfringens) and reference pathogens (Campylobacter, adenovirus, Cryptosporidium oocysts). As such, laboratory cultures of other test microorganisms were added to the inﬂow for the purpose of the current study. Semi-natural stormwater was supplemented with the diluted laboratory-grown cultures of two indicator microorganisms (FRNA coliphages and C. perfringens) in week 14, 26, 27, 31 and 41 (Figure 1). The diluted laboratory-grown cultures of the reference pathogens (Campylobacter, adenovirus, Cryptosporidium parvum), supplied by a National Association of Testing Authorities (NATA), Australia accredited laboratory and Environmental and Public Health Microbiology laboratory in Monash University were added only in weeks 27, 31, 4 of 12 4 of 12 1 d Water 2017, 9, 949 Water 2017, 9, 949 and 41 (Figure 1). The authors’ previous experiences indicated that the chosen approach to prepare semi-natural stormwater not only resulted in achieving typical microbial concentrations found in real stormwater, but also the majority of faecal indicator bacteria (E. coli) remain unattached or attached to very small particles (<3-µm) in semi-natural stormwater similar to the natural stormwater collected from a Melbourne suburb [24]. 41 (Figure 1). The authors’ previous experiences indicated that the chosen approach to prepare semi- natural stormwater not only resulted in achieving typical microbial concentrations found in real stormwater, but also the majority of faecal indicator bacteria (E. coli) remain unattached or attached to very small particles (<3-µm) in semi-natural stormwater similar to the natural stormwater collected from a Melbourne suburb [24]. Figure 1. Dosing and sampling plan. Five indicator microorganism sampling days and three reference pathogen sampling days were within the scope of this study. Remaining sampling days were within the scope of the other parallel study [19]. Figure 1. Dosing and sampling plan. Five indicator microorganism sampling days and three reference pathogen sampling days were within the scope of this study. Remaining sampling days were within the scope of the other parallel study [19]. Figure 1. Dosing and sampling plan. Five indicator microorganism sampling days and three reference pathogen sampling days were within the scope of this study. Remaining sampling days were within the scope of the other parallel study [19]. Figure 1. Dosing and sampling plan. 2.2.1. Semi-Natural Stormwater Preparation Five indicator microorganism sampling days and three reference pathogen sampling days were within the scope of this study. Remaining sampling days were within the scope of the other parallel study [19]. 2.2.2. Dosing Frequency and Volumes 2.2.2. Dosing Frequency and Volumes The volumes used were: (1) 20 L, which is the 1 in 1-month average recurrence interval (ARI) event and equivalent to 1 PV (equivalent loading of 8.84 mm per event) or (2) 40 L which is equal to a 1 in 3-month ARI event and 2 PVs (equivalent loading of 17.68 mm per event). In total, samples were collected from three 40 L dosing events and two 20 L dosing events for the current study. g g y To test the effect of extended antecedent dry weather conditions, columns were left to dry for periods of two, four and six weeks before the subsequent dosing to simulate three dry weather periods. As such, in this study, we refer to two periods: (1) ‘wet weather periods’, where the columns were dosed twice a week and dosing occurred at least once every two weeks; and (2) ‘dry weather periods’, where dosing did not occur for 2 weeks or more. The total of five events sampled for the current study comprised of four wet weather period sampling events and one four-week dry weather period sampling event. To test the effect of extended antecedent dry weather conditions, columns were left to dry for periods of two, four and six weeks before the subsequent dosing to simulate three dry weather periods. As such, in this study, we refer to two periods: (1) ‘wet weather periods’, where the columns were dosed twice a week and dosing occurred at least once every two weeks; and (2) ‘dry weather periods’, where dosing did not occur for 2 weeks or more. The total of ﬁve events sampled for the current study comprised of four wet weather period sampling events and one four-week dry weather period sampling event. 2.2.3. Inflow and Outflow Water Quality Sampling 2.2.3. Inﬂow and Outﬂow Water Quality Sampling 2.2.3. Inflow and Outflow Water Quality Sampling 2.2.3. Inﬂow and Outﬂow Water Quality Sampling The inflow was analysed for E. coli concentration in every dosing event. One inflow sub-sample was collected from each 3–5 L pass and each sub-sample was analysed for E. coli using the Colilert The inﬂow was analysed for E. coli concentration in every dosing event. One inﬂow sub-sample was collected from each 3–5 L pass and each sub-sample was analysed for E. coli using the Colilert 5 of 12 Water 2017, 9, 949 method™[25]. Since the variations between each sub-sample were minimal within a single sampling day, only the geometric mean inﬂow E. coli concentrations are reported for each day. On days where laboratory-seeded microbial cultures were added to the inﬂow (i.e., in week 14, 26, 27, 31 and 41; Figure 1), volume-weighted composites were made from sub-samples taken from the inﬂow. These volume-weighted composites of the inﬂow were analyzed for the indicator microorganisms and pathogens of interest at NATA accredited laboratories using standard methods (FRNA coliphages: US EPA 1602, APHA 9224 and ISO 10705-1; C. perfringens: AS/NZS 4276.17.1:2000; Campylobacter spp.: an 11 tube MPN procedure and the AS 4276.19:2001; adenoviruses: cell culture technique followed by conﬁrmation with polymerase chain reaction (PCR) [26]; Cryptosporidium: USEPA 1623 method with some modiﬁcations; refer to Table S1 in Supplementary Materials for further details on each microbial assay). On the same days, these volume-weighted inﬂow composites were also analysed for total suspended solids, total nitrogen, total phosphorus and heavy metal concentrations at NATA accredited laboratories using standard methods ([27,28] and ICP-MS for metals). g ( ) On the ﬁve sampling days (Figure 1), we collected into two composite samples from the total outﬂow drained from each column. The ﬁrst composite outﬂow sample represented ‘old’ water (i.e., water which remained in the SZ and media pores from a prior event) and the second composite sample represented the ‘new’ water (i.e., water which was freshly ﬁltered during the sampling day). The initial tracer study (See Supplementary Materials Figure S1 for more details on tracer test) demonstrated that during wet weather periods, approximately the ﬁrst 10 L of outﬂow was old water and the remaining volume was the new water. Both old and new water samples were analysed for E. coli concentrations. 2.2.3. Inflow and Outflow Water Quality Sampling 2.2.3. Inﬂow and Outﬂow Water Quality Sampling Weights of all old and new water drained from each column were recorded, and these data were then used to derive a volume-weighted, event mean outﬂow E. coli concentration. Due to resource limitations, it was not possible to analyse old and new water separately for the other target faecal microorganisms. For the other target faecal microorganisms, the old and new water samples were combined (after separate aliquots were taken from the new and old water samples for E. coli analysis). A sub-sample taken from this composite sample was analysed for the reference pathogens and/or the other indicator microorganisms. 2.2.4. Inﬁltration Rate Measurements Inﬁltration rates were measured on all ﬁve outﬂow sampling days (Figure 1) using a method similar to a previous study [29]. The ponding water level was recorded at regular intervals (3 min and 45 s) for approximately one hour. The ﬁrst ponding measurements were taken after all the stormwater had been applied to the systems, to ensure that the columns were saturated to a level similar to that of bioﬁlters in the ﬁeld. It should be noted that the inﬁltration rate obtained using this method does not represent the saturated hydraulic conductivity of the system (as the system is not necessarily saturated when the inﬁltration rates are measured), but rather what would be seen typically under ﬁeld conditions, and is dependent on soil moisture prior to the measurements. The recorded ponding depths over the hour were plotted against time, and the gradient of this graph was taken as the average inﬁltration rate through the column. 3.1. Overall Removal Performance of Indicators and Reference Pathogens Water 2017, 9, 949 Contrary to other studies [13,14], there was no notable differences in indicator concentration reductions between vegetated and un-vegetated controls (p > 0.05; independent samples t-test; Figure 2). Plants have previously been highlighted as a key covariant controlling faecal indicator removal due to advanced adsorption to roots and interactions with exudates and the rhizosphere; as such, our results might instead be reﬂecting the relatively higher importance of other design (e.g., presense of an SZ) and operational conditions (large vs. small inﬂow volumes). Nonetheless, the average E. coli log reduction of 1.2 is comparable to E. coli log reductions (1.2–1.4) observed in the authors’ previous work in ﬁeld conditions [30]. C. perfringens were removed better than E. coli (>2 average; p < 0.05; independent samples t-test) owing to their larger size which promotes physical straining by the media [7]. The inconsistent FRNA coliphage removal (ranging from net leaching to over 3 log removal) for both WS and LC is caused by the large variance in inﬂow concentrations (Supplementary Materials Table S2), variable operational conditions (as discussed below) and also due to the potential interactions between coliform bacteria and phages that can occur within bioﬁltration systems [7]. 3. Results and Discussion 3.1. Overall Removal Performance of Indicators and Reference Pathogens Contrary to other studies [13,14], there was no notable differences in indicator concentration reductions between vegetated and un-vegetated controls (p > 0.05; independent samples t-test; Figure 2). Plants have previously been highlighted as a key covariant controlling faecal indicator removal due to advanced adsorption to roots and interactions with exudates and the rhizosphere; as such, our results might instead be reflecting the relatively higher importance of other design (e.g., presense of an SZ) and operational conditions (large vs. small inflow volumes). Nonetheless, the average E. coli log reduction of 1.2 is comparable to E. coli log reductions (1.2–1.4) observed in the authors’ previous work in field conditions [30]. C. perfringens were removed better than E. coli (>2 average; p < 0.05; independent samples t-test) owing to their larger size which promotes physical straining by the media [7]. 3.1. Overall Removal Performance of Indicators and Reference Pathogens Water 2017, 9, 949 The inconsistent FRNA coliphage removal (ranging from net leaching to over 3 log removal) for both WS and LC is caused by the large variance in inflow concentrations (Supplementary Materials Table S2), variable operational conditions (as discussed below) and also d t th t ti l i t ti b t lif b t i d h th t ithi y All reference pathogen concentrations were reduced by the vegetated bioﬁlters, which is a substantial ﬁnding considering no previous data was available on stormwater bioﬁlter capacity to remove these pathogens and the sampling events were designed to test the bioﬁlters during very challenging operational conditions. However, of the three reference pathogens tested, Campylobacter spp. had the most variable and the lowest average removal rates (0.7 average log reduction). These results are explained by the highly variable inﬂow concentrations (Supplementary Materials Table S2) and the uncertainties associated with the current Australian standard method of analysis of thermophilic Campylobacter [31]. It is encouraging to note that the average Campylobacter spp. log reduction observed in the current study is comparable to the average Campylobacter spp. log reduction observed in the authors’ previous work in ﬁeld bioﬁltration systems [30]. Cryptosporidium oocysts were the most effectively removed reference pathogen by stormwater bioﬁlters (1.7 average log reduction); as for C. perfringens, this is likely due also to its larger size [32]. Adenoviruses were removed, on average, but with a 1 log reduction. This reﬂects its smaller particle size, which prohibits active straining. due to the potential interactions between coliform bacteria and phages that can occur within biofiltration systems [7]. All reference pathogen concentrations were reduced by the vegetated biofilters, which is a substantial finding considering no previous data was available on stormwater biofilter capacity to remove these pathogens and the sampling events were designed to test the biofilters during very challenging operational conditions. However, of the three reference pathogens tested, Campylobacter spp. had the most variable and the lowest average removal rates (0.7 average log reduction). These results are explained by the highly variable inflow concentrations (Supplementary Materials Table S2) and the uncertainties associated with the current Australian standard method of analysis of thermophilic Campylobacter [31]. It is encouraging to note that the average Campylobacter spp. log reduction observed in the current study is comparable to the average Campylobacter spp. log reduction observed in the authors’ previous work in field biofiltration systems [30]. 2.3. Data Analysis Microbial concentrations below the lowest detection limit were taken as the lowest detection limit, and the microbial concentrations above the highest detection limit were taken as the highest detection limit for analysis. Removal performance in terms of log reduction is the difference between the logarithmic (base 10) inﬂow concentration and the logarithmic outﬂow concentration. Log reduction data were found to be normally distributed according to the Shapiro-Wilk test. Therefore, the independent sample t-tests with the signiﬁcance level, α taken as 0.05 was used to test the effect of bioﬁlter vegetation (LC vs. WS) on the removal of the three indicator microorganisms and differences in indicator microorganism log removal rates. 6 of 12 Water 2017, 9, 949 3. Results and Discussion 3.1. Overall Removal Performance of Indicators and Reference Pathogens Water 2017, 9, 949 3.2. Effect of Dry Weather Periods Water 2017, 9, 949 Log reductions before and after four weeks of dry weather are shown in Figure 3a. E. coli log reduction performance decreased drastically after the 4-week dry period (median log reduction from 1.41 to 0.23). Generally, SZ water is lost during extended dry weather periods due to evapotranspiration (Figure 3c shows a 50% reduction in outﬂow volume after the dry period even though the inﬂow volume in the preceding event was the same). This loss of SZ water diminishes the contribution of the ‘old’ SZ water to the efﬂuent, which generally has lower E. coli concentrations because of its longer contact time and hence more microbial die-off [13]. As a result, the outﬂow after four weeks of drying is mainly comprised of freshly treated stormwater, which has relatively higher E. coli concentrations because of its shorter contact time. An increase in inﬁltration rates was also observed after four weeks of dry weather (Figure 3d), possibly due to the creation of cracks and macropores in the ﬁlter media. It is well known that increased inﬁltration rates lead to decreased bacterial adsorption to ﬁlter media/bioﬁlms [33]. Therefore, it is evident that the combined effect of increased inﬁltration rates and decreased SZ water volume has led to a lower E. coli log reduction after the four weeks dry weather period. 3.2. Effect of Dry Weather Periods Log reductions before and after four weeks of dry weather are shown in Figure 3a. E. coli log reduction performance decreased drastically after the 4-week dry period (median log reduction from 1.41 to 0.23). Generally, SZ water is lost during extended dry weather periods due to evapotranspiration (Figure 3c shows a 50% reduction in outflow volume after the dry period even though the inflow volume in the preceding event was the same). This loss of SZ water diminishes the contribution of the ‘old’ SZ water to the effluent, which generally has lower E. coli concentrations because of its longer contact time and hence more microbial die-off [13]. As a result, the outflow after four weeks of drying is mainly comprised of freshly treated stormwater, which has relatively higher E. coli concentrations because of its shorter contact time. An increase in infiltration rates was also observed after four weeks of dry weather (Figure 3d), possibly due to the creation of cracks and macropores in the filter media. 3.2. Effect of Dry Weather Periods Water 2017, 9, 949 It is well known that increased infiltration rates lead to decreased bacterial adsorption to filter media/biofilms [33]. Therefore, it is evident that the combined effect of increased infiltration rates and decreased SZ water volume has led to a lower E. coli log reduction after the four weeks dry weather period. Figure 3. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) before and after the four-week dry period (in weeks 27 and 31) in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent sampling event on week 27, and shaded bars represent sampling event on week 31. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. (Cryptosporidium oocysts concentrations were below detection in all three outflow samples before the four-week dry period, and hence outflow concentration was taken as the limit of reporting, which was 1 oocyst/7 L). Figure 3. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) before and after the four-week dry period (in weeks 27 and 31) in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent sampling event on week 27, and shaded bars represent sampling event on week 31. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. (Cryptosporidium oocysts concentrations were below detection in all three outﬂow samples before the four-week dry period, and hence outﬂow concentration was taken as the limit of reporting, which was 1 oocyst/7 L). Figure 3. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) before and after the four-week dry period (in weeks 27 and 31) in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent sampling event on week 27, and shaded bars represent sampling event on week 31. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. 3.1. Overall Removal Performance of Indicators and Reference Pathogens Water 2017, 9, 949 Cryptosporidium oocysts were the most effectively removed reference pathogen by stormwater biofilters (1.7 average log reduction); as for C. perfringens, this is likely due also to its larger size [32]. Adenoviruses were removed, on average, but with a 1 log reduction. This reflects its smaller particle size, which prohibits active straining. Figure 2. Overall reference pathogen and other indicator microorganism log reduction in LC and WS configurations. WS columns were not tested for any reference pathogens. Box plots for WS represent three sampling events (in week 14, 26 and 41) and the box plots for LC represent five sampling events (in week 14, 26, 27, 31 and 41). Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. Figure 2. Overall reference pathogen and other indicator microorganism log reduction in LC and WS conﬁgurations. WS columns were not tested for any reference pathogens. Box plots for WS represent three sampling events (in week 14, 26 and 41) and the box plots for LC represent ﬁve sampling events (in week 14, 26, 27, 31 and 41). Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. Figure 2. Overall reference pathogen and other indicator microorganism log reduction in LC and WS configurations. WS columns were not tested for any reference pathogens. Box plots for WS represent three sampling events (in week 14, 26 and 41) and the box plots for LC represent five sampling events (in week 14, 26, 27, 31 and 41). Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. Figure 2. Overall reference pathogen and other indicator microorganism log reduction in LC and WS conﬁgurations. WS columns were not tested for any reference pathogens. Box plots for WS represent three sampling events (in week 14, 26 and 41) and the box plots for LC represent ﬁve sampling events (in week 14, 26, 27, 31 and 41). Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. 7 of 12 Water 2017, 9, 949 7 of 12 3.2. Effect of Dry Weather Periods Water 2017, 9, 949 (Cryptosporidium oocysts concentrations were below detection in all three outflow samples before the four-week dry period, and hence outflow concentration was taken as the limit of reporting, which was 1 oocyst/7 L). Figure 3. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) before and after the four-week dry period (in weeks 27 and 31) in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent sampling event on week 27, and shaded bars represent sampling event on week 31. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. (Cryptosporidium oocysts concentrations were below detection in all three outﬂow samples before the four-week dry period, and hence outﬂow concentration was taken as the limit of reporting, which was 1 oocyst/7 L). Water 2017, 9, 949 8 of 12 The other two indicator microorganisms showed different responses to the 4-week dry weather period. The log reduction of C. perfringens was relatively unaffected by the 4-week dry period. One plausible explanation could be that despite the creation of some cracks and macropores, C. perfringens might have been retained by straining due to its large size [7]. Median log reductions in the virus indicator, FRNA coliphage, increased from 1.24 to 1.91 after the four-week dry period. Similar observations were reported in [7] in a previous laboratory experiment. One plausible explanation for this increased FRNA coliphage removal might be the increased FRNA coliphage inactivation under unsaturated conditions [34] prevailed in the column with almost all of the SZ water lost during the 4-week dry weather period (Nearly 9 L decrease in outﬂow volume after a 4-week dry period in Figure 3c). Dry weather seemed to improve the removal of Campylobacter spp.( Figure 3a), but more plausibly the removal rates are instead explained by the differences observed in Campylobacter spp. inﬂow concentrations before and after the dry weather period (Figure 3b) [35]. Cryptosporidium oocyst removal rates decreased by 1.2 log after the dry period. This could imply that oocysts retained from previous events are ﬂushed out of the bioﬁlters after dry weather periods due to cracks and macropores; importantly, even if these persistent oocysts have died during the dry period, the enumeration method employed here is still likely to detect them [36,37]. 3.2. Effect of Dry Weather Periods Water 2017, 9, 949 This effect was not observed for C. perfringens because their enumeration is culture based and hence will not detect dead organisms. Adenoviruses were largely unaffected by the dry weather period, reﬂecting the irrelevance of cracks and macropores (because of the size of the microorganisms) and submerged zone interactions (because of their ability to persist and culture-based enumeration). 3.3. Effect of Event Size Log reductions observed in two different event sizes (20 L vs. 40 L) are shown in Figure 4a. Only FRNA coliphage and Campylobacter log reductions seemed to be notably different during the two events, and these differences in removal rates were most likely due to differences in inﬂow concentrations rather than the event size. Similarly, signiﬁcantly different inﬂow adenovirus concentrations in the two events make it difﬁcult to distinguish the effect of event size on adenovirus removal. On the other hand, inﬂow concentrations of Cryptosporidium oocysts and C. perfringens were comparable in both events, and overall removal rates were relatively unaffected by the event volume. One possible explanation for the relatively consistent removal of these two larger microorganisms is that most of these microbes were retained by straining, which was not affected by the event volume. On the other hand, other microbes such as FRNA coliphages are removed by adsorption and desorption, which are two removal process that can be affected by variation in event volume [18]. The relatively consistent E. coli log reductions observed during both events conﬂicts with the trend of decreasing E. coli log reduction with increasing event volume as reported in the authors’ previous work [19]. A further investigation into E. coli inﬂow and outﬂow concentration revealed that the E. coli concentrations were different in the inﬂows of the two events, and it is likely that this masked any inﬂuence of the increased volume in the 40 L event. Hence, more testing with consistent inﬂow microbial concentrations is required to fully understand how removal is affected by different event volumes. 9 of 12 12 Water 2017, 9, 949 Water 2017, 9, Figure 4. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) during a 20 L (in week 27) and a 40 L (in week 41) event in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent 20 L sampling event on week 27, and shaded bars represent 40 L sampling event on week 41. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. Figure 4. 3.4. Comparison of Indicator and Reference Pathogen Removal 3.4. Comparison of Indicator and Reference Pathogen Removal p f f g Comparing the removal performances of the selected indicator microorganisms and reference pathogens revealed that indicator removal rates fail to consistently represent the removal rates of their corresponding reference pathogens. For instance, the average Campylobacter spp. log reduction rate was lower than that of E. coli (Figure 2). Furthermore, E. coli log reduction decreased after a long dry period while Campylobacter spp. log reduction rate showed an increase (Figure 3a). Similarly, the two microorganisms were affected differently by the event size (Figure 4a) with an increased Campylobacter spp. log reduction with an increased event volume while E. coli log reduction remained unaffected. However, conflicting trends between the two microorganisms may be due to large variances in inflow Campylobacter spp. concentrations. The average Cryptosporidium oocysts log reduction was similar to that of C perfringens However Comparing the removal performances of the selected indicator microorganisms and reference pathogens revealed that indicator removal rates fail to consistently represent the removal rates of their corresponding reference pathogens. For instance, the average Campylobacter spp. log reduction rate was lower than that of E. coli (Figure 2). Furthermore, E. coli log reduction decreased after a long dry period while Campylobacter spp. log reduction rate showed an increase (Figure 3a). Similarly, the two microorganisms were affected differently by the event size (Figure 4a) with an increased Campylobacter spp. log reduction with an increased event volume while E. coli log reduction remained unaffected. However, conﬂicting trends between the two microorganisms may be due to large variances in inﬂow Campylobacter spp. concentrations. The average Cryptosporidium oocysts log reduction was similar to that of C. perfringens. However, the impact of dry weather on these two microorganisms was different. Cryptosporidium oocysts log reduction decreased after a dry weather period while C. perfringens were relatively unaffected. This variation could be due to differences in enumeration methods (culture vs. microscopic). However, both microbes were relatively unaffected by the event volume. Lastly, the average adenovirus log reduction was similar to that of FRNA coliphages, but The average Cryptosporidium oocysts log reduction was similar to that of C. perfringens. However, the impact of dry weather on these two microorganisms was different. Cryptosporidium oocysts log reduction decreased after a dry weather period while C. perfringens were relatively unaffected. This variation could be due to differences in enumeration methods (culture vs. microscopic). However, both microbes were relatively unaffected by the event volume. 3.3. Effect of Event Size Change in log reduction (a), inﬂow concentration (b), total outﬂow volume (c) and inﬁltration rate (d) during a 20 L (in week 27) and a 40 L (in week 41) event in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent 20 L sampling event on week 27, and shaded bars represent 40 L sampling event on week 41. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. Figure 4. Change in log reduction (a), inflow concentration (b), total outflow volume (c) and infiltration rate (d) during a 20 L (in week 27) and a 40 L (in week 41) event in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent 20 L sampling event on week 27, and shaded bars represent 40 L sampling event on week 41. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oo y t Figure 4. Change in log reduction (a), inﬂow concentration (b), total outﬂow volume (c) and inﬁltration rate (d) during a 20 L (in week 27) and a 40 L (in week 41) event in LC columns; Bars represent the median value while error bars represent the minimum and maximum measured or estimated values. Unshaded bars represent 20 L sampling event on week 27, and shaded bars represent 40 L sampling event on week 41. Microbial abbreviations: FRNA: FRNA coliphages; AV = adenovirus; EC = E. coli; CA = Campylobacter; CP = Clostridium perfringens; CR = Cryptosporidium oocysts. 4. Conclusions Two main objectives of this investigation were to test the capacity of bioﬁlters to remove pathogens and to compare the inﬂuence of design characteristics and operational conditions on the removal of faecal indicator microorganisms and reference pathogens. The laboratory-scale column study showed that stormwater bioﬁlters are able to remove reference pathogens (Campylobacter spp., adenoviruses and Cryptosporidium oocysts) and the faecal indicator microorganisms, E. coli and C. perfringens. Out of the three tested common indicators, only C. perfringens and FRNA coliphages showed comparable overall log reductions with their corresponding reference pathogens, but then these indicators and their corresponding reference pathogens showed different responses when the stormwater bioﬁlters were subjected to different operational conditions such as varying antecedent dry weather periods and different event volumes. This highlights that the removal processes of pathogens in stormwater bioﬁlters cannot be accurately characterised by the behaviour of their corresponding indicator microorganisms under all operational conditions. An understanding of the key removal processes of individual microorganisms is critical when optimising stormwater bioﬁltration systems for reducing microorganism levels in stormwater. As such, this study demonstrates that more data on pathogen removal in stormwater bioﬁlters must be collected, and the effects of different design characteristics and operational conditions should be tested. Supplementary Materials: The following are available online at www.mdpi.com/2073-4441/9/12/949/s1, Figure S1: Change of outﬂow KCl concentration (normalised to the inﬂow concentration) with cumulative outﬂow during the tracer test trail in three replicates of WS un-vegetated columns, Table S1: Microbial assay description, Table S2: A summary of inﬂow and outﬂow microbial concentrations of each sampling round. Acknowledgments: Authors wish to acknowledge Monash Water for Liveability and CRC for Water Sensitive Cities for ﬁnancially contributing to this study and the Australian Research Council-Discovery Early Career Researcher Award (grant number DE140100524) for the ﬁnancial support during the manuscript preparation. Authors also received funds from CRC for Water Sensitive Cities for covering the costs to publish in open access. Support from Yali Li, Minna Tom, Anthony Brosinsky, Christelle Schang, Peter Kolotelo, Kun Kim, Ashley Connelly and Ben Evans during the experimental duration is also gratefully acknowledged. Author Contributions: Gayani Chandrasena, David McCarthy and Ana Deletic conceived and designed the experiments; Gayani Chandrasena and Rebekah Henry performed the experiments; Gayani Chandrasena and Anna Lintern analysed the data; Rebekah Henry contributed reagents/materials/analysis tools; Gayani Chandrasena wrote the paper; David McCarthy, Anna Lintern, Ana Deletic and Rebekah Henry reviewed the paper. 4. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. 3.4. Comparison of Indicator and Reference Pathogen Removal 3.4. Comparison of Indicator and Reference Pathogen Removal adenoviruses were relatively unaffected by dry weather periods while FRNA coliphages showed an increased log reduction after a dry weather period. These conflicting responses to dry weather periods could be due to the differences in major removal mechanisms (attachment and die-off) and Lastly, the average adenovirus log reduction was similar to that of FRNA coliphages, but adenoviruses were relatively unaffected by dry weather periods while FRNA coliphages showed an increased log reduction after a dry weather period. These conﬂicting responses to dry weather periods could be due to the differences in major removal mechanisms (attachment and die-off) and enumeration techniques. Likewise, both viruses showed opposite responses when the event volume was changed, but those responses were most likely due to the variances in inﬂow concentrations. Water 2017, 9, 949 10 of 12 10 of 12 These results reinforce the inadequacy of using just a single indicator microorganism to test the capacity of stormwater bioﬁlters and as such, encourages the use of a suite of indicators and reference pathogens. Although more data is required to fully understand reference pathogen behaviour, this is one of the few sets of reference pathogen data in the context of stormwater bioﬁlters. These data provide useful insight into the ability of bioﬁlters in reducing actual pathogen concentrations rather than just indicator microorganisms. Further investigations could broaden our knowledge on the governing removal mechanisms of different pathogen types and how these mechanisms are affected by different bioﬁlter designs and operational conditions. g 2. Urban Water Resources Research Council. Pathogens in Urban Stormwater Systems; American Society of Civil Engineers: Reston, VA, USA, 2014. 1. 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https://openalex.org/W2991466924	https://europepmc.org/articles/pmc6960572?pdf=render	English	null	A Theory of Guilt Appeals: A Review Showing the Importance of Investigating Cognitive Processes as Mediators between Emotion and Behavior	Behavioral sciences	2,019	cc-by	7,500	Review A Theory of Guilt Appeals: A Review Showing the Importance of Investigating Cognitive Processes as Mediators between Emotion and Behavior Aurélien Graton 1,* and Melody Mailliez 2 1 LIP/PC2S, Université Savoie Mont-Blanc and Université Grenoble Alpes, 38000 Grenoble, France 2 ISAE/SUPAERO, Université de Toulouse, 31055 Toulouse, France; melody.mailliez@isae-supaero.fr * Correspondence: aurelien.graton@univ-smb.fr Aurélien Graton 1,* and Melody Mailliez 2 Received: 16 October 2019; Accepted: 16 November 2019; Published: 20 November 2019 Received: 16 October 2019; Accepted: 16 November 2019; Published: 20 November 2019 Abstract: Guilt appeals in the ﬁeld of persuasion are quite common. However, the eﬀectiveness of these messages is sometimes ambivalent. It is widely acknowledged that guilt leads people to engage into prosocial behaviors, but the eﬀects of guilt can also be counter-productive (e.g., reactance-like eﬀects). We argue that the explanations for these contradictions remain unsatisfactory and suggest that taking into account the implications of underlying cognitive—especially attentional—mechanisms would provide a better understanding of these paradoxical outcomes. This article provides a brief review of the literature on the link between guilt and pro-social behaviors and its classical interpretations. We propose a reinterpretation of this link by taking into account speciﬁc attentional processes triggered by the emotion of guilt. Attentional biases are, in our opinion, better predictors of the eﬀectiveness of a message than the amount of emotion induced by the same message. This consideration should guide future research in the ﬁeld of guilt appeals and pro-social behaviors. Implications, in terms of a broader comprehension of the emotion–behavior association in decision making processes, are discussed. Keywords: guilt; emotions; attention; persuasion; cognitive processes; decision making A picture depicts an African woman lying in a desert, holding a stylish handbag. A slogan states “Handbag: 32€. Food for a week: 4€”. Another advertisement shows a famished child, with the following statement “He’s starving, we’re not. It’s time to share”. These two examples taken from the 2000s illustrate how persuasion frequently relies on moral emotions and, especially, guilt appeals. Moral emotions can be depicted as emotions “linked to the interests or welfare either of society as a whole or at least of persons other than the judge or the agent” ([1], p. 853). Guilt is often seen as the most prototypical moral emotion [2], and it is repeatedly used in persuasion campaigns for its prosocial consequences. A wide body of research has focused on its deﬁnition (i.e., the origin of guilt) and behavioral consequences (i.e., restorative behaviors). However, there is a lack of knowledge regarding the processes linking guilt to prosocial actions. Review A Theory of Guilt Appeals: A Review Showing the Importance of Investigating Cognitive Processes as Mediators between Emotion and Behavior The objective of the present article is to shortly review the sometimes paradoxical eﬀects of guilt on persuasion and to propose alternative explanations based on the investigation of underlying cognitive, especially attentional, processes. Although there are other theoretical frameworks to explain these eﬀects, we limit our paper to the contribution of taking these cognitive processes into account. behavioral sciences behavioral sciences 1. Guilt and Prosocial Behaviors: From Reparation to Backlash Eﬀects In this type of persuasion, individuals are typically exposed to messages that both induce feelings of guilt and subsequently provide a way to reduce these feelings by adopting the proposed behavior. As an illustration, Coulter and Pinto [20] designed a study using a commercial message about children’s dental health. The message included “guilt inducing” sentences (e.g., “mothers who neglect their children’s dental hygiene have children with dental problems. It is your responsibility to ensure the oral follow-up of your children, do not let your family down!”), followed by a suggestion for parents to take care of their children’s health with the use of ﬂoss. Results showed that the level of guilt predicted the intention to buy dental ﬂoss. Following the same method, guilt has been used to promote condom purchase [21], anti-alcohol messages [22,23], pro-environmental behaviors [24,25], or to encourage charity giving [26,27]. More generally, guilt appeals are often used by companies or NGOs to promote their cause and products, as depicted at the beginning of this article. However, a closer look at the association between guilt and prosocial behaviors shows that this link is not as systematic as expected. Several studies questioned the link between guilt and reparation (e.g., [28]). In a series of ﬁve experiments, Cryder, Springer, and Morewedge [29] showed that the induction of guilt led to reparative actions only when the victim was present and could witness the restorative behavior. In other cases (e.g., absence of the victim), guilt did not lead to reparation. It therefore seems that guilt does not act as a universal trigger for restorative actions. Coulter and Pinto [20] suggested the existence of an “inverted U” curve describing the inﬂuence of guilt on persuasive messages: On the one hand, a “moderate” level of guilt would lead to greater support for the message. On the other hand, a high level of guilt would be likely to cause a rejection of the persuasive message. Other studies showed that guilt appeals in mass media campaign could be counterproductive [30]. These results are consistent with a meta-analysis conducted by O’Keefe [31] suggesting a non-linear relationship between the level of guilt expressed and the persuasive eﬀect of a message: a high level of guilt appears to be associated with a low level of persuasion, while a low level of guilt is associated with a high level of persuasion. 1. Guilt and Prosocial Behaviors: From Reparation to Backlash Eﬀects It has been largely demonstrated that emotions play a regulatory function for society at large [3,4]. More speciﬁcally, guilt occurs when an individual considers that his or her actions have violated a personal moral norm and caused harm to others [2,5]. Guilt, therefore, includes a sense of personal Behav. Sci. 2019, 9, 117; doi:10.3390/bs9120117 www.mdpi.com/journal/behavsci www.mdpi.com/journal/behavsci 2 of 10 Behav. Sci. 2019, 9, 117 responsibility, with some form of distress, towards others [5–7] (In German, the word “schuld” refers to both “guilt” and “debt”). Guilt could then encourage confessions, apologies, excuses [8], or inhibitions of subsequent behaviors [9]. The objective of these actions is to restore the relationship with others as it existed before the transgressive behavior [6]. However, reparative actions can have a broader scope than strict interpersonal relationships and can be part of the social life: “Prosocial behaviors” beneﬁt individuals other than the self and even society as a whole [10]. The idea of a link between guilt and prosocial behavior is fairly old [11], but the experimental evidence for this association is quite recent. In the 1960s, studies showed that the adoption of transgressive behavior led to a desire to repair the harm caused (e.g., [12,13]). For instance, Regan, Williams, and Sparling [14] conducted an experiment in which participants could be led to believe that they had damaged an experimenter’s camera after he had asked them to take a picture. Participants then met a second accomplice experimenter whose bag was open. More participants who thought they had damaged the camera reported to the accomplice that his/her bag was open. In these studies, restorative behavior was attributed to guilt. However, guilt was not always assessed through experimental manipulation checks, nor was it always explicitly manipulated. At a broader scale, a meta-analysis showed a global eﬀect of negative eﬀects on pro-social behavior [15]. More recent studies have thus focused on better formalizing the speciﬁc causal relationship between guilt and pro-social restorative behavior. Ketelaar and Au [16] proposed two experiments in which participants were asked to play social-dilemma games. During the diﬀerent game turns, it was possible to adopt a cooperative or an individualistic behavior. Participants experiencing guilt showed more cooperative behaviors than those in the control group (see also [17–19]). This causal relationship between guilt and prosocial behavior explains why this emotion is widely used in the ﬁelds of prevention or social marketing. 1. Guilt and Prosocial Behaviors: From Reparation to Backlash Eﬀects According to the theory of “aﬀect as information” (According to this theory, emotional feelings act as sources of information, in the same way as other stimuli in the environment; for a review see [44], guilt would also serve as an informational indicator that the behavior adopted, or soon to be adopted, is not compatible with a social norm of the person concerned (see [16]). Consequently, it is necessary to act in the direction of greater compliance with the standard concerned and to adopt appropriate restorative behavior (e.g., compliance with a persuasion campaign). In the case of social dilemma games, participants would attribute their guilt to poor choices in the game. Guilt would therefore only serve as valid information for those who have violated the moral standard, which would explain the eﬀect of more prosocial behavior for those who feel guilty. “The aﬀect as information” theory may therefore be relevant to explain the consideration that a standard has been breached and that positive action must be performed to restore a deteriorated relationship. Yet, while this theory can shed light on how a person introspectively realizes that reparation must take place, it does not provide an understanding of how and by what means an individual experiencing guilt will actually repair the harm caused. The behavioral consequences of guilt have also been explained in terms of the reciprocity standard. It is a universal human principle that many social behaviors and interpersonal relationships could be based on the expected reciprocity of behaviors. This rule has been formalized in psychology in diﬀerent ways, particularly under the name of “reciprocity standard” [45]. Many studies have considered guilt and the desire not to experience this emotion (aversion to guilt) as a consequence of the norm of reciprocity (e.g., [46],). Within this theoretical framework, moral emotions and guilt, in particular, would serve as a warning that the reciprocity standard has been violated, i.e., in the statement “I wouldn’t want others to harm me, so I should not harm them”, anticipated guilt acts as a “mediator” between moral transgression and behavior, which is close to the “aﬀect as information” theories mentioned above. Taking into account this norm of reciprocity may help to understand the occurrence of guilt, but not the processes by which this emotion leads to reparation, especially since this link is not systematic. 1. Guilt and Prosocial Behaviors: From Reparation to Backlash Eﬀects These paradoxical results echo the concept of “reactance” deﬁned by Brehm ([32], see also [33]). This psychological defense mechanism occurs when an individual perceives his freedom of action to be threatened. An individual in a state of reactance will behave in such a way as to 3 of 10 Behav. Sci. 2019, 9, 117 restore his freedom (or, at least, his sense of freedom), for example, by performing behaviors that are contrary to those required. As an illustration, Graton and colleagues [18] showed that guilt induction led to prosocial behavior when persuasion messages contained subtle reparation proposals. When the message was presented in a blatant or overly explicit way, guilt led to reactance-like behaviors (i.e., opposed to the persuasive message request) showing that guilt by itself is not suﬃcient to trigger altruistic behaviors. In summary, there is no systematic link between guilt and prosocial behaviors in persuasion. It also seems that the inconsistencies observed in the literature are not only due to the intensity of guilt but also to other potential factors (e.g., [25]. We argue that the current explanations for these discrepancies are not suﬃcient. A ﬁrst explanation relies on the “unpleasant” nature of guilt. As a result of this aspect, individuals who experience guilt may experience distress and general discomfort [2]. Like other negative emotions (e.g., sadness, disgust), the goal for a person experiencing such a feeling is usually to “get out” as fast as possible [34–36] and to regain a positive emotional state. Prosocial actions make it possible to return to this positive emotional state by carrying out a “positive” action. However, the mere valence of guilt is not enough to explain why the associated behavioral tendency would be prosocial behavior, i.e., speciﬁc restorative behavior, and not, for example, relaxing while watching a TV show. Depending on the context, several other negative emotions can also lead to prosocial behaviors, like shame [37–40] embarrassment [41] (or regret (e.g., [42]). Conversely, other negative emotions do not lead to the behavioral tendency of reparation (e.g., fear leads, in the ﬁrst place, to running away; see also [43]). 2. What Cognitive Processes Are Involved in Guilt Appeals? The shift from emotion to behavior involves physiological and cognitive intermediate processes. For example, fear is often considered to indicate a danger to the individual and leads to a state of readiness to ﬂee by directing the attention of the person in danger both to the origin of the danger (e.g., a snake) and to escape possibilities, such as a hideout (see [51]). Under this perspective, a large body of research has examined the role of attentional mechanisms associated with emotions (e.g., [52–56]). Other studies have shown that emotions make information relevant to the achievement of a behavior being more “accessible” (e.g., [57,58]). Within this context, the “feeling is for doing” approach (see [43]) argues for a need to be speciﬁc when investigating the impact of emotions on decision making. However, the authors also pointed out that the link between emotion and behavior is not enough to understand the speciﬁcity of an emotion and that “it is time to move beyond the mere documentation of behavioral results of emotions to direct tests of the proposed mechanism underlying these eﬀects. Although many studies demonstrate congruence between observed decisional eﬀects and emotional goals, this does not conclusively attest for the idea that goal activation as a result of emotional states causes these eﬀects.” ([59], p. 24). In fact, little work has been done to understand the cognitive mechanisms at play in the guilt–prosocial behavior relationship. A better understanding of these processes could also provide a better understanding of the global nature of the relationship between emotions and behavior. It has been shown that diﬀerent types of cognitive processing can mediate the emotion–behavior relationship. As highlighted by the “feeling is for doing” approach, the allocation of speciﬁc attention modiﬁed by the emotional state might represent a relevant candidate to explain the mediation of the emotion–behavior relationship. In other words, attention involves the exclusion or abstraction of certain objects in order to treat others more eﬀectively [60]. Attention is thus a corollary of the inability to process, visually or cognitively, all the information available in one’s environment [61] at one speciﬁc moment. Our objective here is not to detail the diﬀerent components and speciﬁcities of attention (for a review, see, for instance, [62]) but to highlight how selective attention can account for paradoxical eﬀects of guilt appeals. 1. Guilt and Prosocial Behaviors: From Reparation to Backlash Eﬀects Other theories, like cognitive dissonance (see [47]), have been used to explain the eﬀects of guilt in the consumer decision-making process (e.g., [48]). However, they do not take into account the way guilt could interact with other factors and, in particular, they are insuﬃcient to interpret its sometimes paradoxical consequences (e.g., “backﬁre” or reactance eﬀects). 4 of 10 Behav. Sci. 2019, 9, 117 Finally, it has also been argued that these paradoxical eﬀects could be explained by the very nature of guilt. Some authors have addressed the explanation that guilt is not an emotion, but a mere cognitive assessment of causing harm ([49], see also [50]). Following this assessment, diverse emotions could emerge, e.g., sadness or empathy over causing harm or fear of punishment or shame because of external views of the transgression. These emotions would, in turn, trigger diﬀerent kinds of behavior and explain the non-linear eﬀects of guilt. In our opinion, taking into account attentional processes could make it possible to better understand this “cocktail of emotions” potentially triggered by the emotion, or concept, of guilt. Depending on the context, guilt mixed with too much “shame” would, for instance, direct attention to the self and not toward reparation possibilities. In other cases, prototypical guilt would lead to attention being paid to the means provided for repair. One way to deepen the understanding of guilt’s role in persuasion is, therefore, in our view, to take cognitive mechanisms into account chronologically prior to behavior. 2. What Cognitive Processes Are Involved in Guilt Appeals? g pp Many studies have highlighted the existence of attentional biases towards negative stimuli or those associated with the notion of threat for people experiencing anxiety [52,53,56]. For example, Mogg and Bradley [53] used an attentional probe task to show that anxiety disorders led to more attention being directed to negative stimuli than depression. These eﬀects may be explained by the need to detect stimuli perceived as threatening more quickly in order to get prepared for fast action (escape). These results were replicated with other tasks, such as the “emotional Stroop test [63]. The literature on attention and anxiety shows a greater allocation of attention to the source of the anxiety feeling. In the context of an action-oriented conception of emotion, it is conceivable that the emotional state is also directed towards the means at disposal to prepare this action (e.g., the possibility to escape for fear). To date, few studies have examined the attention mechanisms speciﬁc to social and more 5 of 10 Behav. Sci. 2019, 9, 117 complex emotions, such as guilt. Graton and Ric [64] showed that guilt increased attention towards items related to reparation, but this did not imply persuasion messages or real means provided to repair. This attentional selectivity towards the means of performing the action associated with the emotional state may allow for a more accurate interpretation of the behavioral eﬀects of guilt. As seen above, most studies relying on guilt appeals use fairly similar methodologies: The ﬁrst part of a message seeks to make the target feel guilty (via a suﬃciently strong image or slogan). The second part of the same persuasion message makes it possible to engage in pro-social behavior (donation, involvement in health behavior, etc.) and thus reduce feelings of guilt by “repairing”. The problem with this type of message is that it is diﬃcult to anticipate the risks of backlash and reactance. Meta-analyses showing inverted U-type curves (e.g., [31]) indicate a “threshold of guilt” at which too much emotional feeling causes reactance. However, they do not show the process by which this emotional “saturation” occurs, leading to a large number of persuasive messages that are relatively disappointing in their eﬀects, even though the message intends to induce “little” guilt (see [65]). Another way to address these threshold eﬀects is to take attention into account. 2. What Cognitive Processes Are Involved in Guilt Appeals? Social marketing measurement tools have long been interested in these attentional processes (see [66]), particularly in the area of health and nutrition messages [67]. For example, studies have used eye-tracking techniques (for a review of these techniques, see [68]) to highlight the attentional processes involved in exposure to anti-tobacco campaigns for adolescents [69]. Wedel and Pieters [70] also showed that attention to a prevention message was increased when the number of words in the message was reduced. Oddly enough, attention measurement techniques are rarely used to study the relationships between the emotional state induced by a message and adherence to it. We support the idea that a process such as reactance is not the mere result of “too much guilt”, as can be explained in the current literature, but the result of an interaction between attentional processes activated by the emotional state and the possibility of reparation oﬀered by the message (see, for example, [18]). In other words, it seems more relevant to us to measure the “amount” of attention allocated to a message caused by an emotion rather than the amount of emotion induced by the same message. From this attentional process could come a feeling of cognitive saturation that could lead to rejection of the message (i.e., reactance). 3. Reinterpretation of Studies on Guilt and Prosocial Behavior An approach based on emotionally triggered cognitive processes has two consequences: On the one hand, it renders possible a reinterpretation of studies investigating the link between guilt and pro-social behavior. On the other hand, and in a more theoretical way, it also calls the concept of “moral emotion” attached to guilt into question. In this way, a closer look at the Ketelaar and Au [16] studies shows that guilt can be induced via two diﬀerent methods. In a ﬁrst experiment, the source of the emotion had a direct link with the possibilities of reparation proposed afterwards (i.e., “integral”, or “endogenous”, emotion). In this case, the participants were led to feel guilty towards a partner during a cooperative game. The second part of the experiment allowed them to “repair” the harm caused by favoring this partner. In a second study, guilt was “incidental”, or “exogenous”, i.e., guilt whose origin was unrelated to the subsequent prosocial behaviors. They then participated in a social dilemma game. In both cases, guilt did cause restorative behavior. If guilt was strictly aimed at restoring the relationship degraded by the harm caused, only “integral” guilt should have caused reparation. Reparation options following “incidental” guilt should not lead to restorative or prosocial behavior, since they do not directly serve to help the victim. The results of Ketelaar & Au [16] are consistent with the “feeling is for doing” approach and a broader conception of guilt directed towards the action of repairing. In this context of a general orientation towards reparation, guilt may have served as a “cognitive trigger” by directing the attention of “guilty” participants to the possibilities of reparation oﬀered to them. It should be noted that reparation was lacking when participants were identiﬁed as “prosocial” by nature, as opposed to “selﬁsh” participants, the only ones to whom guilt inﬂuenced behavioral choices. This diﬀerence is also consistent with a concept of guilt that directs attention towards reparation; this attentional selectivity 6 of 10 Behav. Sci. 2019, 9, 117 caused by guilt would be necessary only for participants who are not, by nature, altruistic. In contrast, it is useless for individuals who are normally oriented towards altruism and cooperation. Even more interesting is the analysis of studies showing a deﬁciency in the guilt–prosocial behavior association. De Hooge et al. [28] asked participants to allocate a sum of money for a birthday gift (Experiment 1) to several other individuals. 3. Reinterpretation of Studies on Guilt and Prosocial Behavior Depending on the experimental conditions, participants could have been led to feel guilty towards one of these persons. In these cases, the aggrieved party obtained a larger gift, while third parties were penalized. The idea of a systematic and linear relationship between guilt and restorative behavior, even for “incidental” guilt, is not consistent with these results, in which case, third parties should also have received a larger gift, which was possible in the experimental situation. It is, however, plausible that guilt acted as a cognitive guide to direct the participants’ attention here to the origin/source of the guilty feeling. This focus on priority reparation (i.e., the injured person) could then be at the expense of other individuals. This approach based on attention allows a better understanding of the paradoxical eﬀects (like inverted U curve) of guilt appeals. Cotte, Coulter, and Moore [71] showed that a strong induction of guilt led to increased awareness of the manipulative intentions of the author of an advertising message, which, in turn, led to reactance and the absence of pro-social behavior. As previously stated, an interpretation based on a traditional deﬁnition of guilt (i.e., harm caused leading to a willingness to repair) could not explain these results. Alternatively, they are compatible with the idea that guilt would trigger greater acuity in the direction of restorative opportunities in participants’ environment. As such, the manipulative intent of a message would itself become more obvious, and the message would become less eﬀective. Similarly, Graton et al. [18] showed that, in resource-dilemma games, guilt interacted with the way reparation opportunities were presented. When these possibilities were subtly presented, guilt led to restorative behavior. On the other hand, explicit instructions (e.g., “it is necessary to mobilize to save the planet!”) provoked backlash behaviors. Again, a strict “feeling is for doing” reading of guilt cannot be suﬃcient to account for the interaction between emotion and type of instructions. According to this functionalist approach of emotions, a main eﬀect of guilt should have been obtained as well, resulting from a previously activated amount of guilt. In a more subtle manner, guilt probably acted here as a “cognitive facilitator”, directing participants’ attention to opportunities for repair. When these possibilities were already prominent, it is possible that the person experiencing guilt may have felt “constrained” in his or her choices. 4. Extension to Other Emotions: Mediators of the Emotion–Behavior Relationship Taking cognitive processes into account as a mediator seems to be a way to better understand the emotion–behavior relationship. In the “feeling is for doing” approach, the transition from emotion to action through intermediate processes, such as attention, remains poorly studied. For example, attentional biases towards aggressive faces have been found for angry people compared to a control group [76] but little research has directly examined an anger-attentional bias towards stimuli related to the attack. This attentional bias towards objects related to aggressiveness (e.g., weapons, verbs such as “attack”, “strike”) could be an intermediate step between anger and aggressive behavioral tendencies. Similarly, emotions like disgust have mainly been studied in terms of increased attention to the source of this emotion (e.g., diﬃculty to disengage confronting a disgusting face, see [77]). It is, however, imaginable that some behaviors frequently associated with disgust (e.g., object rejection) may be preceded by attentional processes directed at “how” to achieve this behavior (e.g., stimuli representing a “garbage can” or “sink” to get rid of the object causing disgust, see [78]). Here again, “attentional probe” or eye-tracking experiments could highlight a possible attentional bias mediating the relationship between emotion and behavior. In accordance with the “feeling is for doing” theory, studies must now be carried out to better understand the nature of the association between emotion and behavior. We think that a large number of emotionally associated behaviors can potentially be explained by attentional biases directed at objects facilitating the motivated behavior. From this evidence may emerge a better understanding of the emotion–behavior relationship and a more precise use of emotional appeals in social marketing. Taking cognitive processes into account as a mediator seems to be a way to better understand the emotion–behavior relationship. In the “feeling is for doing” approach, the transition from emotion to action through intermediate processes, such as attention, remains poorly studied. For example, attentional biases towards aggressive faces have been found for angry people compared to a control group [76] but little research has directly examined an anger-attentional bias towards stimuli related to the attack. This attentional bias towards objects related to aggressiveness (e.g., weapons, verbs such as “attack”, “strike”) could be an intermediate step between anger and aggressive behavioral tendencies. 5. Conclusions It has been well established that emotions act as preparation for action (e.g., “feeling is for doing”, see [43]). This emotional–behavioral link is frequently studied in the literature, but the mechanisms underlying it remain poorly understood. As highlighted in the present paper, research has experimentally demonstrated the link between guilt and prosocial behaviors [16,17,19,29], leading to a large number of messages using guilt as a method of persuasion. However, reducing guilt to an emotion systematically leading to restorative actions does not theoretically allow a series of studies showing the imperfection of this guilt–restorative association to be taken into account, especially in the ﬁeld of persuasion [20,71,79]. We propose the hypothesis that prosocial behavioral tendencies will go along with attentional biases. These biases may, depending on the case, facilitate prosocial behavior or, on the other hand, cause reactance. This consideration of the underlying cognitive processes of attention not only allows for a better interpretation of otherwise ambiguous results (e.g., inverted U-shaped curves, [72]) but also opens new research possibilities. It is the entire chain—source of emotion–cognitive processes–prosocial behavior—that must be taken into account in future research to understand the real role of emotions in ﬁelds like persuasion or health communication. Author Contributions: G.A. contributed in initial conception of the work, drafting the article, revision of the article and ﬁnal approval of the version to be published. M.M. contributed in drafting the article, critical revision and revision of the article and ﬁnal approval of the version to be published. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3. Reinterpretation of Studies on Guilt and Prosocial Behavior This feeling of deprivation of liberty is characteristic of the reactance and “backlash” eﬀects observed in all these experiments, as well as those frequently mentioned as illustrating the paradoxical eﬀects of guilt in the ﬁeld of persuasion (see [72]). The interaction of attention to repair opportunities and overly explicit proposals may have caused this impression of “forced choice” and reduced ﬂexibility. We therefore believe that future research into studying the link between guilt appeals and persuasion will have to rely more often on attention measurement methods such as eye-tracking. There are, however, circumstances where traditional deﬁnitions of guilt are not relevant and the origin of guilt does not lie in the violation of a personal norm. For instance, guilt can be felt without actual transgression of norms and without having caused harm to others. These situations are described in the case of vicarious guilt [73]. In these cases, guilt is felt indirectly and results from acts and transgressions committed by others. In other contexts, the behavioral response resulting from guilt is automatically triggered in response to learned behaviors, which may range from reaction to one’s mother’s disapproving expression over one’s behavior to other scripted and automatic behaviors. These situations have been summarized in several emotional models such as the “compass of shame” model ([74], see also [75]). This model describes, for instance, how shame-guilt may lead to some sort of self-directed anger that could, in turn, lead to paradoxical eﬀects. In these examples, attention processes can nevertheless be present as mediating processes, even if the behavioral responses are more automatic and scripted.” Behav. Sci. 2019, 9, 117 7 of 10 3. Frijda, N.H.; Mesquita, B. The social roles and functions of emotions. 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https://openalex.org/W4362477695	https://figshare.com/articles/journal_contribution/Supplementary_Figure_5_from_Human_Umbilical_Cord_Blood_Derived_Mesenchymal_Stem_Cells_Producing_IL15_Eradicate_Established_Pancreatic_Tumor_in_Syngeneic_Mice/22499560/1/files/39958534.pdf	English	null	Supplementary Figure 5 from Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells Producing IL15 Eradicate Established Pancreatic Tumor in Syngeneic Mice	null	2,023	cc-by	210	Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ cells were determined by flow cytometry at 10 days after MSC-IL-15 Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ cells were determined by flow cytometry at 10 days after MSC-IL-15 Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ regulatory T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. (B) Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ regulatory T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. (B) Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ regulatory T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. (B) Supplementary figure 5 (A) Intratumor infiltration of CD4+FoxP3+ regulatory T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. (B) mRNA levels of Foxp3 in tumor tissue were detected by qRT-PCR. (C) MSC-GFP with tumor cells was co-injected, and 7 days later, MSC-IL-15 was administrated. Tumor growth was recorded over time. , P<0.05; , P<0.01. mRNA levels of Foxp3 in tumor tissue were detected by qRT-PCR. (C) MSC-GFP with tumor cells was co-injected, and 7 days later, MSC-IL-15 was administrated. Tumor growth was recorded over time. , P<0.05; *, P<0.01. Tumor growth was recorded over time. , P<0.05; **, P<0.01.
https://openalex.org/W2786387883	https://pubs.rsc.org/en/content/articlepdf/2018/ra/c8ra00016f	English	null	Nitrogen-doped hierarchical porous carbon derived from a chitosan/polyethylene glycol blend for high performance supercapacitors	RSC advances	2,018	cc-by	6,663	Introduction nitrogen functional groups under long-term/harsh working condition.27,28 In addition, the subsequently introduced nitrogen functional groups may block up the pores thus reducing the ion- accessible surface area.25,29 Although using nitrogen-enriched materials as precursors directly is able to overcome these weaknesses, they also exhibit some defects, such as uncontrol- lable pore structure and expensive template,30,31 which limits its performance optimization and large-scale applications. Considering these problems, synthesis of NHPC using the low- cost raw materials by a facile approach is very attractive. Supercapacitors, possessing a high power density, superior lifetime, fast charge–discharge ability and excellent safety property, have attracted great attention as next-generation energy storage devices.1–6 A variety of materials have been selected to build high performance supercapacitors to obtain high energy storage capability, such as carbonaceous materials, transition metal oxides and conducting polymers.7–11 Of them, carbon materials are thought to be appropriate electrode materials for supercapacitors due to many advantages such as low-cost, easy accessibility and excellent electrical conduc- tivity.12–15 However, the relatively low specic capacitance is still hindering their use,16 so it is essential to develop carbon elec- trode materials with high specic capacitance. Chitosan, the deacetylated derivative of chitin, is the second most popular natural polymer aer cellulose.32–34 Because of a large number of amino-groups, chitosan can be used to synthesize N-doped carbon materials with excellent super- capacitor performance.33,35 However, in contrast to cellulose- based carbons, the research on the preparation of porous carbons using chitosan is still relatively few so far. There are only a few reports about the preparation of N-doped carbons from chitosan,36,37 and the pore structure of the obtained carbons is poorly developed, which would greatly hamper their application in supercapacitors.38–40 So adding appropriate substances to regulate pore structure is desired. PEG is a water soluble polymer, has good compatibility with chitosan, they can be mixed into homogeneous solutions.41–43 During the high temperature carbonization, PEG was removed by thermal degradation, the obtained samples possess ample pore struc- tures, and the pore structure can be regulated by adjusting the ratio of chitosan and PEG. aSchool of Materials and Chemical Engineering, Zhongyuan University of Technology, Zhengzhou, 451191, PR China. E-mail: doctorpan0152@163.com bCenter for Advanced Materials Research, Zhongyuan University of Technology, Zhengzhou, 451191, PR China. E-mail: mlwzzu@163.com Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 This article is licensed under a Creative Commons Attribution 3.0 U Nitrogen-doped hierarchical porous carbon (NHPC) materials were synthesized by using a chitosan/ polyethylene glycol (PEG) blend as raw material through a facile carbonization–activation process. In this method, chitosan was used as a nitrogen-containing carbon precursor, low cost and large-scale commercial PEG was employed as a porogen. The physical and electrochemical properties of the resultant NHPC were aﬀected by the ratio of chitosan and PEG. The sample obtained by the ratio of 3 : 2 exhibits a high speciﬁc surface area (2269 m2 g1), moderate nitrogen doping (3.22 at%) and optimized pore structure. It exhibits a high speciﬁc capacitance of 356 F g1 in 1 M H2SO4 and 271 F g1 in 2 M KOH at a current density of 1 A g1, and over 230 F g1 can be still retained at a high current density of 20 A g1 in both electrolytes. Additionally, the assembled symmetric supercapacitors show an excellent cycling stability with 94% (in 1 M H2SO4) and 97% (in 2 M KOH) retention after 10 000 cycles at 1 A g1. These results indicate that the chitosan/PEG blend can act as a novel and appropriate precursor to prepare low-cost NHPC materials for high-performance supercapacitors. Received 2nd January 2018 Accepted 5th February 2018 Received 2nd January 2018 Accepted 5th February 2018 DOI: 10.1039/c8ra00016f rsc.li/rsc-advances RSC Advances RSC Advances Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: RSC Adv., 2018, 8, 7072 Received 2nd January 2018 Accepted 5th February 2018 DOI: 10.1039/c8ra00016f rsc.li/rsc-advances PAPER hed on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. licensed under a Creative Commons Attribution 3.0 Unported Licence. Nitrogen-doped hierarchical porous carbon derived from a chitosan/polyethylene glycol blend for high performance supercapacitors Cite this: RSC Adv., 2018, 8, 7072 This journal is © The Royal Society of Chemistry 2018 RSC Advances PAPER View Article Online View Journal \| View Issue RSC Advances PAPER View Article Online View Journal \| View Issue Introduction This provides us a good way to In general, improvement of the specic capacitance of carbon materials is usually by introducing heteroatoms (e.g., O, B, N) and regulating pore structure.17–21 Structural doping with heteroatoms, especially nitrogen, could improve the capacitance of carbon materials by introducing the pseudo-capacitance.22–24 In order to introduce nitrogen, postprocess, for example mela- mine immersion and ammonia heat treatment, is the most commonly used method.25,26 However, these methods may lead to a cumbersome process, pores structure collapse and unstable This journal is © The Royal Society of Chemistry 2018 7072 \| RSC Adv., 2018, 8, 7072–7079 RSC Advances View Article Online Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances View Article Online Paper synthesize NHPC materials for high-performance super- capacitors and there are no other studies have been reported. microscope (SEM) equipped with an energy dispersive X-ray spectroscopy (EDX) system. High-resolution transmission elec- tron microscopic (HRTEM) images were obtained with a JEOL JEM2100F microscope. The surface chemical properties of the samples were characterized by X-ray photoelectron spectroscopy (XPS, Escalab 250, USA). X-ray diﬀraction (XRD) patterns were conducted using a X-ray powder diﬀractometer (Bruker D8 Advance) with Cu-Ka irradiation. Raman spectra were acquired with a Raman spectrometer (LabRAM HR Evolution). Ther- mogravimetry (TG) was carried out on a TG209F1 under nitrogen ow. N2 adsorption–desorption isotherms were measured at 77 K using ASAP 2420 V2.09 A. The specic surface area was measured using the Brunauer–Emmett–Teller (BET) method and the pore size distribution (PSD) was determined using the classical Barrett–Joyner–Halenda (BJH) model. synthesize NHPC materials for high-performance super- capacitors and there are no other studies have been reported. Herein, we used chitosan/PEG blend as raw materials to fabricate the NHPC by a facile method. It was found that the porous structure and specic capacitance of NHPC could be adjusted by changing the ratio of chitosan and PEG. The prepared NHPC exhibited an excellent electrochemical perfor- mance in both acidic and alkaline medium due to the high specic surface area and hierarchical porous structure. Herein, we used chitosan/PEG blend as raw materials to fabricate the NHPC by a facile method. It was found that the porous structure and specic capacitance of NHPC could be adjusted by changing the ratio of chitosan and PEG. s Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Electrochemical measurements Working electrodes were prepared by pressing a disk (1 cm diameter) containing 80 wt% of active material, 10 wt% of acetylene black and 10 wt% of polytetrauoroethylene onto nickel foam (used in KOH solution) and stainless steel mesh (used in H2SO4 solution). The active mass was 2.0–3.0 mg per electrode. The electrochemical performances of as-prepared electrodes were studied in 1 M H2SO4 and 2 M KOH solution using a three-electrode and two-electrode system on a CHI660E electrochemical workstation (Chenhua Instruments Co. Ltd., Shanghai). In the three-electrode system, platinum foil and Ag/ AgCl electrode were used as the counter and reference elec- trodes, respectively. Cyclic voltammetry (CV), galvanostatic charge–discharge (GCD) and Electrical Impedance Spectroscopy (EIS) measurements were performed. The specic capacitance (Cg) can be calculated by using the formula of Cs ¼ I Dt/(m DV), where I is the discharge current (A), Dt is the discharge time (s), m is the mass (g) of the active materials, and DV is the potential window (V) during discharge.44,45 The specic capaci- tance of the two-electrode symmetrical supercapacitor cell can Preparation of NHPC Chitosan and PEG (Mw ¼ 6000) with a total of 5 g were dissolved in 95 g acetic acid aqueous solution that was obtained by dissolving 2 g acetic acid in 93 g de-ionized water at 50 C for 1.5 h with stirring to obtain a uniform solution. The solution was added onto a glass plate to form a solid lm aer the evaporation of solvent. Then the as-produced lm was peeled oﬀfrom the glass plate and carbonized at 800 C under N2 atmosphere for 2 h with an increasing rate of 2 C min1. Aer cooled down to room temperature naturally, the carbonized lm was ground into powder. Then the powder was blend with KOH solution with a power/KOH weight ratio of 1 : 3. The mixture was dried at 110 C to evaporate the water. Aer that, the dried mixture was heated at 800 C for 2 h at 5 C min1 under a N2 atmosphere before being cooled down to room temperature. The product obtained was washed with 1 M HCl solution and de-ionized water until a neutral pH. Finally, the washed powder was dried at 60 C under vacuum overnight to get the NHPC. The sample was signied as the PEG-x%, where x% stands for the ratio of PEG/(PEG + CS). The synthesis process is schematically presented in Fig. 1. This journal is © The Royal Society of Chemistry 2018 Introduction The prepared NHPC exhibited an excellent electrochemical perfor- mance in both acidic and alkaline medium due to the high specic surface area and hierarchical porous structure. Characterization of the prepared materials Fig. 3 (a) XPS survey, (b) C 1s, (c) N 1s, and (d) O 1s of PEG-40%. Fig. 2a–f show the SEM images of the NHPC materials. It can be seen that all samples possess pore structures, and such a porous structure renders the electrolyte able to penetrate into the bulk particles to form three-dimensional channels for ions' trans- portation. It is obviously observed that the microstructure and morphology are seriously aﬀected by the PEG content. With the increase of PEG in the preparation process, the pores of samples are increase. In contrast, PEG-50% has the 3D network structure with plentiful and loose pores randomly. Fig. 2g shows the HRTEM images of PEG-40% samples. It can be seen that PEG- 40% mainly exhibits an amorphous structure, and some locally ordered structure could be observed, as marked by the red arrows, indicating partial graphitization of the synthesized materials. hydroxyl), and O]C–O (carboxyl) groups are determined. The XPS results demonstrated the eﬀective doping of nitrogen by chitosan in the preparation process. The as-prepared carbon materials were further analyzed by XRD and Raman patterns to conrm the graphitized structure. All XRD patterns in Fig. 4a show the diﬀraction peaks at 2q around 25 and 43, corresponding to (002) and (100) crystal- lographic planes, respectively. From the picture it can be seen that PEG-50% had a low order degree, which is in good agree- ment with SEM analysis (Fig. 2). Fig. 4b presents the Raman patterns of PEG-30%, PEG-40% and PEG-50% samples. All three samples display G (around 1580 cm1) and D (around 1340 cm1) bands, reection of ordered and disordered struc- tures,23 respectively. And the narrower G-band and the lower intensity of ID/IG oen imply higher ordered structures and graphitizatio degree.24,32,45 The values of ID/IG for PEG-30%, PEG- 40% and PEG-50% were 0.93, 0.94 and 1.07, respectively, this result demonstrates that the degree of graphitization decreases with the PEG content increases, and PEG-50% sample possesses much lower degree of graphitization than others, which is identical with the results of above XRD analysis. The element composition of the PEG-40% sample was determined by XPS measurement (Fig. 3). The strong signals in the survey XPS spectra reveal the existence of three peaks at 284.8, 400.9 and 532.6 eV, corresponding to C 1s, N 1s and O 1s, respectively (Fig. 3a). High resolution XPS measurements were performed to investigate the atom binding states. Characterization The morphologies and component of the NHPC materials were characterized by a Zeiss Merlin Compact scanning electron Fig. 1 Schematic illustration of the preparation of NHPC materials. Fig. 1 Schematic illustration of the preparation of NHPC materials. RSC Adv., 2018, 8, 7072–7079 \| 7073 This journal is © The Royal Society of Chemistry 2018 This journal is © The Royal Society of Chemistry 2018 This journal is © The Royal Society of Chemistry 2018 View Article Online Fig. 3 (a) XPS survey, (b) C 1s, (c) N 1s, and (d) O 1s of PEG-40%. Paper RSC Advances be calculated by using the formula of Ccell ¼ I Dt/(M DV), where Ccell is the total cell specic capacitance (F g1), and M is the total mass (g) of active materials in both electrode. The energy and power densities (E, W h kg1 and P, W kg1) were calculated according to the equation of E ¼ 1/2CcellV2 and P ¼ E/ t, in which Ccell is the specic capacitance of the device, V is the voltage decrease in discharge, and t is the discharge time. The long-term cycling performance of electrodes were measured by a symmetrical supercapacitor in 1 M H2SO4 and 2 M KOH solution at a current density of 1 A g1 on a Land Battery Tester (LAND electronics Co. Ltd, Wuhan) at ambient temperature. Characterization of the prepared materials In case of C 1s, four peaks at 284.8, 285.4, 286.4 and 288.8 eV corresponding to Csp2, C–O/C–N, C]O and O–C]O groups was observed. The N 1s was determined by 398.5, 400.3 and 401.2 eV, which can be attributed to pyridinic N, pyrrolic N and graphitic N, respectively.45 In case of O 1s, three peaks at 531.8, 532.7, and 533.6 eV, corresponding to C]O (carbonyl), C–O (epoxy and The thermal decomposition curves of PEG-0% to PEG-50% solid lms and pure PEG were shown in Fig. 4c. The pure PEG Fig. 2 SEM images of (a) PEG-0%, (b) PEG-10%, (c) PEG-20%, (d) PEG-30%, (e) PEG-40%, (f) PEG-50% and (g) HRTEM image of the PEG-40% sample. Fig. 2 SEM images of (a) PEG-0%, (b) PEG-10%, (c) PEG-20%, (d) PEG-30%, (e) PEG-40%, (f) PEG-50% and (g) HRTEM image of the PEG-40% sample. This journal is © The Royal Society of Chemistry 2018 This journal is © The Royal Society of Chemistry 2018 This journal is © The Royal Society of Chemistry 2018 7074 \| RSC Adv., 2018, 8, 7072–7079 RSC Advances View Article Online RSC Advances View Article Online Paper Paper Fig. 4 XRD patterns (a) and Raman spectra (b) of the carbon materials. Thermal decomposition curves (c) of diﬀerent solid ﬁlms. result can be further conrmed by using BJH model to calculate the pore size distribution. And the hierarchical porous structure is conform to the ideal supercapacitor electrode materials that containing macropores, mesopores and micropores for ion buﬀering reservoir, ion transport and enhancement of charge storage,33 respectively. The data on specic surface area and pore volume are listed in Table 1. With the increase of PEG content, the total pore volume was increase, moreover, the mesopores and macropores volume was increase and the micropores volume was decrease. Compared with PEG-40%, the total pore volume of PEG-50% has a slight increase due to the high content of PEG. When the content of PEG is too high, a large number of PEG gather together and leave large pores aer decomposition, and large pore size may lead to pores structure collapse seriously in the process of post treatment and more pore volume was destroyed when grinding into powder. With the increase of PEG content, the specic surface area increased rstly and then decreased, this is the results of combined action of pore volume and pore size. Characterization of the prepared materials With the increase of PEG content, the pore volume was increase, although the pore size would also increase, the pore volume increased substantially, so the specic surface area will increase. The pore volume increased little when the PEG content reaches a certain value, the eﬀect less than the pore size, therefore the specic surface area decreased rapidly with the increase of PEG content. From the above results, it can be seen that diﬀerent proportion can inuence the specic surface area and pore volume. Sample PEG-40% has the most suitable pore size and size distribution, which is expected to enhance its behavior as a capacitor. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 XRD patterns (a) and Raman spectra (b) of the carbon materials. Thermal decomposition curves (c) of diﬀerent solid ﬁlms. exhibited only one stage around 360–440 C due to the thermal degradation of PEG. When the temperature reaches to 800 C, the weight loss of PEG was 99.2%, indicating that PEG was almost complete decomposition. The PEG-0% exhibited two stages around 80–150 C and 250–350 C due to the evaporation of water and thermal degradation of chitosan, respectively. The other samples showed three distinct stages from 30 to 900 C, which were attributed to the evaporation of water (around 80– 150 C), thermal degradation of chitosan (around 250–350 C), and thermal degradation of PEG (around 360–440 C). It can be seen that carbon residue increases during following the increasement of chitosan, which is in good agreement with above analysis. N2 adsorption–desorption isotherms were performed to examine the specic surface area and the pore size and distri- bution in the samples. As shown in Fig. 5a, all isotherms exhibit type IV with uptake at low pressure and small hysteresis loops ranging from 0.5 to 1.0, which imply the coexistence of micro- pores, mesopores, and macropores in these samples.8 This RSC Adv., 2018, 8, 7072–7079 \| 7075 Electrochemical performance (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 1 M H2SO4 aqueous electrolyte. (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Fig. 6c and 7c show the GCD curves of each sample at the current density of 1 A g1. The curves show a typical triangular shape with slight curvature, and the deviation from linearity is due to the pseudo faradic reactions during the charging–dis- charging process, which is consistent with the CV curves. Compared with other samples, PEG-40% has the much larger capacitance owing to its high specic surface area (2269 m2 g1) and moderate N-doped (3.22% obtained by EDX). Its Cs is as high as 356 and 271 F g1 in 1 M H2SO4 and 2 M KOH, respectively. Fig. 6d and 7d show the capacitance retention for current density from 1 to 20 A g1, the specic capacitances progressively decrease with the increase of current density due to the increasing diﬀusion limitation. However, PEG-40% can still retain over 230 F g1 at a high current density of 20 A g1 in both electrolytes, which means a good rate performance for the supercapacitor. The performances of diﬀerent N-doped porous carbons are listed in Table 2. Overall, the performance of the as- prepared carbon materials in this work is superior to those proposed in the literature. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 This article is licensed under a Creative Commons Attribution 3.0 Unported Fig. 6 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 1 M H2SO4 aqueous electrolyte. Electrochemical performance (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Fig. 6 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 1 M H2SO4 aqueous electrolyte. (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Fig. 8 shows the Nyquist plot of the samples at the frequency range from 0.01 Hz to 100 kHz to further investigate the capacitive property of the electrodes. The Nyquist plots were measured in a three-electrode system at the open circuit voltage. And the plot of PEG-40% is modeled and interpreted with the assistant of an appropriate electric equivalent circuit. The Nyquist plot could be divided to three parts, including an uncompleted semicircle part at high frequency, an inclined portion about 45 at the middle frequency and a linear part at low frequency. The high-frequency intercept with the real axis of PEG-40% gives the resistance value (Rs) of 1.6 U (in 1 M H2SO4) and 0.29 U (in 2 M KOH) which includes the electrolyte Fig. 7 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 2 M KOH aqueous electrolyte. (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Fig. 8 Nyquist plots measured in 1 M H2SO4 (a) and 2 M KOH (b). The inset shows a magniﬁed view of the high frequency region of the impedance spectra. Fig. 7 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 2 M KOH aqueous electrolyte. (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. scanning rates were measured. The shapes of CV curves can be well retained even under the high scan rate (Fig. Electrochemical performance 6b and 7b), indicating a superb capacitive behavior. Fig. 8 Nyquist plots measured in 1 M H2SO4 (a) and 2 M KOH (b). The inset shows a magniﬁed view of the high frequency region of the impedance spectra. Table 2 Comparison of performances of diﬀerent N-doped porous carbons Materials SBET (m2 g1) Cg (F g1) Cycle performance Reference K800 2435 197 (0.2 A g1) 92% aer 10 000 cycles at 1 A g1 6 RGH3-1 173 258 (0.1 A g1) 97% aer 4000 cycles at 1 A g1 16 N-PGCNS0.2–1–2 2130 337 (0.5 A g1) 88% aer 5000 cycles at 2 A g1 24 NCNF3 763 251 (0.1 A g1) 99% aer 2000 cycles at 5 A g1 25 NHPC-800 1542 242 (0.2 A g1) 94% aer 10 000 cycles at 1 A g1 32 3DHCG 1511 320 (1 A g1) 96% aer 2000 cycles at 2 A g1 45 PEG-40% 2269 356 (1 A g1) 94% aer 10 000 cycles at 1 A g1 This work 271 (1 A g1) 97% aer 10 000 cycles at 1 A g1 This work 7076 \| RSC Adv., 2018, 8, 7072–7079 This journal is © The Royal Society of Chemistry 2018 Table 2 Comparison of performances of diﬀerent N-doped porous carbons 7076 \| RSC Adv., 2018, 8, 7072–7079 Electrochemical performance Electrochemical properties of the resultant NHPC materials were measured using a standard three-electrode conguration in both acidic and alkaline solution. Fig. 6a and 7a show the typical CV curves of samples at a scan rate of 10 mV s1 in 1 M H2SO4 and 2 M KOH, respectively. In both cases, the covered area of PEG-40% sample is larger than the other samples, it illustrates that PEG-40% sample possesses the highest capaci- tance, which was consistent with the physical characteristics. And all the samples present a rectangular-like shape with obvious redox peaks due to the introduction of N heteroatom in carbon materials. The pseudocapacitance in acidic electrolyte is more obvious than that in basic electrolyte, probably owing to the Lewis base behavior of the nitrogen functionalities in the carbons.44 In order to further investigate the capacitance behavior of PEG-40% material, the CV curves at diﬀerent Fig. 5 (a) N2 adsorption–desorption isotherms at 77 K and (b) PSD curves of PEG-30%, PEG-40% and PEG-50% samples. Fig. 5 (a) N2 adsorption–desorption isotherms at 77 K and (b) PSD curves of PEG-30%, PEG-40% and PEG-50% samples. Table 1 Porous property of PEG-30%, PEG-40% and PEG-50% samples Sample SBET (m2 g1) Vtotal (cm3 g1) Vmicro Vmeso Vmacro Pore size (nm) PEG-30% 2052 0.429 0.146 0.226 0.057 2.7 PEG-40% 2269 0.516 0.107 0.302 0.107 3.5 PEG-50% 1436 0.520 0.091 0.309 0.120 3.6 This journal is © The Royal Society of Chemistry 2018 RSC Adv., 2018, 8, 7072–7079 \| 7075 Table 1 Porous property of PEG-30%, PEG-40% and PEG-50% samples Sample SBET (m2 g1) Vtotal (cm3 g1) Vmicro Vmeso Vmacro Pore size (nm) PEG-30% 2052 0.429 0.146 0.226 0.057 2.7 PEG-40% 2269 0.516 0.107 0.302 0.107 3.5 PEG-50% 1436 0.520 0.091 0.309 0.120 3.6 Paper View Article Online View Article Online RSC Advances Paper Fig. 6 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 1 M H2SO4 aqueous electrolyte. (a) CV curves at a scan rate of 10 mV s1; (b) CV curves of PEG-40% electrode material at diﬀerent scan rates; (c) GCD curves at a current density of 1 A g1; (d) speciﬁc capacitances at diﬀerent current densities. Fig. 7 Electrochemical performances of the PEG-x% materials based electrode measure in a three-electrode system in 2 M KOH aqueous electrolyte. This journal is © The Royal Society of Chemistry 2018 View Article Online Paper Fig. 10 Ragone plot comparing the energy density and power density of PEG-40% symmetric supercapacitor with other reported carbon materials in an aqueous electrolyte. RSC Advances RSC Advances resistance, the active material resistance, and the active mate- rial interface resistance. The lower value of Rs is associated with better electrode conductivity. The diameter of the semi-circle is a charge-transfer resistance (Rct), which is attributed to the charge transfer at the interface of the electrode and electrolyte, and a smaller semicircle means smaller charge transfer resis- tance. The Rct of PEG-40% electrode is 0.03 U (in 1 M H2SO4) and 0.13 U (in 2 M KOH), which is lower than those of other electrode materials. The 45 slope region at middle frequency can be attributed to the ions diﬀusion/transport from the electrolyte to the pore on the surface of samples. The short lengths of these slopes indicate that the electrolyte ions diﬀuse fast in this carbon framework. The almost vertical line repre- sents the dominance of ideal double-layer charge/discharge behaviors at low frequencies. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 10 Ragone plot comparing the energy density and power density of PEG-40% symmetric supercapacitor with other reported carbon materials in an aqueous electrolyte. To further study the electrochemical behavior of the PEG- 40% electrode as a real capacitor in 1 M H2SO4 and 2 M KOH solution, a symmetrical two-electrode conguration was con- structed. It can be observed that the fabricated symmetric supercapacitor shows near-rectangular shapes in both acid and basic solution (Fig. 9a and b), even at high scan rates, indicating its ideal capacitive behavior.45 Moreover, the GCD curves in Fig. 9c and d display good symmetrical linear curves, revealing its high electrochemical stability. The specic capacitance of the symmetric supercapacitors calculated from GCD curves is 55 F g1 and 42 F g1 (220 F g1 and 168 F g1 for single elec- trode) at 0.5 A g1, 91% and 86% capacitance is retained even at 5 A g1 in 1 M H2SO4 and 2 M KOH, respectively. Stability testing was conducted under 1 A g1 for 10 000 cycles. Fig. 9e and f show the cycling stability of the symmetric supercapacitors in 1 M H2SO4 and 2 M KOH, respectively. This journal is © The Royal Society of Chemistry 2018 The capacitance reten- tion is about 94% and 97% of its initial value aer 10 000 cycles, indicating the excellent cycling stability of PEG-40%. To further demonstrate the device performance aer cycles, two super- capacitors were connected in series to light a red LED. As shown in Fig. 9e and f, aer charging to 2.2 V at a current density of 1 A g1, the device could power a red LED, and even aer 10 min the red LED still remained bright, indicating its excellent elec- trochemical properties even aer 10 000 cycles. Energy and power densities are key parameters to evaluate the performance of a certain material when applied as elec- trodes for supercapacitors. The Ragone plot of PEG-40% symmetric supercapacitor in 1 M H2SO4 and 2 M KOH aqueous solutions calculated from discharge curves at diﬀerent current densities is displayed in Fig. 10, as well as its comparison with the representative porous carbon-based supercapacitors. The results showed that the energy density of the supercapacitors utilizing PEG-40% as electrode material could reach 7.58 W h kg1 with a corresponding specic power density of 0.25 kW kg1 in 1 M H2SO4 solution, and an energy density of 5.79 W h kg1 could be obtained with 0.25 kW kg1 of specic power in 2 M KOH electrolyte, which is higher than those of commercially available supercapacitors (3–5 W h kg1)24 and most carbon-based/N-doped carbonaceous aqueous capacitors reported in previous articles. Fig. 9 Electrochemical performance of PEG-40% symmetrical supercapacitors in 1 M H2SO4 and 2 M KOH, respectively. (a, b) CV curves at diﬀerent scanning rates; (c, d) GCD curves at diﬀerent current densities; (e, f) cycling stability at a 1 A g1. This journal is © The Royal Society of Chemistry 2018 There are no conicts to declare. There are no conicts to declare. 21 N. Phattharasupakun, J. Wutthiprom, P. Suktha, 21 N. Phattharasupakun, J. Wutthiprom, P. Suktha, P. Iamprasertkun, N. Chanlek, C. Shepherd, Acknowledgements E. Hadzifejzovic, M. G. Moloney, J. S. Foord and M. Sawangphruk, Electrochimica. Acta, 2017, 238, 64–73. E. Hadzifejzovic, M. G. Moloney, J. S. Foord and E. Hadzifejzovic, M. G. Moloney, J. S. Foord and M. Sawangphruk, Electrochimica. Acta, 2017, 238, 64–73. M. Sawangphruk, Electrochimica. Acta, 2017, 238, 64–73. 22 G. Ma, Z. Zhang, H. Peng, K. Sun, F. Ran and Z. Lei, J. Solid State Electrochem., 2016, 20, 1613–1623. 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Conclusions In summary, NHPC materials have been fabricated by the carbonization of chitosan/PEG blend and activation with KOH. Beneting from its hierarchical porous structure and numerous pseudocapacitive functional groups, the carbon materials exhibits an excellent performance. As an example, the PEG-40% sample has micro, meso-, and macro- hierarchical porous struc- ture and possesses a high specic surface area of 2269 m2 g1. It can deliver high specic capacitances of 356 F g1 (in 1 M H2SO4) Fig. 9 Electrochemical performance of PEG-40% symmetrical supercapacitors in 1 M H2SO4 and 2 M KOH, respectively. (a, b) CV curves at diﬀerent scanning rates; (c, d) GCD curves at diﬀerent current densities; (e, f) cycling stability at a 1 A g1. This journal is © The Royal Society of Chemistry 2018 RSC Adv., 2018, 8, 7072–7079 \| 7077 View Article Online RSC Advances s Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 19 A. Elmouwahidi, E. Bail´on-Garc´ıa, A. F. P´erez-Cadenas, F. J. Maldonado-H´odar and F. Carrasco-Mar´ın, Electrochim. Acta, 2017, 229, 219–228. Paper and 271 F g1 (in 2 M KOH) at the current density of 1 A g1, retain 65% and 86% at 20 A g1. Furthermore, the assembled symmetric supercapacitors show an excellent cycling stability with 94% (in 1 M H2SO4) and 97% (in 2 M KOH) retention aer 10 000 cycles at 1 A g1. The outstanding capacitive behavior is attributed to the unique features including a high specic surface area, reasonable pore size and pore size distribution, and moderate nitrogen doping. 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This journal is © The Royal Society of Chemistry 2018 7078 \| RSC Adv., 2018, 8, 7072–7079 Paper This journal is © The Royal Society of Che Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:14 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 February 2018. Downloaded on 10/24/2024 5:48:1 This article is licensed under a Creative Commons Attribution 3.0 Unporte RSC Adv., 2018, 8, 7072–7079 \| 7079 This journal is © The Royal Society of Chemistry 2018
https://openalex.org/W3127372883	https://www.frontiersin.org/articles/10.3389/fphar.2020.610556/pdf	English	null	Qu-Zhuo-Tong-Bi Decoction Alleviates Gouty Arthritis by Regulating Butyrate-Producing Bacteria in Mice	Frontiers in pharmacology	2,021	cc-by	8,767	Qu-Zhuo-Tong-Bi Decoction Alleviates Gouty Arthritis by Regulating Butyrate-Producing Bacteria in Mice College of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou, China Qu-zhuo-tong-bi decoction (QZTBD) is a traditional Chinese medicine prescription used to treat hyperuricemia and gout with no obvious adverse effects. However, the mechanism by which QZTBD treats gout has not been fully explored. Here, we investigated the effects of QZTBD on gouty arthritis and its therapeutic mechanism from the perspective of the gut microbiome. Our results demonstrated that QZTBD was effective for reducing serum uric acid level and attenuating paw edema and mechanical allodynia. QZTBD promoted the abundance of butyrate-producing bacteria and the production of SCFAs. Further study revealed that QZTBD restored the intestinal barrier function, modulated the expression of GPR43 and ABCG2, suppressed the activity of key glycolysis-related enzymes, and inhibited the generation of intestinal inﬂammatory factors. These ﬁndings suggested that QZTBD is an effective therapeutic drug for gouty arthritis. Butyrate-producing bacteria and its metabolites SCFAs might act as a potential target of QZTBD. Reviewed by: Reviewed by: Rui Rui Wang, Shanghai University of Traditional Chinese Medicine, China Jinjun Shan, Nanjing University of Chinese Medicine, China Reviewed by: Rui Rui Wang, Shanghai University of Traditional Chinese Medicine, China Jinjun Shan, Nanjing University of Chinese Medicine, China Correspondence: Chengping Wen wengcp@163.com Tiejuan Shao tiejuanshao@zcmu.edu.cn †These authors have contributed equally to this work Correspondence: Chengping Wen wengcp@163.com Tiejuan Shao tiejuanshao@zcmu.edu.cn INTRODUCTION †These authors have contributed equally to this work †These authors have contributed equally to this work Gout is a purine metabolism disorder and chronic inﬂammatory disease with increasing prevalence and incidence, particularly in developed countries (Kuo et al., 2015). Severe pain, swelling, warmth, and redness at the gouty joint characterize the disorder (Dalbeth et al., 2016; So and Martinon, 2017; Yang et al., 2019). These traits develop following massive deposition of monosodium urate (MSU) crystals in joints and surrounding tissues. Establishing reliable treatments that target serum urate is essential for effective gout management. Allopurinol and febuxostat (FBST) are the ﬁrst-line urate- lowering therapies (Dalbeth et al., 2016). However, common side effects of using these drugs in the clinical treatment of gout include skin rash, nausea, vomiting, diarrhea, abnormal liver function, and chronic renal toxicity. Strategies to achieve new treatment targets and to improve the quality of gout care are needed. Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 28 September 2020 Accepted: 30 December 2020 Published: 02 February 2021 Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Keywords: Qu-zhuo-tong-bi decoction, gouty arthritis, gut microbiota, SCFAs, gut homeostasis ORIGINAL RESEARCH published: 02 February 2021 doi: 10.3389/fphar.2020.610556 Edited by: Houkai Li, Shanghai University of Traditional Chinese Medicine, China Citation: Traditional Chinese medicine (TCM) has been widely used in China for thousands of years. It is believed to be a valuable form of medicine with better efﬁcacy and fewer side effects. Qu-zhuo-tong- bi decoction (QZTBD), an empirical prescription for gout treatment, has deﬁnite clinical effects on lowering serum urate levels and preventing the recurrence of gout attacks with no serious adverse effects. This prescription has obtained a national invention patent in China (Patent No.: ZL201010209598.7). Its multiple functions, including reinforcing renal function, promoting blood circulation, and relieving pain, have been demonstrated elsewhere (Chen et al., 2016; Lv Wen X, Lou Y, Song S, He Z, Chen J, Xie Z, Shi X, Wen C and Shao T (2021) Qu-Zhuo-Tong-Bi Decoction Alleviates Gouty Arthritis by Regulating Butyrate- Producing Bacteria in Mice. Front. Pharmacol. 11:610556. doi: 10.3389/fphar.2020.610556 February 2021 \| Volume 11 \| Article 610556 1 Frontiers in Pharmacology \| www.frontiersin.org QZTBD for Gouty Arthritis Wen et al. TABLE 1 \| The compositions of Qu-zhuo-tong-bi decoction (QZTBD). Chinese name Latin name Scientiﬁc name Weigh(g) Parts used Tu Fu Ling Rhizoma Smilacis Glabrae Smilax glabra Roxb 60 Rhizome Bi Xie Dioscoreae Spongiosae Rhizoma Dioscorea spongiosa J. Q. Xi, M. Mizuno et W. L. Zhao 30 Rhizome Yu Mi Xu Corn Stigma Zea mays L 15 Stigma Yi Yi Ren Coicis Semen Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf 30 Seed Xi Xian Cao Siegesbeckiae Herba Siegesbeckia orientalis L 18 Herb Jiang Huang Curcumae Longae Rhizoma Curcuma longa L 12 Rhizome Sang Ji Sheng Taxilli Herba Taxillus chinensis (DC.) Danser 15 Branch, Leaf Yan Hu Suo Corydalis Rhizoma Corydalis yanhusuo (Y. H. Chou and Chun C. Hsu) W. T. Wang ex Z. Y. Su and C. Y. Wu 18 Tuber Fo Shou Citri Sarcodactylis Fructus Citrus medica L. var. sarcodactylis Swingle 12 Fruit TABLE 1 \| The compositions of Qu-zhuo-tong-bi decoction (QZTBD). by ﬁltration. The ﬁltrate was further concentrated using a rotary evaporator and dried using a freeze dryer. The ﬁnal aqueous extracts of QZTBD were homogenized and stored in an air-tight container at −20°C. FBST and MSU were purchased from Hangzhou Zhuyangxin Pharmaceutical Co., Ltd (Hangzhou, China) and Sigma (MO, United States), respectively. et al., 2019). However, its underlying mechanism of its anti-gouty arthritis effect has not yet been fully explored. A growing number of studies have shown that intestinal ﬂora can participate in purine metabolism. Quality Control and Content Determination of QZTBD of QZTBD The main contents of QZTBD were analyzed by high- performance liquid chromatography (HPLC) for quality control of QZTBD. 0.2 g of dried QZTBD extract was resuspended in deionized water, sonicated for 30 min at 35 kHz and 25°C, and then centrifuged at 3,500 rpm for 5 min. The supernatant was ﬁltered through a 0.22 μm PES ﬁlter before HPLC analysis. The HPLC analysis was carried out by Waters e2695 + 2489 HPLC system with C18-AR column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid (B). The elution program was as follows: 0–10 min 5–25% A; 10–20 min 25–30% A; 20–40 min 30–90% A; 40–42 min 90-5% A; 42–52 min 5-5% A. The ﬂowrate was 1.0 ml/min, the detection wavelength was 220 nm, and the column temperature was maintained at 25°C. MATERIALS AND METHODS Astilbin, tetrahydropalmatine, and quercetin are the main bio- active components of Smilax glabra Roxb., Corydalis yanhusuo (Y.H. Chou and Chun C. Hsu) W.T. Wang ex Z.Y. Su and C.Y. Wu, and Taxillus chinensis (DC.) Danser respectively. Rutin is the common component of Chinese medicinal herbs in the QZTBD. These four components were selected as the standards for quality control of QZTBD. All four chemicals (HPLC ≥98%) were purchased from Shanghai Yuanye Bio-technology Co., Ltd. (Shanghai, China) and were analyzed by the same elution program. The detection wavelength was at 220 nm, 360 nm, 360 nm, and 210 nm, respectively. Citation: Gut excretion of urate is critical for regulating serum urate (Ichida et al., 2012). Decreased gut urate excretion induced by a microbial imbalance is an important cause of gout (Guo et al., 2016). The gut microbiome has attracted more and more attention as a promising target for the treatment of gout. An increasing number of traditional Chinese herbal ingredients have been proven to manipulate the intestinal microbiota community structure. A fundamental way for TCM to take effect is the regulation of intestinal ﬂora. To investigate the effect of QZTBD on intestinal ﬂora and its potential mechanism in the treatment of gout, a high-fat diet (HFD) and MSU crystal- induced animal model was established. After treating the mouse model with QZTBD and FBST, the structure of intestinal ﬂora was analyzed and the content changes of primary SCFAs in feces were detected. Our results showed that QZTBD can effectively attenuate gouty symptoms by restoring the gut microbiota and promoting the production of SCFAs, which is different from FBST. QZTBD and Chemicals procedures were approved by the Laboratory Animal Management and Welfare Ethical Review Committee of Zhejiang Chinese Medical University (Permission number: ZSLL-2018-0012). After 1 week of acclimatization, the mice were randomly divided into four groups: control group, gouty arthritis model group, QZTBD group, and FBST group. Each group was comprised of seven animals. The control group was fed with a standard diet, and 40 μL of PBS was injected into the right hind footpad every 10 days. The animals in the other three groups were fed with HFD (10% yeast extract) and injected with MSU crystals (1 mg MSU crystals in 40 μL PBS/mouse) every 10 days to simulate gouty arthritis (Lin et al., 2020). Six-week therapy started at the induction of the gouty arthritis model. Drug dosages were calculated based on the conversions from clinical adult dosages and the body weight of mice. According to our previous study, the QZTBD group and the FBST group were administered by gavage with 18.0 g/kg/day QZTBD and 5.2 mg/kg/day FBST respectively (Liu et al., 2019). The control group and model group were given equal volumes of distilled water. Immunohistochemical Analysis of Intestinal GPR43 and Tight-Junction Proteins Immunohistochemical Analysis of Intestinal GPR43 and Tight-Junction Proteins Formalin-ﬁxed, parafﬁn-embedded colon tissue sections were prepared for immunohistochemistry (IHC) assays. Primary antibodies, including anti-ZO-1 (Afﬁnity Biosciences, OH, United States, #AF5145), anti-Occludin (Afﬁnity Biosciences, OH, United States, #DF7504), and anti-GPR43 (Bioss Inc., Mass, United States, #BS-13536R), were respectively applied to incubate with sections overnight at 4°C. After being incubated with anti-rabbit IgG for 60 min at room temperature, the stained sections were washed and visualized brown with DAB (C-0003, Bioss, Beijing, China) and scanned by a digital pathological section scanner (NDP, Hamamatsu, Japan). The staining was manually evaluated by two independent certiﬁed pathologists using NDP view 2.0 software. The claw region of mice was separated and ﬁxed with 10% paraformaldehyde in PBS, decalciﬁed for 3 weeks with EDTA, embedded in parafﬁn wax, and then stained with hematoxylin and eosin for conventional morphological evaluation. Determination of Fecal Short-Chain Fatty Acids The short-chain fatty acids (SCFAs) were extracted with anhydrous ether from acidiﬁed fecal water extract. The analysis was carried out using a gel ﬁltration chromatography- mass spectrometry (GPC-GC/MS-2010, Shimadzu, Kyoto, Japan) with a Rtx-Wax capillary column (Shimadzu, Kyoto, Japan). A split injection of 1 µL sample was made at a ratio of 10:1, with a column helium ﬂow rate of 1.2 ml/min. Ion source temperature was 230°C, while the injector temperature was 260°C. The column initial temperature was 100°C. It was then increased by 8°C/min to 140°C, which was held for 2 min. Column temperature was increased again by 60°C/min to 200°C and held there for 3 min. The scan mode was Scan and SIM mode. Identiﬁcation of the primary SCFAs, including acetate, propionate, and butyrate, were carried out according to the retention time. SCFA standards acetate (#S5636), propionate (#P1880), and butyrate (#303410) were purchased from Sigma Aldrich. All samples were analyzed in triplicate. QZTBD and Chemicals QZTBD is composed of nine Chinese medicinal herbs (Table 1): Smilax glabra Roxb. (60 g, Batch No: 180701), Dioscorea spongiosa J. Q. Xi, M. Mizuno et W. L. Zhao (30 g, Batch No: 180701), Zea mays L. (15 g, Batch No: 180801), Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf (30 g, Batch No: 181001), Siegesbeckia orientalis L. (18 g, Batch No: 181001), Taxillus chinensis (DC.) Danser (15 g, Batch No: 180301), Curcuma longa L. (12 g, Batch No: 181101), Corydalis yanhusuo (Y. H. Chou and Chun C. Hsu) W. T. Wang ex Z. Y. Su and C. Y. Wu (18 g, Batch No: 180501), and Citrus medica L. var. sarcodactylis Swingle (12 g, Batch No: 180301). All herbal materials were purchased from Zhejiang Chinese Medical University Medical Pieces., LTD. (Hangzhou, China). The above plant samples were stored in a speciﬁc Herbarium room. QZTBD was prepared using a traditional decoction method. The mixed medicine was boiled with distilled water for 45 min, and the supernatant was collected Animals and Drug Administration Twenty-eight speciﬁc pathogen-free male C57BL/6 mice (4–6 weeks, 15 ± 3 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in the Laboratory Animal Center of Zhejiang Chinese Medical Animal Care (AAALAC) under standard environmental conditions (12 h light-dark cycles and 25 ± 1°C). Experimental February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 2 QZTBD for Gouty Arthritis Wen et al. procedures were approved by the Laboratory Animal Management and Welfare Ethical Review Committee of Zhejiang Chinese Medical University (Permission number: ZSLL-2018-0012). units (OTUs) based on a 97% similarity cutoff using QIIME 2 (https://qiime2.org/). Alpha diversity and beta diversity analysis were performed by R software (1.2.5033). Bray-Curtis dissimilarity matrix was developed with the normalized sequences. Hierarchical cluster analysis (HCA), principal coordinates analysis (PCoA), and distance between two groups were performed based on the Bray-Curtis dissimilarity matrix. The linear discriminant analysis (LDA) effect size (LEfSe) method was applied to reveal the effect of each differentially abundant taxon and distinguish the key phenotypes responding to the QZTBD treatment, with a set logarithmic LDA score of 2.0. Spearman’s correlation coefﬁcient was conducted between microﬂora and gout symptoms using the function “cor.test” in the statistical software R and visualized with a heatmap. procedures were approved by the Laboratory Animal Management and Welfare Ethical Review Committee of Zhejiang Chinese Medical University (Permission number: ZSLL-2018-0012). Evaluation of Gouty Symptoms At the end of the experiment, the blood was obtained by retro- orbital bleeding. The serum was then isolated. The concentration of serum uric acid (SUA) was detected strictly according to the operation steps of the TBA-40FR automatic biochemical analyzer (Toshiba Co., Ltd., Japan). Footpad swelling was measured as reported previously (Lv et al., 2019). In brief, the footpad thickness for each mouse was measured by a digital caliper (Minet Industrial Co., Ltd., China) before stimulus and 4 h, 24 h, 48 h, and 72 h after the administration of MSU. Footpad swelling was evaluated as an increase in footpad thickness, which was calculated as the difference between the initial thickness and the test thickness observed at different time points. The pain threshold was measured by measuring the mechanical withdrawal threshold (MWT) with the von Frey monoﬁlaments (Danmic Global Llc Co., Ltd., Unites States) (Yu et al., 2016; Lv et al., 2019). All the above behavioral tests were conducted by an investigator blinded to experimental conditions. Analysis of Gut Microbiota Feces were collected from the middle segment of the colon, and total DNA was extracted from stool samples using the QIAmp DNA microbiome kit (Qiagen, German) according to the manufacturer’s protocol. Gut microbiota abundance and diversity were analyzed by sequencing of 16S rRNA gene (V3- V4 region) with Illumina MiSeq platform. High-quality reads were selected and library size across samples were normalized to the same number to exclude the bias caused by different sequencing depth. The effective reads were aligned with the SILVA database and clustered into operational taxonomic Real-Time Quantitative PCR (qPCR) Total RNAs from colon tissue were extracted with Trizol reagent (Thermo Fisher Scientiﬁc, United States). The RNA was reverse transcribed into cDNA using random hexamers February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 3 QZTBD for Gouty Arthritis Wen et al. primers with HiFiScript cDNA Synthesis Kit (CWBIO, China) according to the manufacturer’s instructions Each reaction level. qPCR was performed with Bio-Rad iQ5 PCR sys (Bio-Rad United States) using the SYBR Premix ExTaq FIGURE 1 \| HPLC analysis of Qu-zhuo-tong-bi decoction (QZTBD). QZTBD (A) and standard substance (B). Identiﬁcation of main compounds in QZTBD as follows: Astilbin (1), Rutin (2), Tetrahydropalmatine (3), and Quercetin (4). primers with HiFiScript cDNA Synthesis Kit (CWBIO, China) according to the manufacturer’s instructions. Each reaction was performed at least in triplicate and normalized to β-actin level. qPCR was performed with Bio-Rad iQ5 PCR system (Bio-Rad, United States) using the SYBR Premix ExTaq K (Takara Bio Inc., China). The CT value of each well wa FIGURE 1 \| HPLC analysis of Qu-zhuo-tong-bi decoction (QZTBD). QZTBD (A) and standard substance (B). Identiﬁcation of main compounds in QZTBD as follows: Astilbin (1), Rutin (2), Tetrahydropalmatine (3), and Quercetin (4). Frontiers in Pharmacology \| www.frontiersin.org February 2021 \| Volume 11 \| Article 61055 4 FIGURE 1 \| HPLC analysis of Qu-zhuo-tong-bi decoction (QZTBD). QZTBD (A) and standard substance (B). Identiﬁcation of main compounds in QZTBD as FIGURE 1 \| HPLC analysis of Qu-zhuo-tong-bi decoction (QZTBD). QZTBD (A) and standard substance (B). Identiﬁcation of ma follows: Astilbin (1), Rutin (2), Tetrahydropalmatine (3), and Quercetin (4). FIGURE 1 \| HPLC analysis of Qu-zhuo-tong-bi decoction (QZTBD). QZTBD (A) and standard substance (B). Identiﬁcation of main compounds in QZTBD as follows: Astilbin (1), Rutin (2), Tetrahydropalmatine (3), and Quercetin (4). primers with HiFiScript cDNA Synthesis Kit (CWBIO, China) according to the manufacturer’s instructions. Lactic Acid Assay The concentrations of serum and fecal lactic acid were determined using a lactic acid assay kit (Njjcbio, China; #A019-2-1) according to the manufacturer’s instructions. The absorbance of the samples was measured at 530 nm on a spectrophotometer. The concentrations of lactic acid were calculated according to the standard curve. Analysis of Gut Microbiota Each reaction was performed at least in triplicate and normalized to β-actin primers with HiFiScript cDNA Synthesis Kit (CWBIO, China) according to the manufacturer’s instructions. Each reaction was performed at least in triplicate and normalized to β-actin level. qPCR was performed with Bio-Rad iQ5 PCR system (Bio-Rad, United States) using the SYBR Premix ExTaq Kit (Takara Bio Inc., China). The CT value of each well was primers with HiFiScript cDNA Synthesis Kit (CWBIO, China) according to the manufacturer’s instructions. Each reaction was performed at least in triplicate and normalized to β-actin level. qPCR was performed with Bio-Rad iQ5 PCR system (Bio-Rad, United States) using the SYBR Premix ExTaq Kit (Takara Bio Inc., China). The CT value of each well was February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 4 QZTBD for Gouty Arthritis Wen et al. TABLE 2 \| Herbal retention times and sample contents of components in QZTBD. Constituents Retention times (min) Sample contents (mg/g) Astilbin 13.180 0.1174 Rutin 13.427 0.0063 Tetrahydropalmatine 20.277 0.0140 Quercetin 25.013 0.0109 and 25.075 min, respectively. Through the calculation of the regression curve, the concentrations of these four components in QZTBD were 0.1174, 0.0063, 0.0140, and 0.0109 mg/g, respectively (Table 2). QZTBD Alleviated Gouty Symptoms Effectively y In order to evaluate the effects of QZTBD on gout mice, we monitored the animal’s SUA levels, footpad swelling degree, and pain threshold. Compared with the control group, the SUA level in the model group was signiﬁcantly increased. As expected, both QZTBD and FBST (the drug commonly used for lowering SUA level and was used as a positive control) effectively decreased the level of SUA (Figure 2A). The animals that received an injection of MSU crystals showed an increase in footpad swelling and a decrease in mechanical pain threshold (Figures 2B,C). FBST showed a better performance in ameliorating MSU-stimulated swelling (Figure 2B), while QZTBD manifested a better anti- allodynic effect over the observation period (Figure 2C). HE analysis indicated that the model group demonstrated increased inﬂammatory cells’ inﬁltration and hyperplasia synovial. These pathological states were ameliorated to some degree by treatment with QZTBD and FBST (Figure 2D). There was no signiﬁcant difference in spleen, kidney, liver indices, and CREA levels between the control group and the QZTBD group (Supplementary Figure S1), indicating the safety of QZTBD decoction on male C57BL/6 mice. These results suggest that QZTBD can effectively attenuate gouty symptoms induced by a high-fat diet and MSU injection without obvious toxicity. determined by instrument software. The average of the values was calculated. The relative quantiﬁcation was analyzed by the 2−ΔΔCt method. The primer sequences were shown in Supplementary Table S1. QZTBD Regulated the Microbial Community Structure Mounting evidence has demonstrated that gut dysbiosis is a pivotal factor of gout and that TCM can regulate the composition and function of gut microbiota. We next sequenced the bacterial V3-V4 region of the 16S rRNA gene to proﬁle gut microbiota composition between the normal and gout mice and evaluated the inﬂuence of QZTBD on the intestinal ﬂora. A total of 1261461 tags were generated, with an average of 45,052 tags per sample. The rarefaction curves of all samples tended to be ﬂat, indicating the amount of sequencing data is reasonable and the depth of sequencing is appropriate (Supplementary Figure S2). The estimated richness as indicated by ACE and Chao indexes were signiﬁcantly lower in gout mice. Microbial diversity, which was assessed via Shannon and Simpson indexes, showed a robust decrease in the model group (Figure 3A). Both QZTBD and FBST could promote microbial richness and diversity. Hierarchical clustering analysis revealed FBST treatment mice have a similar microbial community to gout mice, while QZTBD-treated mice’s microbiomes are structured differently (Figure 3B). PCoA analysis (Figure 3C) showed that samples could be separated based upon the microbial Western Blotting Analysis The intestinal tissue was homogenized in RIPA protein lysis buffer (Thermo Fisher Scientiﬁc, United States) and the concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Protein was separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% milk in TBST buffer for 1 h at room temperature, probed with the primary antibodies overnight at 4°C, and then incubated with HRP-coupled secondary antibodies for 1 h at room temperature. The primary antibodies used in this study were anti-PFKFB3 (1:5000 dilution, Abcam, #ab181861) and anti-LDH (1:7500 dilution, Abcam, #ab52488). β-actin (1:1,000 dilution, Absin, #abs830031) was used to ascertain that equal amounts of protein were loaded. The gray value analysis of gel quantiﬁcation was performed using ImageJ software. Statistical Analysis y SPSS 20.0 was used to analyze the statistical difference of the experimental data. GraphPad Prism 7.0 software was used for drawing. The measurement data were expressed as mean ± SEM. The normality of the data and the homogeneity of variance between groups were tested using one-way ANOVA or the Wilcoxon rank-sum test. p-value below 0.05 was considered statistically signiﬁcant. SPSS 20.0 was used to analyze the statistical difference of the experimental data. GraphPad Prism 7.0 software was used for drawing. The measurement data were expressed as mean ± SEM. The normality of the data and the homogeneity of variance between groups were tested using one-way ANOVA or the Wilcoxon rank-sum test. p-value below 0.05 was considered statistically signiﬁcant. Quantitative Analysis of the Chemical Constituents of QZTBD The characteristic chromatogram of the standard and QZTBD were shown in Figure 1. The retention times of astilbin, rutin, tetrahydropalmatine, and quercetin were 13.180, 13.427, 20.277, February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 5 Wen et al. QZTBD for Gouty Arthritis i Fi 3D d d h di K Ph t R di t th QZTBD FIGURE 2 \| QZTBD alleviated gouty symptoms effectively. Effects of QZTBD on SUA (A), footpad swelling index (B), footpad pain threshold (C), and HE analysis of the footpad (D). “#” represents p < 0.05 in the comparison with the control group; “# #” represents p < 0.01 in the comparison with the control group and “# # #” represents p < 0.001 in the comparison with the control group; “” represents p < 0.05 in the comparison with the model group; “” represents p < 0.01 in the comparison with the model group and “*” represents p < 0.001 in the comparison with the model group. N 7/group. Wen et al. QZTBD for Gouty Arthritis FIGURE 2 \| QZTBD alleviated gouty symptoms effectively. Effects of QZTBD on SUA (A), footpad swelling index (B), footpad pain threshold (C), and HE analysis of the footpad (D). “#” represents p < 0.05 in the comparison with the control group; “# #” represents p < 0.01 in the comparison with the control group and “# # #” represents p < 0.001 in the comparison with the control group; “” represents p < 0.05 in the comparison with the model group; “” represents p < 0.01 in the comparison with the model group and “” represents p < 0.001 in the comparison with the model group. N 7/group. Key Phenotypes Responding to the QZTBD Treatment in Gout community structure. Figure 3D demonstrated the distance between the two groups. The intragroup distance was smaller than the intergroup distance, indicating the microbiota composition of mice within the same group is more similar to mice from a different group. QZTBD can regulate the structure of intestinal ﬂora in a unique way. Based on 16S rRNA gene sequencing, we observed that Firmicutes, Bacteroidetes, Epsilonbacteraeota, and Proteobacteria are the four dominant phyla (Figure 4A). Compared to the control group, the relative abundance of Firmicutes and Bacteroidetes decreased in the February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 6 QZTBD for Gouty Arthritis Wen et al. FIGURE 3 \| QZTBD regulated the microbial community structure. Effects of QZTBD on microbial richness and diversity (A); Hierarchical clustering analysis on OTU level (B); PCoA score plots (C) and distance between the two groups (D) based on Bray-Curtis dissimilarities. “” represents p < 0.05; “*” represents p < 0.01. N 7/group. FIGURE 3 \| QZTBD regulated the microbial community structure. Effects of QZTBD on microbial richness and diversity (A); Hierarchical clustering analysis on OTU level (B); PCoA score plots (C) and distance between the two groups (D) based on Bray-Curtis dissimilarities. “” represents p < 0.05; “*” represents p < 0.01. N 7/group. Frontiers in Pharmacology \| www.frontiersin.org February 2021 \| Volume 11 \| Article 610556 7 QZTBD for Gouty Arthritis Wen et al. FIGURE 4 \| Key phenotypes responding to the QZTBD treatment in gout. Differences of gut microbiota at the phylum level (A); LEfSe analysis between the control group and the model group (B); Analysis of correlation between characteristic intestinal ﬂora and gout symptoms is shown through a heatmap. Positive correlations are displayed in red and negative correlations in blue. The intensity of the color is proportional to the correlation coefﬁcient (C); Relative abundance of characteristic intestinal ﬂora after QZTBD and FBST treatment (D); LEfSe analysis between Model group and QZTBD group (E). “” represents 0.2 < r ≤0.4; “” represents 0.4 < r ≤0.7; “” represents r > 0.7 in Figure 4C. “ns” represents not signiﬁcant; “” represents p < 0.05, “” represents p < 0.01 and “” represents p < 0.001 in Figure 4D. N 7/group. FIGURE 4 \| Key phenotypes responding to the QZTBD treatment in gout. Key Phenotypes Responding to the QZTBD Treatment in Gout As shown in Figure 4C, Butyrivibrio, Butyricicoccus, Lachnospira, Eubacterium, and Faecalibaculum, which are known as butyrate producers, were negatively correlated with SUA level and paw edema, while they were positively correlated with pain threshold. The bacteria those cultivated in gout mice were perfectly positively correlated with gout assessment indicators. The above results propose the importance of gut ﬂora, particularly butyrate-producing bacteria, in the pathogenesis of gout. These taxa provide a new potential treatment target for gout. We next checked the changes of these characteristic ﬂora after drug treatment (Figures 4D,E and Supplementary Figure S3). Both QZTBD and FBST inhibited the growth of Lachnospiraceae_A2 (a bacterium enriched in gout mice) and boosted the abundance of Muribaculum (a bacterium strongly correlated with SCFAs). Moreover, QZTBD notably increased the abundance of Butyricicoccus while FBST mainly produced an increase in abundance of Lachnospiraceae_GCA- 900066575. These differences in genera compound our earlier ﬁnding that QZTBD and FBST play different roles in gut microbial regulation. Key Phenotypes Responding to the QZTBD Treatment in Gout Differences of gut microbiota at the phylum level (A); LEfSe analysis between the control group and the model group (B); Analysis of correlation between characteristic intestinal ﬂora and gout symptoms is shown through a heatmap. Positive correlations are displayed in red and negative correlations in blue. The intensity of the color is proportional to the correlation coefﬁcient (C); Relative abundance of characteristic intestinal ﬂora after QZTBD and FBST treatment (D); LEfSe analysis between Model group and QZTBD group (E). “” represents 0.2 < r ≤0.4; “” represents 0.4 < r ≤0.7; “” represents r > 0.7 in Figure 4C. “ns” represents not signiﬁcant; “” represents p < 0.05, “” represents p < 0.01 and “” represents p < 0.001 in Figure 4D. N 7/group. February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 8 QZTBD for Gouty Arthritis Wen et al. FIGURE 5 \| QZTBD inﬂuences the fecal SCFAs levels. Acetate (A), Propionate (B) and Butyrate (C). “ns” represents not signiﬁcant; “” represents p < 0.05 and “*” represents p < 0.01. N 7/group. FIGURE 5 \| QZTBD inﬂuences the fecal SCFAs levels. Acetate (A), Propionate (B) and Butyrate (C). “ns” represents not signiﬁcant; “” represents p < 0.05 and “*” represents p < 0.01. N 7/group. main SCFAs, especially of butyrate (p < 0.05). FBST clearly improved fecal acetate level (p < 0.05), but its application had no effect on the contents of propionate and butyrate. These metabolic features were compatible with the results of microbial composition. They further demonstrated that abnormally reduced intestinal levels of SCFAs or SCFA- producing bacteria are usually closely related to gout, which has been commonly found in patients who were diagnosed with inﬂammatory bowel disease, type 2 diabetes, obesity, and autoimmune diseases. model group, while the amounts of Epsilonbacteraeota and Proteobacteria increased. QZTBD restored the abundance of Bacteroidetes, but FBST mainly altered the abundance of Firmicutes, suggesting these two drugs have different regulatory mechanisms (Figure 4A). LEfSe analysis showed that 31 bacterial genera were depleted in gout mice, while 18 genera were enriched in these same samples (Figure 4B). It is worthy to note that the abundance of SCFAs-producing bacteria was signiﬁcantly reduced in the gut of gout mice (Figure 4B). A series of Spearman’s correlations were further conducted to elucidate the association between characteristic ﬂora and symptoms of gout. QZTBD Improved Intestinal Mucosal Barrier and Intestinal Urate Excretion Considering SCFAs are essential for the maintenance of the intestinal mucosal barrier function, the reduction of SCFAs may have an impact on the permeability of the intestinal mucosa by modulating the expression of tight junction (TJ)- related proteins. The levels of ZO-1 and Occludin were determined by IHC staining and qPCR. Consistent with our expectations, both protein and mRNA expression levels of ZO-1 (Figures 6A,B) and Occludin (Figures 6C,D) in the colon were signiﬁcantly reduced in the model group. QZTBD up-regulated both mRNA and protein levels of these two TJ- related proteins. However, no signiﬁcant expression change was detected in FBST-treated mice. Given the association between intestinal barrier dysfunction and inﬂammation, we next examined the expression of related inﬂammatory factors in intestinal tissues. The mRNA levels of NLRP3, IL-1β, and TNF-α in the model group were remarkably higher than those in the control group, and dramatically fell in QZTBD group. FBST also lowered the mRNA expression of NLRP3, IL-1β, and TNF-α despite its inability to repair the intestinal barrier (Figure 6E). These results suggest that QZTBD enhances intestinal barrier function by increasing the expression of TJ- related proteins and inhibiting intestinal inﬂammation. Furthermore, mRNA expression level of ABCG2, a well- known urate transporter, was decreased in the gouty mice. QZTBD increased the expression of ABCG2 and improved the intestinal urate excretion (Figure 6F). QZTBD Inﬂuenced the Fecal SCFAs Levels Taking into account the decline of SCFAs/butyrate-producing bacteria, we next tested the concentration of the most abundant SCFAs, including acetate, propionate, and butyrate, in the feces of mice by GC-MS (Supplementary Figure S4). As shown in Figure 5 and Supplementary Table S2, as compared with the control group, the concentrations of acetate, propionate, and butyrate in the model group were dramatically decreased (p < 0.01). QZTBD signiﬁcantly increased the levels of all these three February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 9 QZTBD for Gouty Arthritis Wen et al. FIGURE 6 \| QZTBD improved intestinal mucosal barrier. Expression of ZO-1 in the colon was measured by immunohistochemistry (A) and qRT-PCR (B); Expression of Occludin in the colon was measured by immunohistochemistry (C) and qRT-PCR (D); The mRNA expression of inﬂammatory factors in the intestinal tissues (E); The mRNA expression of ABCG2 in the intestine (F). “ns” represents not signiﬁcant; “” represents p < 0.05, “” represents p < 0.01 and “” represents p < 0.001. QZTBD Modulated the Expression of GPR43 and Energy Homeostasis in the Intestine and Energy Homeostasis in the Intestine Given the effect of SCFAs is derived from their ability to stimulate G-protein coupled receptors, we proceeded to examine the expression of GPR43. As described in Figures 7A,B, mRNA and protein levels of GPR43 in the colon were reduced in the gouty mice. QZTBD enhanced both mRNA and protein levels of GPR43 while FBST was unable to restore the levels of GPR43. On the other hand, mounting evidence has demonstrated that SCFAs are the primary energy sources for colonocytes and SCFAs alter the metabolic rate by stimulating GPR43. To reconcile these ﬁndings, we next examined the metabolic phenotype of intestinal cells. An elevated expression of glucose transporter type 1 (GLUT 1), which augments glucose uptake and glycolytic ﬂux, was observed in gouty mice (Figure 7C). mRNA levels of key genes related to glycolysis, including PFK1 (phosphofructokinase 1), PFKFB3 (6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3), and LDH (lactic acid dehydrogenase), were upregulated in the model group (Figure 7C). Western blotting analysis further veriﬁed the upregulation of PFKFB3 and LDH protein levels in gouty mice (Figure 7D). Moreover, the concentration of lactic acid, the dead- end product of glycolysis, was increased in both serum and feces, revealing the association of glycolysis and gout (Figure 7E). Both QZTBD and FBST inhibited the expression of GLUT1, PFK1, and PFKFB3, yet they resulted in different impacts on gene expression of LDH and lactic acid level (Figures 7C–E). We therefore speculated that SCFA deﬁciency is closely tied to enhanced glycolysis and increased susceptibility to gout. Inhibition of glycolysis is a potential target for gout treatment and QZTBD can effectively down-regulate the expression of glycolysis related enzymes. y SCFAs are important mediators that gut microbiota use to affect the host’s physiology and immunity. GPR43 is one of the primary receptors of SCFAs. Signaling through GPR43 plays a signiﬁcant role in the anti-inﬂammatory effects of SCFAs (Maslowski et al., 2009; Haslberger and Terkeltaub, 2015). Viera et al. demonstrated that germ free mice show attenuated MSU crystal induced inﬂammation which was mediated by acetate, and acetate acts via the macrophage receptor GPR43 to modulate inﬂammasome activation and IL-1β production (Vieira et al., 2015). They also concluded that acetate alleviates the inﬂammatory response to MSU crystals by inducing caspase- dependent apoptosis of neutrophils and enhancing the production of anti-inﬂammatory mediators (Vieira et al., 2017). The study was limited to examining the effects of butyrate on gout. QZTBD Improved Intestinal Mucosal Barrier and Intestinal Urate Excretion N 7/group. FIGURE 6 \| QZTBD improved intestinal mucosal barrier. Expression of ZO-1 in the colon was measured by immunohistochemistry (A) and qRT-PCR (B); Expression of Occludin in the colon was measured by immunohistochemistry (C) and qRT-PCR (D); The mRNA expression of inﬂammatory factors in the intestinal tissues (E); The mRNA expression of ABCG2 in the intestine (F). “ns” represents not signiﬁcant; “” represents p < 0.05, “” represents p < 0.01 and “” represents p < 0.001. N 7/group. February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 10 QZTBD modulated the expression of GPR43 and metabolic phenotype. Expression of GPR43 in the colon were measured by immunohistochemistry T-PCR (B); The mRNA expression of key glycolytic enzymes (GLUT1, PFK1, PFKFB3, and LDH) in the intestinal tissues (C); The protein expression of PFKFB3 the colon were measured by Western blotting (D). The concentration of lactic acid in serum and feces (E). “ns” represents not signiﬁcant; “” represents ” represents p < 0.01 and “” represents p < 0.001. N 7/group. QZTBD for Gouty Arthritis QZTBD for Gouty Arthritis Wen et al. FIGURE 7 \| QZTBD modulated the expression of GPR43 and metabolic phenotype. Expression of GPR43 in the colon were measured by immunohistochemistry A) and qRT-PCR (B); The mRNA expression of key glycolytic enzymes (GLUT1, PFK1, PFKFB3, and LDH) in the intestinal tissues (C); The protein expression of PFKFB3 and LDH in the colon were measured by Western blotting (D). The concentration of lactic acid in serum and feces (E). “ns” represents not signiﬁcant; “” represents 0 05 “” t 0 01 d “” t 0 001 N 7/ FIGURE 7 \| QZTBD modulated the expression of GPR43 and metabolic phenotype. Expression of GPR43 in the colon were measured by immunohistochemistry (A) and qRT-PCR (B); The mRNA expression of key glycolytic enzymes (GLUT1, PFK1, PFKFB3, and LDH) in the intestinal tissues (C); The protein expression of PFKFB3 and LDH in the colon were measured by Western blotting (D). The concentration of lactic acid in serum and feces (E). “ns” represents not signiﬁcant; “” represents p < 0.05, “” represents p < 0.01 and “*” represents p < 0.001. N 7/group. February 2021 \| Volume 11 \| Article 610556 11 Frontiers in Pharmacology \| www.frontiersin.org QZTBD for Gouty Arthritis Wen et al. QZTBD Modulated the Expression of GPR43 and Energy Homeostasis in the Intestine Cleophas et al. reported that butyrate inhibited the pro-inﬂammatory responses induced by the combination of urate crystals and the long-chain fatty acid palmitate (C16.0) in PBMCs from healthy donors and gout patients, acting as an inhibitor of class I HDACs (Cleophas et al., 2016). In this study, we found the concentrations of all three primary SCFAs in feces and the expression of GPR43 in the intestine were signiﬁcantly decreased in gout mice. The inhibition of SCFAs-GPR43 signaling leads to exacerbated or unresolving inﬂammation in models of gout arthritis, which was observed in colitis and asthma models as well (Maslowski et al., 2009). QZTBD increased the production of SCFAs, especially the production of butyrate, and the expression of GPR43. The regulation of SCFAs-GPR43 signaling was one of the critical ways for QZBTD to alleviate gouty arthritis, which was different from the means by which FBST takes effect. QZTBD Improved Intestinal Mucosal Barrier and Intestinal Urate Excretion regulating the production, degradation, and excretion of uric acid (Zhang et al., 2018). The intestine has been proven to be an important place for urate excretion (Ichida et al., 2012), and accumulating evidence suggests that the gut microbiome is involved in the pathogenesis of gout (Ma, 2020). Initiation of responses to MSU crystal in gout model required microbiota, and germ-free mice or mice treated with antibiotics had no response to the injection of MSU crystals (Vieira et al., 2015). Intestinal ﬂora usually affect purine metabolism by secreting metabolic enzymes such as xanthine oxidase (Chiaro et al., 2017), or producing speciﬁc metabolites such as SCFAs (Haslberger and Terkeltaub, 2015). A remarkable reduction of Faecalibacterium prausnitzii and the inhibition of butyrate biosynthesis was observed in gout patients (Guo et al., 2016). Our previous study showed that the genera Lachnospiraceae NC2004 group, Lachnospiraceae UCG_005, Ruminococcaceae NK4A214 group, and Ruminococcaceae UCG_011, which are associated with SCFAs production, were depleted in gout patients (Shao et al., 2017). In this study, we veriﬁed the depletion of butyrate producers in gout mice, which were strongly inversely correlated with symptoms in gout mice. Targeting the gut microbiome, especially SCFAs producing bacteria, is a potential novel strategy for characterizing and treating gout (Ma, 2020). QZTBD can restore the gut microbiota ecosystem and promote the growth of SCFA-producing bacteria such as Butyricicoccus. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION QZTBD increased the level of microbiota-derived butyrate by promoting the proliferation of butyrate-producing ﬂora, thereby further activating the expression of SCFAs receptor GPR43, increasing the expression of intestinal tight junction related proteins, facilitating the metabolism of colonocytes toward oxidative phosphorylation, inhibiting downstream inﬂammatory pathways and eventually ameliorating gout. Red arrow indicates upregulation, green arrow indicates downregulation. glycolysis (GLUT1, PFK1, PFKFB3, and LDH), the concentration of lactic acid was also enhanced in both feces and serum, indicating an increase in glycolysis in the mice model with gouty arthritis. Furthermore, there was a signiﬁcant reduction of oxygen consumption and an obvious increment of HIF-1α expression in the intestine of gout mice, which further conﬁrmed that the alteration of gut microbiota and intestinal metabolic phenotype associated with the development of gout (Supplementary Figure S5A). The inhibition of the glycolysis pathway has been proposed as a new target for the treatment of gout ﬂare and other IL-1β- related inﬂammation (Wen et al., 2012; Mian et al., 2019). QZTBD effectively inhibits the expression of genes involved in glycolysis, reduces the concentration of fecal and serum lactate, and restores the intestinal oxygen consumption (Supplementary Figure S5B), revealing a possible biochemical mechanism underlying this clinical prescription. Although increased intestinal permeability has been reported to trigger an immune response and low-grade inﬂammation, disrupted intestinal barrier function is associated with the development of various diseases, including intestine inﬂammatory diseases, extra-intestinal autoimmune diseases, and metabolic disorders (Chelakkot et al., 2018; Zaiss et al., 2019). We are the ﬁrst to report on the altered expression of intestinal TJ-related proteins in mice with gouty arthritis. Gut microbiota and their metabolites are closely related to intestinal barrier function. Eeckhaut et al. reported that patients with inﬂammatory bowel disease have lower numbers of Butyricicoccus bacteria. Administration of this bacteria result in a protective effect by strengthening the intestinal barrier function (Eeckhaut et al., 2013) and lowering intestinal TNF-α and IL-12 levels. Butyrate could prompt improvements to intestinal barrier function by inducing the expression of tight junction proteins such as ZO-1 and Occludin (Zaiss et al., 2019). Our results demonstrated that QZTBD enhances the expression of TJ-related proteins and inhibits the production of inﬂammatory cytokines by promoting the growth of Butyricicoccus. Moreover, it was reported that butyrate could upregulate the expression of urate transporter ABCG2 (Ferrer-Picón et al., 2020). Deﬁciency of butyrate is associated with the dysfunction of ABCG2. DISCUSSION QZTBD is an empirical prescription that has been developed for treating gouty arthritis for many years. Our previous research demonstrated that the potential anti-gouty arthritis effect of QZTBD could be attributed to the inhibition of the activation of the NLRP3 inﬂammasome and the production of downstream proinﬂammatory cytokines (Lv et al., 2019). The present study further veriﬁed its analgesic and anti- inﬂammatory effects and explored its potential mechanism from the perspective of the gut microbiome. We found that QZTBD can effectively reduce the symptoms of gout arthritis with comparable effects to FBST. It does so without obvious harmful side effects on animals, suggesting its clinical value for gout treatment. Our results indicated that QZTBD may exert its therapeutic effects by restoring the composition of gut microbiota and promoting the generation of SCFAs. Through these changes, QZTBD treatment attenuates intestinal mucosal barrier function, prompts intestinal uric acid excretion, inhibits glycolysis, and suppresses the production of intestinal pro-inﬂammatory cytokines (Figure 8). Uric acid is the ﬁnal metabolite of purine metabolism in the human body and the homeostasis of uric acid is achieved by February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 12 QZTBD for Gouty Arthritis Wen et al. FIGURE 8 \| Proposed mechanism of QZTBD in the treatment of gouty arthritis. In gout mice, the deﬁciency of gut butyrate-producing bacteria resulted in the decrease of intestinal butyrate content. Dropped butyrate inhibited the expression of GPR43 and intestinal tight junction related proteins, and further shifted the metabolism of colonocytes toward glycolysis and activated downstream inﬂammatory pathways. QZTBD increased the level of microbiota-derived butyrate by promoting the proliferation of butyrate-producing ﬂora, thereby further activating the expression of SCFAs receptor GPR43, increasing the expression of intestinal tight junction related proteins, facilitating the metabolism of colonocytes toward oxidative phosphorylation, inhibiting downstream inﬂammatory pathways and eventually ameliorating gout. Red arrow indicates upregulation, green arrow indicates downregulation. FIGURE 8 \| Proposed mechanism of QZTBD in the treatment of gouty arthritis. In gout mice, the deﬁciency of gut butyrate-producing bacteria resulted in the decrease of intestinal butyrate content. Dropped butyrate inhibited the expression of GPR43 and intestinal tight junction related proteins, and further shifted the metabolism of colonocytes toward glycolysis and activated downstream inﬂammatory pathways. CONCLUSION Our current results demonstrated that the anti-gouty arthritis effect of QZTBD, an empirical TCM prescription in clinic and a potential therapeutic decoction for the prevention and treatment of gout, may be attributed to the restoration of gut dysbiosis and enhancement of SCFAs formation, the renovation of the intestinal barrier function, the suppression of the key glycolysis-related enzymes, and the inhibition of the production of inﬂammatory factors. Another interesting aspect regarding the enhancement of glycolysis contributes to the development of gout. Glycolysis is a metabolic pathway characterized by low oxygen consumption, high glucose utilization, and high lactate release (Litvak et al., 2018). A very recent study reported the observation that MSU crystals lead to a metabolic rewiring toward the aerobic glycolysis pathway because of an increased expression of GLUT1 in the plasma membrane and an up-regulation of glucose uptake on macrophages (Renaudin et al., 2020). 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ACKNOWLEDGMENTS The animal study was reviewed and approved by Experimental procedures were approved by Laboratory Animal Management and Welfare Ethical Review Committee of Zhejiang Chinese Medical University (Permission number: ZSLL-2018-0012). We appreciate the technical support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University. FUNDING accession number(s) can be found in the article/Supplementary Material. accession number(s) can be found in the article/Supplementary Material. This research was supported by grants from the National Natural Science Foundation of China (81873145 and 82074248 and 81873269). DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 13 QZTBD for Gouty Arthritis Wen et al. SUPPLEMENTARY MATERIAL TS and XW were responsible for the design of the work. XW, YL, SS, ZH, JC, ZX, and XS were responsible for the acquisition and analysis of data. TS and XW drafted the manuscript. CW and TS approved the ﬁnal version to be published. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020.610556/ full#supplementary-material. Yu, J., Tang, Y.-Y., Wang, R.-R., Lou, G.-D., Hu, T.-T., Hou, W.-W., et al. (2016). A critical time window for the analgesic effect of central histamine in the partial sciatic ligation model of neuropathic pain. J. Neuroinﬂammation 13 (1), 163. doi:10.1186/s12974-016-0637-0 Frontiers in Pharmacology \| www.frontiersin.org February 2021 \| Volume 11 \| Article 610556 REFERENCES Combined effects of MSU crystals injection and high fat-diet feeding on the establishment of a gout model in C57BL/6 mice. Adv. Rheumatol. 60 (1), 52. doi:10.1186/s42358- 020-00155-3 February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 14 Wen et al. QZTBD for Gouty Arthritis Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Copyright © 2021 Wen, Lou, Song, He, Chen, Xie, Shi, Wen and Shao. 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. Zaiss, M. M., Jones, R. M., Schett, G., and Paciﬁci, R. (2019). The gut-bone axis: how bacterial metabolites bridge the distance. J. Clin. Invest. 129 (8), 3018–3028. doi:10.1172/jci128521 Zhang, Y., Jin, L., Liu, J., Wang, W., Yu, H., Li, J., et al. (2018). Effect and mechanism of dioscin from Dioscorea spongiosa on uric acid excretion in animal model of hyperuricemia. J. Ethnopharmacol. 214, 29–36. doi:10.1016/j. jep.2017.12.004 February 2021 \| Volume 11 \| Article 610556 Frontiers in Pharmacology \| www.frontiersin.org 15
https://openalex.org/W3128847091	https://link.springer.com/content/pdf/10.1007/s42452-021-04226-y.pdf	English	null	Optimal distributed generation location and sizing for loss minimization and voltage profile optimization using ant colony algorithm	SN applied sciences/SN Applied Sciences	2,021	cc-by	7,258	Abstract A system of power generation whereby the generating equipment is located close to the point of usage, thereby reducing losses and operation cost is called distributed generation (DG). However, it is imperative that DGs are sited such that the quality of power delivered is optimized and the total real power loss within the system minimized. This paper proposes an approach for optimum sizing and siting of DGs sizing in a power distribution system using Ant Colony Optimization (ACO) algorithm. To validate the algorithm the IEEE 30 bus standard test system was employed. A 92% decrease in real power loss within the system relative to the value before the connection of DGs was observed, while the minimum bus voltage increased from 0.656 per unit to 0.965 per unit. The results obtained from ACO are further verified by creating an ETAP model of the IEEE 30 bus system and simulating the impact of DG on the system. A significant reduction in total real power losses within the system and improvement in voltage profile was observed when the DGs are placed at the ACO derived sites relative to at other locations. Therefore, Ant Colony Algorithm can be used in deriving the optimum sites and sizes of DGs in a power distribution system. Keywords Ant colony optimization (ACO) · Distributed generation (DG) · Renewable energy · Power flow · Voltage quality * Moses Omolayo Petinrin, mo.petinrin@ui.edu.ng; layopet01@yahoo.com \| 1Department of Mechanical Engineering, University of Ibadan, Ibadan, Nigeria. 2Department of Computer Science, City University of Hong Kong, Kowloon, Hong Kong. 3Department of Mathematical Sciences, Kings University, Ode‑Omu, Osun State, Nigeria. 4Department of Electrical and Electronic Engineering, Federal Polytechnic Ede, Ede, Nigeria. 5Power Equipment and Electrical Machinery Development Institute, Okene, Kogi State, Nigeria. Research Article Optimal distributed generation location and sizing for loss minimization and voltage profile optimization using ant colony algorithm Adeseye Amos Ogunsina1 · Moses Omolayo Petinrin1 · Olutomilayo Olayemi Petinrin2,3 · Emeka Nelson Offornedo1 · Joseph Olawole Petinrin4 · Gideon Olusola Asaolu5 Received: 18 July 2020 / Accepted: 15 January 2021 / Published online: 1 February 2021 © The Author(s) 2021 OPEN 1 Introduction thermal systems which harnesses solar energy from the sun and transform it into a suitable form of energy such as heat and electricity are prime candidates for distributed energy generation [3]. Other forms of renewable energy technologies applied in distributed generation systems are small hydro power systems, wind turbines, biogas and biothermal systems and so on. Also, due to the harmful effect of fossil fuel usage on the environment such as the greenhouse effect and global warming, there has been a significant increase in the usage of renewable energy technologies. Distributed Generators (DGs) are power generation sys‑ tems whose output are not connected to a central grid structure for transmission over long distances to the point of usage, rather they are located at the point of usage, with limited or no requirement for transportation [1, 2]. Due to their modularity and portability and little or no require‑ ment for the transportation of the energy resources (fuel) from point of production to the point of usage, renewable energy technologies such as solar photovoltaic and solar * Moses Omolayo Petinrin, mo.petinrin@ui.edu.ng; layopet01@yahoo.com \| 1Department of Mechanical Engineering, University of Ibadan, Ibadan, Nigeria. 2Department of Computer Science, City University of Hong Kong, Kowloon, Hong Kong. 3Department of Mathematical Sciences, Kings University, Ode‑Omu, Osun State, Nigeria. 4Department of Electrical and Electronic Engineering, Federal Polytechnic Ede, Ede, Nigeria. 5Power Equipment and Electrical Machinery Development Institute, Okene, Kogi State, Nigeria. SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y V l ( ) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article Thus, due to the variable nature of solar and other renewable energy resources, as their penetration rate into the grid increases, they pose some technical challenges on the system such as feeder overloading, harmonic pol‑ lution and so on. Also, high capital cost, low reliability and low efficiency limits the widespread usage of these technologies [1]. Besides, voltage flickers and power fluc‑ tuations due to variations in solar irradiation are some of the adverse effects of high penetrated PV in power sys‑ tems [4]. In their research, Miller and Ye [5] evaluated the impact of using DG as a significant portion of the total energy resources by creating a model of Jeju Island, Korea and made use of three distributed generation systems to assess the potential negative power quality effects of high penetration of DG. 1 Introduction Also, Ajan and Nirmala [6] modelled a stand-alone and grid-tied PV system using Simulink which was used to evaluate the relationship between PV system parameters and the output. The various Voltages, Current and Power levels are recorded. The various advantages of optimally allocated DGs in a distribution network such as improvement in overall system voltage, reduction in total system power loss, improvement in system power quality and frequency regulation are highlighted in [7, 8]. advantages of these type of algorithms in solving opti‑ mization problems include their simplicity and flexibility. They however only derive a near-optimal solution rather than an exact one which is often within acceptable lim‑ its for most problems [18]. A metaheuristic cost–benefit approach for the siting and sizing of DGs to ensure the peak demand forecast is optimally met was presented by El-khattam et al. [19]. Kuri et al. [20] applied genetic algo‑ rithm for optimal positioning and sizing of DGs in a distri‑ bution network, they took into consideration the number of planning years in setting the optimization objectives and constraints. Golshan and Arefifar [21] used an algo‑ rithm based on Tabu Search (TS) in solving a comprehen‑ sive planning objective for determining the size, loca‑ tions and mode of operation of Reactive Power Sources (RPSs) and Distributed Generation Resources (DGRs) in a power system. Gandomkar et al. [22], presented a hybrid of simulated annealing and genetic algorithm for solving the problem of DG siting in power distribution networks. Kalkhambkar et al. [23–25] had also implemented the grey wolf algorithm to minimize energy loss in energy storage and renewable distributed generations. Celli et al. [26] proposed a tool to plan and operate DGs for deferment of upgrades in transmission and distribution systems and so on. In the proposed algorithm, multi-objective program‑ ming and genetic algorithm (GA) were utilized in a finding the optimum location and number of distributed energy resources to be placed in a network. Similarly, a method for reduction in real power loss, improvement in voltage profile and release of substation in a power system based on analysis of voltage sensitivity index was presented by Gopiya et al. [27]. However, to derive maximum benefits from DG, the generators must be appropriately sized and located within the power distribution network [7, 9, 10]. To achieve this objective, various researchers have proposed different approaches. 1 Introduction These methods are classified based on the driving algorithms into metaheuristic, analytical or numer‑ ical based methods. Analytical techniques for solving the DG allocation problem are presented in [11–13]. Several authors have also published research studies on opti‑ mal allocation of DG and reactive power sources such as capacitors in a distribution system using numerical based techniques [14]. One of such study carried out by Fawzi et al. [15] made use of Dynamic programming. Resener et al. [16] applied Mixed-integer linear programing (MILP). However, these analytical and numerical based methods are often computationally intensive as all possible combi‑ nations of DG sites are required to be evaluated to derive the optimum solution. This often limits the number of vari‑ ables considered in most researches applying these meth‑ ods. Also, due to the non-linear nature of the problem, the linear programming methods often perform poorly in find‑ ing the optimal solutions [17]. In simplifying the problem of DG location and sizing, most researchers in the literatures often either fix the num‑ ber of DGs or the sizes. The proposed method in this paper eliminates this restriction by allowing the algorithm to heuristically search through the entire search space with‑ out restricting the possible number of DGs or the capac‑ ity of the DGs in deriving the optimum combination of the number, the site and size of DGs to minimize the real and reactive power loss in the system. Due to the relative novelty of ant colony optimization (ACO) algorithm com‑ pared to other population-based optimization algorithm and also because ACO was initially designed for discrete problems, not many researches on the suitability of ACO for DG allocation problem has been done. However, ACO has been chosen over other metaheuristic based approach for solving the DG allocation problem for its ability to derive the global optimum solution with minimal number of iterations and its better comparison with other similar algorithms [28, 29]. This study helps to further explore the application of the ACO algorithm in discrete and continu‑ ous problem domain. The ACO algorithm will be applied to Metaheuristic population-based algorithms such as genetic algorithm, firefly, grey wolf, bee colony, particle swarm and ant colony algorithms have an advantage over the numerical and analytical based methods because it doesn’t have to cover the entire possible search space in deriving the optimal solution. Vol:.(1234567890) 4 Implementation of the algorithm (4) nDG ∑ i=1 PDGi + jQDGi ≤PS∕S + jQS∕S (5) max { Sij or Sji } ≤Sijmax (6) nDG ≤nDGmax The Ant Colony Optimization (ACO) algorithm applied for this study was as presented in [33]. The main steps of the applied ACO algorithm are outlined below: The Ant Colony Optimization (ACO) algorithm applied for this study was as presented in [33]. The main steps of the applied ACO algorithm are outlined below: (4) max { Sij or Sji } ≤Sijmax (5) 2 Problem formulation The total real power loss within the distribution system and the deviation of the bus voltages from the nominal voltage magnitude, is given by equations below [30]: Plosses = 1 2 N ∑ i=1 N ∑ j=1 ℜ{yij }[ \|\|Vi\|\| 2 + \|\|\|Vj\|\|\| 2 −2\|\|Vi\|\| \|\|\|Vj\|\|\| cos 훿ij ] (1) (2) VD = NB ∑ i=1 (Vi −1)2 (2) where ℜ(∙) denotes the real component of a complex number,yij is the admittance of the ijth branch, N is the number of buses, Vi and Vj denotes the bus voltages, 훿ij is 훿i −훿j. j The objective function to be minimized to derive the best DG allocation for minimum power losses and enhanced voltage profile using ACO is formulated as: (3) min f = PLoss + w ⋅vD (3) The inequality constraints of the minimization algo‑ rithm are outlined below: 1 Introduction This helps to minimize the computational resources required and allows for consid‑ eration of a large number of variables and buses. Other Vol:.(1234567890) Vol:.(1234567890) Vol:.(1234567890) Research Article SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y (8) 훿min i ≤훿i ≤훿max i optimally site and size DGs in a power distribution system such that the total real power loss within the system is minimized while the system voltage profile is significantly enhanced. In order to demonstrate the suitability of the proposed algorithm, it was applied on the IEEE 30-Bus Distribution System. 훿min i ≤훿i ≤훿max i (8) where: nDG is the number of DG sources, PDGi is the real power output of the ith DG source while QDGi is its reac‑ tive power output, Sij is the apparent power flow in the ijth branch and Sijmax is the maximum apparent power flow in the ijth branch. The remainder of the paper is organized as follows: Sect. 2 provides the mathematical formulation of the problem of real power loss and voltage profile deviation of a power distribution system. The proposed Ant Colony Optimization algorithm for solving the DG optimization problem is presented in Sect. 3, 4. Section 5 presents the model of the case study power distribution system. Analy‑ sis and simulation results are presented in Sect. 6. Finally, the conclusion is drawn in Sect. 7. 3 Optimization algorithm In this study, the Ant Colony Optimization (ACO) is pro‑ posed for solving the DG allocation problem. ACO is an algorithm that is inspired by the biological behaviour of ants, which enable them to discover the shortest route between their nest and food. Ants do not communicate directly with one another, but they exchange informa‑ tion using what is known as pheromones [31]. When an ant traverses a path in search for food, it lays pheromones on the path, as more ants pass through the shorter path between the nest and the food per unit time relative to the longer path, the quantity of pheromone laid on the shorter path becomes higher than that on the longer path, thereby increasing the probability that an ant will choose the shorter path. The likelihood that an ant will choose a given path, therefore, depends on the quantity of phero‑ mone laid on the path relative to other paths which in turn depends on the number of ants that had previously chosen the same path [28, 32]. The ACO algorithm pro‑ cedure comprises majorly of three steps: In the first step, the candidate variable values are randomly generated and the pheromone trail is initialized. Secondly, each ant uses the probabilistic state transition rule to find solution to the problem. Thirdly, the pheromone value at each edge is updated, first by evaporating a proportion of the phero‑ mone at all the edges, thereafter the amount of phero‑ mone on paths whose solutions are of high fitness values is reinforced. 4.3 Ants’ solution construction In this step, m ants construct a solution from the gener‑ ated candidate variable values. An ant k selects a variable with an index l(k) i for the ith variable from the candidate variable values according to (11). (9) x(j) i = li + ui −li m + 휗 ( j −1 + randj i ) (9) where (m + ϑ) is the number of initialized randomly gener‑ ated solution for variable i, randj i is a uniformly distributed random number between 0 and 1, i = 1, 2, …, n, while j = 1, 2, …, (m + ϑ). The initial buses for siting the DGs are uni‑ formly randomly chosen from the total number of buses. The randomly generated initial solutions are evaluated and ranked according to their fitness values. The solution with the highest fitness value is captured as the initial global best solution. (11) l(k) i = ⎧ ⎪ ⎨ ⎪⎩ arg max 𝜏(1) i , 𝜏(2) i , ..., 𝜏(m) i , if q < q0 L(k) i , otherwise (11) where q is a uniform random number based on which an ant either selects a variable with the highest pheromone value among the ones generated in the previous iteration or randomly chooses an index L(k) l ∈{0, 1, ..., m + gi} . The probability distribution for the random variable choice is given in (12). Four sources are utilized in generating the variable values from which the ants choose during each iteration, they are: the best solutions derived from all previous itera‑ tions, which are also referred to as the global best solution; solutions generated from a random exploration process across the entire solution space; solutions derived from a dynamic exploitation of the solution space around the global best solution; and finally from the values selected by the ants in the last iteration of the algorithm. In each iteration of the ACO algorithm, the m number ants create m solutions of the problems which serves as candidate val‑ ues for the next iteration of the algorithm. The best solu‑ tion from the previous iteration is thereafter compared to the current global best solution in terms of their fitness value, and the global best solution is updated accordingly. 4.4 Pheromone update The solutions generated by all the ants in step 3 are evalu‑ ated at the end of each iteration of the algorithm and are sorted in descending order of their fitness value. Thereaf‑ ter, the amount of pheromones on a predetermined num‑ ber of variable values are evaporated according to (13). 4.1 Generation of the candidate variable values Vol.:(0123456789) (6) (7) The solution variables are the real and reactive power generation capacities of the DGs and the bus number at which the generators will be connected. The number of DG is initialized to a certain number. Initially, the candi‑ date variable values for the generators’ real and reactive (6) (7) The solution variables are the real and reactive power generation capacities of the DGs and the bus number at which the generators will be connected. The number of DG is initialized to a certain number. Initially, the candi‑ date variable values for the generators’ real and reactive nDG ≤nDGmax nDG ≤nDGmax Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article power capacity, which are real continuous variables, are randomly sampled subject to the equality and inequality constraints as: 4.3 Ants’ solution construction (12) p(j) i = 휏(j) i ∑m+gi u=0 휏(u) i , j = 0, 1, ..., m + gi (12) The solution derived by each ant from the step above is denoted by x(k) = (x (l(k) 1 ) 1 , x (l(k) 2 ) 2 , ..., x(l(k) n ) n ). 4.2 Dynamic exploitation process (13) 휏(j) i ←(1 −휌) ⋅휏(j) i + 휌⋅Tmin (13) This involves the searching of the solution space around x0 = (x0 1, x0 2, ..., x0 n) , the global best solution, in the interval [ x(0) i −ri, x(0) i + ri ] , i = 1, 2, ..., n . The value of the variables in the global best solution is either increased, decreased or left unchanged according to the following equations: where 휌 is a real number between 0 and 1 which defines the applied rate of pheromone evaporation per iteration of the algorithm, Tmin is a predefined constant representing the minimum pheromone value. Also, to ensure conver‑ gence of the algorithm towards the global minima, the pheromone values of the best 휓 solutions’ variable values are reinforced as (10) ̂xi = ⎧ ⎪ ⎨ ⎪⎩ min(x(0) i + ri ⋅𝜎i, ui), 0 ≤q < 1∕3 x(0) i , 1∕3 ≤q < 2∕3 max(x(0) i −ri ⋅𝜎i, li), 2∕3 ≤q < 1 (10) (14) 휏(j) i ←(1 −훼) ⋅휏(j) i + 훼⋅Tmax (14) where 훼 is the defined rate of pheromone reinforcement and it is any number between 0 and 1. The implementa‑ tion flowchart for the ACO algorithm is shown in Fig. 1. where 휎i ∈(0, 1] and q ∈[0, 1) are uniform random values, i = 1, 2, ..., n. Then the resulting solution ̂x = (̂x1, ̂x2, ..., ̂xn) . If this process generates a solution with a better fitness value than the global best solution, the recorded global best solution is replaced with the derived solution. The process is repeated for ϑ number of times. Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article 5 Case study Fig. 1 Implementation flowchart of the ACO algorithm [33] 5.1 Test system The proposed ACO algorithm was implemented in MATLAB R2016a and executed on an Intel® Pentium® N3540 2.16 GHz personal computer, while the power flow algorithm for eval‑ uating the fitness value of each solution generated by the ants was implemented using Matpower® library in MATLAB. It was tested on an IEEE 30-bus system. The total loads of the 30-bus system are 189.2 MW and 107.2 MVar, with its bus and branch parameters given in [34]. A schematic diagram of the test feeder is shown in Fig. 2. The following assumptions were made in formulating the proposed method: • Each bus within the system can take a maximum of one DG unit; • The total DG penetration within the distribution system will not exceed 100% of the total load; • The size of a single DG unit connected to a bus in the algorithm is allowed to can range from zero (no DG connected to the bus) to the total system load (100% DG penetration with only 1 DG source); • All the DGs have unlimited capability to supply real and reactive power according to the demand; • All the buses, except bus 1 which is the reference bus, has the capacity for DG connection; • Continuous values are assumed for the size of the DGs. 6.1 ACO results The voltage magnitude across all the buses in the distri‑ bution system before and after ACO optimized DGs siting are presented in Fig. 3. It shows a significant improvement in the system voltage profile. In the base case, with the system only fed from the grid via Bus 1, there is a decline in the per unit (pu) voltage magnitude at the buses as we progress along the system from bus 1 to bus 30. However, with the DGs sited at the optimum buses as derived using ACO, there is a significant improvement in the voltage profile of the system with all the buses approaching the nominal voltage. Before DG siting, the least voltage mag‑ nitude across all the buses was obtained at bus 26 with a per unit voltage of 0.656, however, after DG siting, this value increased by 47% to 0.965 per unit. Fig. 6 ETAP derived voltage profile across all the system buses sized and sited DGs, the losses reduced drastically across all the branches, with values approaching zero as close as possible. 5.2 Validation In order to validate the impact of results of the ACO algo‑ rithm, a model of the IEEE 30 bus system was developed using the Electrical Transient Analyzer Programme (ETAP) and load flow analysis of the system under the ACO derived optimal DG number, sites and sizes were carried out. The system was initially connected to the grid at bus 1 and the voltage profile at each of the 30 buses as well as the total system losses were recorded. Thereafter, 6 num‑ ber generators were connected to the system, at first, the generators were connected at bus numbers 7, 8, 12, 19, 21 and 30 derived as the optimum sites for the DGs from the ACO analysis, the observed losses and system voltage profile are compared to the values obtained under grid power supply. To further revalidate the selection of the optimum site by the ACO algorithm, three additional case studies with the DGs sited at randomly selected buses with in the modelled system were evaluated. The IDs of the buses at which the DGs were sited under the various case studies are outlined in Table 1. The system voltage profile Fig. 1 Implementation flowchart of the ACO algorithm [33] Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article Fig. 2 ETAP Model of IEEE 30-Bus System Fig. 2 ETAP Model of IEEE 30-Bus System Fig. 2 ETAP Model of IEEE 30-Bus System Vol:.(1234567890) Vol:.(1234567890) Vol:.(1234567890) 452-021-04226-y Research Article 0 1 2 3 4 5 6 7 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Real Power Loss (MW) Branch Number Before DG Aer DG Fig. 4 Real power loss across the branches before and after DG installation 0 5 10 15 20 25 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Reacve Power Loss (MVar) Branch Number Before DG Aer DG Fig. 5.2 Validation 4 Real power loss across the branches before and after DG installation 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Voltage Magnitude (pu) Bus Number Before DG Aer DG Branch Number Fig. 4 Real power loss across the branches before and after DG installation 0 5 10 15 20 25 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Reacve Power Loss (MVar) Branch Number Before DG Aer DG Fig 5 Reactive power loss across the branches before and after DG 0 5 10 15 20 25 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Reacve Power Loss (MVar) Branch Number Before DG Aer DG Fig. 5 Reactive power loss across the branches before and after DG installation 0.5 0.6 0.7 0.8 0.9 1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Voltage (pu) Bus IDs Before DG Optimum Site Case 1 Case 2 Case 3 Fig. 6 ETAP derived voltage profile across all the system buses Bus Number Fig. 3 Voltage magnitude at each bus before and after DG installa‑ tion Branch Number Fig. 5 Reactive power loss across the branches before and after DG installation and the total system losses were thereafter analyzed and the results presented. 0.5 0.6 0.7 0.8 0.9 1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Voltage (pu) Bus IDs Before DG Optimum Site Case 1 Case 2 Case 3 Fig. 6 ETAP derived voltage profile across all the system buses 5.2 Validation 5 Reactive power loss across the branches before and after DG installation SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s4 Table 1 DG sites under different case studies for ETAP simulation Case studies DG sites (Bus ID) ACO optimized sites 7, 8, 12, 19, 21 and 30 Case 1 1, 2, 13, 19, 22 and 27 Case 2 1, 7, 12, 21, 23 and 27 Case 3 1, 2, 12, 23, 26 and 30 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Voltage Magnitude (pu) Bus Number Before DG Aer DG Fig. 3 Voltage magnitude at each bus before and after DG installa‑ tion SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452 021 04226 y Table 1 DG sites under different case studies for ETAP simulation Case studies DG sites (Bus ID) ACO optimized sites 7, 8, 12, 19, 21 and 30 Case 1 1, 2, 13, 19, 22 and 27 Case 2 1, 7, 12, 21, 23 and 27 Case 3 1, 2, 12, 23, 26 and 30 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Voltage Magnitude (pu) Bus Number Before DG Aer DG Fig. 3 Voltage magnitude at each bus before and after DG installa‑ tion 0 1 2 3 4 5 6 7 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Real Power Loss (MW) Branch Number Before DG Aer DG Fig. 4 Real power loss across the branches before and after DG installation 0 5 10 15 20 25 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Reacve Power Loss (MVar) Branch Number Before DG Aer DG 0 1 2 3 4 5 6 7 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Real Power Loss (MW) Branch Number Before DG Aer DG Fig. 6.2 Comparison of ACO with ETAP Similarly, as illustrated in Fig. 4 and Fig. 5, in the base case when the only power source of the network was situ‑ ated at bus 1, we observed significant real and reactive power losses from bus 1 to 10, however, with the optimally The analysis of the distribution system voltage profile as derived from ETAP under the various distributed genera‑ tion arrangement are illustrated in Fig. 6. The ACO results Vol.:(0123456789) Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article 0 5 10 15 20 25 30 35 40 Before DG ACO Optimized DG Sites Case 1 Case 2 Case 3 Reactive Power Loss (MVar) Real Power Loss (MW) 0 5 10 15 20 25 30 35 40 Before DG ACO Optimized DG Sites Case 1 Case 2 Case 3 Reactive Power Loss (MVar) Real Power Loss (MW) Fig. 7 Total real and reactive power losses under different case studies as presented in Fig. 3 with the obtained results from ETAP shown in Fig. 6 for optimized site plot are in good correla‑ tion with an average difference of 0.52%. It is also shown in Fig. 6 that there is a significant improvement in the volt‑ age profile when the system is fed by DGs compared to when it is only fed from the grid. Also, while the voltage at some of the buses under the other three case studies are better than when the DGs are sited at the ACO optimized sites, the best overall voltage profile is obtained at the ACO optimized sites. as presented in Fig. 3 with the obtained results from ETAP shown in Fig. 6 for optimized site plot are in good correla‑ tion with an average difference of 0.52%. It is also shown in Fig. 6 that there is a significant improvement in the volt‑ age profile when the system is fed by DGs compared to when it is only fed from the grid. Also, while the voltage at some of the buses under the other three case studies are better than when the DGs are sited at the ACO optimized sites, the best overall voltage profile is obtained at the ACO optimized sites. Table 2 presents the summary of results of the ACO optimized sites with the DG sites of randomly selected buses simulated in ETAP. 6.2 Comparison of ACO with ETAP It revealed that the total real and reactive power losses within the system reduced by 92% and 97%, respectively when the distribution system is fed by six DGs sited at the ACO optimized locations relative to the values under grid power supply. Also, the highest reduction in losses was obtained with the DGs sited at the ACO derived sites as against when they are sited at other locations within the distribution system, as obtained in Case 1, 2 and 3. This is also illustrated in Fig. 7 which shows a significant reduction in the total real and reactive power losses across the system when DGs are sited at various locations within the system under the four case studies, however, the least losses are obtained when the DGs are sited at the ACO derived optimum buses. Fig. 7 Total real and reactive power losses under different case studies the ACO method presented achieving this with a much smaller search space [12]. The superior performance of the method could be attributed to the fact that the algorithm is not excessively constrained with the optimum combi‑ nation of DG power factors, the number, the capacity and the location of DGs heuristically derived by the algorithm coupled with the superior capacity of ACO to converge as the global minima compared with other metaheuristic algorithms. The results showed that the ACO based approach pre‑ sented in this paper was able to achieve a 92% reduction in real power loss and a 97% reduction in reactive power loss within the distribution system. Compared to other reviewed literatures on the subject, this method resulted in the highest reduction in real and reactive power losses. A 67.01% reduction in losses was derived using Differen‑ tial evolution technique [35]. Using a hybrid of GA and an analytical solution, a reduction in system losses of 83.74% was derived in [36]. Using closed-form analytical expres‑ sions, a maximum reduction in real power losses of 95% and 94% reduction in the reactive power losses, which is comparable with the result obtained in this study with 7 Conclusion The impact of the optimally sized and sited DGs was also significantly higher than the figures achieved in most past research work on the same subject. results obtained showed that the ACO based approach resulted in a significant reduction in the total real and reac‑ tive power losses as well as improvement in the voltage profile of the power distribution network as a result of the DGs. The impact of the optimally sized and sited DGs was also significantly higher than the figures achieved in most past research work on the same subject. g j 7. Kazemi A, Sadeghi M (2009) Sitting and sizing of distributed generation for loss reduction. In proceedings of the asia-pacific power and energy engineering conference, APPEEC pp. 1–6l 8. Ghosh N, Sharma S, Bhattacharjee S (2012) A load flow based approach for optimum allocation of distributed generation units in the distribution network for voltage improvement and loss minimization. Int J Comput Appl 50:15–22. https://doi. org/10.5120/7847-1075 In the future, this research work will be extended by considering and modelling the different types of renewa‑ ble energy sources in formulating the problem rather than using a generic DG source with the capacity to generate any amount of real power and supply or sink any amount of reactive power as presented in this work. Furthermore, more work still needs to be done to solve the planning and dispatch problem associated with a high penetration of renewable energy DGs in a power distribution system due to the variable nature of renewable energy sources. g 9. 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H2Open J 3 135-188. https://doi.org/10.2166/h2oj.2020.128 7 Conclusion The proper placement and sizing of DGs in a power distri‑ bution system plays a significant role in reducing the total real and reactive losses as well as improves the voltage profile within the system. In this paper, an optimization method for determining the optimal sites and capacities of distributed generation resources in a power distribution network using Ant Colony Optimization was presented. The problem consists of two types of variable, the DG placement problem which is discrete in nature and gen‑ erators’ sizing which is of a continuous variable type. The Vol:.(1234567890) Table 2 Voltage profile and total system losses Before DG ACO Opti‑ mized Sites Case 1 Case 2 Case 3 Average bus voltages 0.8460 0.9914 0.9809 0.9878 0.9718 % Increase in average bus voltages 17.19% 15.95% 16.76% 14.87% Standard deviation of bus voltages 0.0429 0.0083 0.0118 0.0093 0.0201 Min bus voltage 0.8069 0.9626 0.9607 0.9698 0.9409 Max bus voltage 1 1 1 1 1 Total real power loss (MW) 8.862 0.675 2.057 1.383 2.799 Total reactive power loss (MVar) 35.756 1.111 5.746 3.149 7.291 % Reduction in real power loss − 92% − 77% − 84% − 68% % Reduction in reactive power loss − 97% − 84% − 91% − 80% Before DG ACO Opti‑ mized Sites Case 1 Case 2 Case 3 Average bus voltages 0.8460 0.9914 0.9809 0.9878 0.9718 % Increase in average bus voltages 17.19% 15.95% 16.76% 14.87% Standard deviation of bus voltages 0.0429 0.0083 0.0118 0.0093 0.0201 Min bus voltage 0.8069 0.9626 0.9607 0.9698 0.9409 Max bus voltage 1 1 1 1 1 Total real power loss (MW) 8.862 0.675 2.057 1.383 2.799 Total reactive power loss (MVar) 35.756 1.111 5.746 3.149 7.291 % Reduction in real power loss − 92% − 77% − 84% − 68% % Reduction in reactive power loss − 97% − 84% − 91% − 80% Table 2 Voltage profile and total system losses Vol:.(1234567890) SN Applied Sciences (2021) 3:248 \| https://doi.org/10.1007/s42452-021-04226-y Research Article 6. 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https://openalex.org/W3153416365	https://journal.unpak.ac.id/index.php/apik/article/download/3287/2221	Indonesian	null	STRATEGI MARKETING PUBLIC RELATIONS DINAS PARIWISATA DAN KEBUDAYAAN KOTA BOGOR DALAM MEMPERKENALKAN BATIK BOGOR	Jurnal penelitian sosial ilmu komunikasi	2,021	cc-by-sa	3,803	Abstrak Tujuan penelitian ini adalah untuk mengetahui analisis humas dinas pariwisata dan budaya Kota Bogor dalam memperkenalkan Batik Bogor. Penelitian ini memiliki rumusan masalah Batik Bogor yang belum banyak diketahui oleh masyarakat khususnya masyarakat Bogor sendiri. Selain itu mengetahui faktor pendukung dan faktor penghambat dalam mengenalkan Batik Bogor. Dalam penelitian ini metode penelitian yang digunakan adalah deskriptif kualitatif berupa kata-kata tertulis atau lisan dari orang-orang dan perilaku yang diamati dalam memperoleh data menggunakan wawancara, observasi, dan dokumentasi. Teori yang digunakan dalam penelitian ini adalah Maketing Public Relations dan informan kunci adalah Undang Sulaeman sebagai bagian kemitraan dan pengembangan ekonomi kreatif dan Sri Hartati sebagai perajin Batik Bogor. Kata kunci: Batik Bogor; budaya Bogor; strategi marketing public relations. Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik STRATEGI MARKETING PUBLIC RELATIONS DINAS PARIWISATA DAN KEBUDAYAAN KOTA BOGOR DALAM MEMPERKENALKAN BATIK BOGOR Sinta Devi Lestari1, Sardi Duryatmo2, Prasetyo Adinugroho3 1,2,3 Universitas Pakuan, Bogor, Indonesia )Surel korespondensi: devilestarisinta@gmail.com 1,2,3 Universitas Pakuan, Bogor, Indonesia )Surel korespondensi: devilestarisinta@gmail.com Kronologi Naskah: diterima 28 Januari 2021; direvisi 10 Maret 2021; diputuskan 21 Maret 2021 Kronologi Naskah: diterima 28 Januari 2021; direvisi 10 Maret 2021; diputuskan 21 Maret 2021 Pendahuluan oleh masyarakat luas khususnya masyarakat Bogor itu sendiri karena produksinya masih bisa dihitung jari, tidak dengan kota lain seperti Pekalongan, produksinya sudah massal. Masyarakat Bogor perlu mengetahui bahwa ada batik khas Bogor, maka Dinas Pariwisata dan Kebudayaan Kota Bogor membina perajin Batik Bogor di kampung batik Cibuluh untuk terus mengembangkan dan menciptakan motif batik yang di ambil dari ikon Kota Bogor serta menguatkan promosi produknya. Indonesia merupakan negara yang kaya akan budaya, salah satu di antaranya adalah batik. Batik merupakan salah satu ciptaan intelektual manusia yang menjadi ciri khas dari suatu daerah. Kekayaan intelektual ini telah menjadi bagian dari budaya masyarakat Indonesia. Banyak motif batik yang memiliki nilai seni yang cukup tinggi dan mempunyai nilai filosofi di berbagai daerah yang ada di Indonesia. Batik sebagai warisan budaya, kekayaan seni masa lampau yang telah menjadikan negara Indonesia memiliki ciri yang khas di mancanegara. Salah satu warisan batik yang masih bertahan dan menjadi kekayaan khas adalah batik tulis. p y Hermawan (2013) mengemukakan bahwa promosi adalah salah satu komponen prioritas dari kegiatan pemasaran yang memberitahukan kepada konsumen bahwa perusahaan meluncurkan produk baru yang menggoda konsumen untuk melakukan kegiatan pembelian. Promosi adalah sarana dimana perusahaan berusaha untuk menginformasikan, membujuk, dan mengingatkan konsumen baik secara langsung atau tidak langsung tentang produk dan merek yang mereka jual Kotler(2010). Batik tulis merupakan kain yang dihias dengan tekstur dan corak batik menggunakan tangan. UNESCO telah menetapkan Batik Indonesia sebagai warisan kemanusiaan untuk budaya lisan dan Nonbendawi (Masterpiece of the Oral and Intangible Heritage of Humanity) sejak 2 Oktober 2009. Sejak saat itu, pemerintah menetapkan 2 Oktober sebagai Hari Batik Nasional melalui keputusan Presiden Nomor 33 Tahun 2009. Kota Bogor mungkin lebih terkenal dengan wisata kulinernya. Namun Kota yang dijuluki sebagai kota hujan ini ternyata memiliki ciri khas lain yang perlu diperkenalkan lebih luas, yaitu batik. Duncan (2010), menyatakan bahwa ada tiga pendekatan Marketing Public Relations yang menggabungkan antara strategi pemasaran tradisional dan dimensi megamarketing yang membutuhkan komunikasi dari bagian yang bukan merupakan bagian pemasaran traditional chain. Untuk kegiatan promosi dengan melakukan pendekatan marketing public relations ada 3 taktik yang digunakan sebagai alat perancangan strategi dari program yang telah terencana dan sedang terlaksana, yaitu bertujuan menarik (pull strategy), mendorong (push strategy), dan upaya untuk mempengaruhi (pass strategy) kesadaran masyarakat akan produk yang ditawarkan. Abstract The purpose of this study was to determine the analysis of the marketing public relations department of tourism and culture of the city of Bogor in introducing Batik Bogor. This research has a problem formulation that is not well known Bogor batik by the public, especially the Bogor community itself. In that addition to knowing the supporting factors and inhibiting factors in introducing Batik Bogor. in this study the research method used was descriptive qualitative form of written or oral words from people and the observed behavior in obtaining data used interviews, observation, and documentation. The theory used in this study is the marketing public relations and the key informant is Undang Sulaeman as the partnership and creative economy developmeint section and Sri Hartati as Bogor batik craftsman. Keywords: Batik Bogor; Bogor culture; Marketing strategy public relations. 11 11 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? Batik Bogor di kampung batik Cibuluh di kampung batik Cibuluh. Bertanggung jawab penuh dalam kegiatan, produksi, dan pemasaran Batik Bogor. 3 Lina Sobariah sebagai Seksi Pemasaran Ekonomi Kreatif Bertanggung jawab dalam proses pemasaran bidang ekonomi kreatif. Batik Bogor di kampung batik Cibuluh di kampung batik Cibuluh. Bertanggung jawab penuh dalam kegiatan, produksi, dan pemasaran Batik Bogor. 2. Bagaimana faktor pendukung dan penghambat Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? 2. Bagaimana faktor pendukung dan penghambat Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? Metode Penelitian Pendekatan yang dilakukan oleh peneliti yaitu pendekatan kualitatif. Peneliti menggunakan pendekatan kualitatif karena pendekatan kualitatif membahas secara mendalam untuk lebih mengetahui fenomena-fenomena. Metode penelitian kualitatif juga lebih bersifat subjektif dan tidak melalui perhitungan statistik. Penelitian ini bersifat fenomenologis karena untuk menunjukan pada pengalaman subjektif dari berbagai jenis dan tipe subjek yang ditemui dan berusaha memahami arti peristiwa dan kaitan- kaitannya terhadap orang-orang dalam situasi tertentu Moleong (2011). Pendahuluan Kota Bogor mempunyai batik khas Bogor dari tahun 2008, sudah cukup lama tetapi sampai saat ini masih banyak yang tidak mengetahui bahwa Kota Bogor mempunyai batik asli. Bahkan masyarakat asli Bogor pun masih ada yang belum mengetahuinya. Banyak faktor yang menyebabkan Batik Bogor kurang dikenal, karena kurangnya promosi yang dilakukan pemerintah kepada masyarakat. Batik Bogor masih kalah pamor dengan batik- batik khas daerah lain di Jawa Barat. Berdasarkan uraian latar belakang yang telah dipaparkan diatas, maka penulis merumuskan suatu permasalahan yang akan diteliti sebagai berikut: Kota Bogor memiliki sentra yang berlokasi di Kampung Neglasari, Kelurahan Cibuluh, Kecamatan Bogor Utara. Batik Bogor belum banyak dikenal 1. Bagaimana strategi marketing public relations Dinas Pariwisata dan 12 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Tabel : Informan Tabel : Informan Nama Alasan peneliti Willyana sebagai konsumen Batik Bogor Sebagai konsumen Batik Bogor yang telah memakai Batik Bogor. Tabel : Informan Nama Alasan peneliti Willyana sebagai konsumen Batik Bogor Sebagai konsumen Batik Bogor yang telah memakai Batik Bogor. Lofland dalam Moleong (2007), sumber data utama dalam penelitian kualitatif adalah kata-kata dan tindakan, selebihnya adalah data tambahan seperti dokumen dan lain-lain. Berkaitan dengan hal ini maka sumber data yang digunakan dalam penelitian ini berupa : Penentuan subjek penelitian ini menggunakan sampling purposive. Teknik ini mencangkup orang-orang yang diseleksi atas dasar kriteria tertentu yang dibuat peneliti berdasarkan tujuan penelitian. Berikut adalah objek penelitian marketing public relations Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor. Adapun data primer dalam penelitian ini merupakan data yang dikumpulkan dari lapangan dengan melakukan wawancara kepada informan kunci atau subjek penelitian dan pihak-pihak yang terkait dengan perihal strategi marketing public relations terhadap Batik Bogor yang mampu memberikan informasi yang dibutuhkan peneliti. Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? 2. Bagaimana faktor pendukung dan penghambat Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor? Tabel 1: Informan Kunci Tabel 1: Informan Kunci No Nama Alasan Peneliti 1 Undang Sulaeman sebagai Kepala seksi Kemitraan dan Pengembangan Ekonomi Kreatif Bertanggung jawab dalam kepengurusan dibidang ekonomi kreatif, bertugas menyusun strategi , membina para pelaku ekonomi kreatif dan bertanggung jawab penuh dalam segala kegiatan yang dilakukan untuk pengembangan ekonomi kreatif di Kota Bogor 2 Sri Hartati sebagai Perajin Beliau sebagai pelopor Batik Bogor Tabel 1: Informan Kunci No Nama Alasan Peneliti 1 Undang Sulaeman sebagai Kepala seksi Kemitraan dan Pengembangan Ekonomi Kreatif Bertanggung jawab dalam kepengurusan dibidang ekonomi kreatif, bertugas menyusun strategi , membina para pelaku ekonomi kreatif dan bertanggung jawab penuh dalam segala kegiatan yang dilakukan untuk pengembangan ekonomi kreatif di Kota Bogor 2 Sri Hartati sebagai Perajin Beliau sebagai pelopor Batik Bogor Data sekunder dalam penelitian ini merupakan data yang diperoleh dari studi kepustakaan atau buku literatur, publikasi nasional, majalah, internet, database perusahaan dan lain-lain mengenai informasi-informasi yang terkait dengan penelitian. Pencarian data ini perlu dilakukan dengan pertimbangan bahwa data-data tersebut dapat menjadi jembatan dari fakta dan realitas yang terjadi di lapangan sehingga diperoleh validitas data serta pengetahuan yang lebih terhadap objek penelitian. Teknik pengumpulan data kualitatif, maka peneliti melakukan beberapa cara, 13 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik promosi, pengukuran efektivitas promosi dan menentukan anggaran promosi. yaitu, wawancara mendalam dengan cara tanya jawab dengan tatap muka antara pewawancara dengan informan atau orang yang diwawancarai dengan atau tanpa menggunakan pedoman (guide) wawancara, dimana pewawancara dan informan terlibat dalam kehidupan sosial yang relatif lama Bungin (2007). Observasi yaitu, wawancara mendalam dengan cara tanya jawab dengan tatap muka antara pewawancara dengan informan atau orang yang diwawancarai dengan atau tanpa menggunakan pedoman (guide) wawancara, dimana pewawancara dan informan terlibat dalam kehidupan sosial yang relatif lama Bungin (2007). Observasi Penelitian ini menggunakan observasi secara langsung di lokasi penelitian tentang pelaksanaan Strategi Marketing Public Relations, sehingga mendapatkan yang akurat. Mengumpulkan data-data pendukung dari arsip dan dokumen dinas terkait fokus penelitian. Dinas pariwisata dan kebudayaan kota Bogor menentukan target sasaran untuk memperkenalkan Batik Bogor ini kepada masyarakat luas, target sasaran awal yaitu masyarakat Bogor itu sendiri. Dinas Pariwisata dan Kebudayaan Kota Bogor menggunakan media online yaitu melalui media sosial instagram. Sebelum itu Dinas Pariwisata dan Kebudayaan Kota Bogor menentukan pesan apa yang akan disampaikan kepada masyarakat terkait Batik Bogor ini. Tabel 1: Informan Kunci Disparbud membuat sebuah konten yang menarik dan unik serta kekinian. Selain itu promosi yang dilakukan melalui media online, Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan promosi secara langsung dengan langsung berkunjung ke instansi- instansi. Penelitian ini menggunakan observasi secara langsung di lokasi penelitian tentang pelaksanaan Strategi Marketing Public Relations, sehingga mendapatkan yang akurat. Mengumpulkan data-data pendukung dari arsip dan dokumen dinas terkait fokus penelitian. Hasil dan Pembahasan Strategi Marketing Public Relations Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor Pull Strategy Strategy Marketing Public Relations Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor menggunakan strategi push, pull, pass. Strategi untuk menarik masyarakat melalui kegiatan yang akan dilakukan oleh Dinas Pariwisata dan Kebudayaan Kota Bogor yaitu menggelar sebuah event atau pameran Batik Bogor juga melakukan publikasi. Hasil dan Pembahasan Penelitian ini tentang kegiatan memperkenalkan Batik Bogor kepada masyarakat melalui pendekatan marketing public relations. Strategi marketing public relations dibutuhkan bagi suatu perusahaan untuk memperkenalkan produknya agar dapat meningkatkan penjualan melalui komunikasi yang kredibel dalam menyampaikan informasi. Strategy Marketing Public Relations Dinas Pariwisata dan Kebudayaan Kota Bogor dalam memperkenalkan Batik Bogor menggunakan strategi push, pull, pass. Adapun strategi yang dilakukan Dinas Pariwisata dan Kebudayaan Kota Bogor yaitu dengan membuat sebuah event atau pameran Batik Bogor. Sebelum mengadakan sebuah event/pameran Batik Bogor tentu Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan identifikasi ide masukan dari pelaku batik budayawan juga dari organisasi pecinta fashion seperti KCBI, melakukan rapat bersama pemerintah kota yang akan mendukung kegiatan yang dilakukan juga melakukan sosialisasi terkait Batik Bogor bagian dari khazanah batik nusantara dan sosialisasi dilakukan rutin, baik saat akan mengadakan event ataupun tidak. Dalam memperkenalkan Batik Bogor tentu adanya perencanaan dasar disetiap kegiatan yang akan dilakukan. Batik Bogor sendiri belum dikenal oleh masyarakat luas, tentu hal tersebut membutuhkan sebuah strategi untuk memperkenalkan Batik Bogor kepada masyarakat. Ada beberapa strategi yang dilakukan oleh Dinas Pariwisata dan Kebudayaan Kota Bogor yaitu strategi untuk mempromosikan Batik Bogor, melakukan publikasi, membuat sebuah event atau pameran adapun membuat sebuah acara amal dan melakukan sponsorship. Dalam strategi tersebut adanya perencanaan dasar yang perlu di siapkan, mulai dari menentukan target sasaran, merencanakan pesan, memilih media, menentukan durasi Selain itu strategi dalam memperkenalkan Batik Bogor dengan mengadakan acara amal yang dilakukan oleh kampung batik Cibuluh yang di fasilitasi oleh Dinas Pariwisata dan Kebudayaan kota Bogor, tujuan diadakan acara amal tersebut agar masyarakat yang kebal terhadap iklan dapat melihat kampung batik Cibuluh dan Disparbud yang membawa Batik Bogor dan melaksanakan kegiatan mencanting gratis, maka masyarakat secara langsung melihat 14 Jurnal Penelitian Sosial Ilmu Komunikasi Volume 5, nomor 1, April 2021, hlm. 11 - 18 https://journal.unpak.ac.id/index.php/apik e-ISSN: 2656-8306 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik bahwa ada kegiatan mencanting Batik Bogor dan masyarakat pun akan mengetahui bahwa Bogor memiliki batik khas melalui acara amal dengan mencanting gratis bersama anak-anak jalanan. efektif bila dilakukan terus menerus dan juga dengan pesan yang dapat dimengerti oleh masyarakat. Selain promosi melalui media sosial instagram promosi yang dilakukan yaitu kampung Cibuluh yang sudah dicanangkan sebagai kampung batik dijadikan salah satu bagian promosi Batik Bogor. Push Strategy Kegiatan yang dilakukan yaitu adanya program peduli tentu sekaligus untuk memperkenalkan Batik Bogor itu sendiri yaitu mencanting dengan anak-anak jalanan di kampung mongol, dilapas dan dibapas. Kegiatan yang dilakukan akan membuat masyarakat mengenal Batik Bogor melalui kegiatan amal, kegiatan amal tentu membuat masyarakat awam dapat mengenal Batik Bogor melalui kegiatan amal tersebut. selain mengadakan kegiatan amal. Sponsorship sebagai dukungan keuangan atau dukungan lainnya agar pihak yang menerima sponsor dapat merasakan manfaat yang positif, kegiatan sponsorship juga sangat bermanfaat bagi pihak yang memberi dana sponsor. Manfaat utama yang dirasakan oleh pihak sponsor adalah dengan semakin dikenalnya Batik Bogor oleh masyarakat atau audiens yang datang melihat acara. Dinas Pariwisata dan Kebudayaan Kota Bogor juga kampung batik Cibuluh melakukan sponsorship ke setiap acara yang diadakan di Bogor salah satunya yaitu acara MOKA (mojang jajaka) Kota Bogor, sponsorship dilakukan agar Batik Bogor dari brand yang terdapat di kampung batik Cibuluh dapat dikenal oleh masyarakat yang datang ke acara yang dilaksanakan. Selain acara MOKA , disetiap acara Komunitas Cinta Berkain (KCBI) juga menjadi sponsor. Tujuan utama nya adalah untuk memperkenalkan dan mempromosikan Batik Bogor juga untuk memperkenalkan kampung batik Cibuluh kepada masyarakat bahwa kampung batik adalah tempat produksi batik di Bogor. pameran di inakraf dan pameran adiwasta, juga pameran diluar negeri di Vietnam Sesudah pameran di Vietnam pameran dilakukan di Dubai. Adapun publikasi yang dilakukan untuk menyampaikan informasi atau pemberitahuan pada masyarakat luas dengan tujuan informasi tersebut dapat tersampaikan pada orang yang dituju. Dinas Pariwisata melakukan publikasi tentang Batik Bogor tentu untuk memperkenalkan Batik Bogor kepada masyarakat terutama masyarakat Bogor itu sendiri karena masih banyak yang belum mengetahui bahwa Bogor mempunyai batik khas. Maka Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan publikasi melalui media online juga media cetak. Melalui media online yaitu media sosial salah satunya instagram disparbud membuat sebuah postingan dengan gambar Batik Bogor dengan memberikan pesan mengenai Batik Bogor dan juga dilengkapi dengan deskripsi dan kata-kata yang menarik juga mencantumkan macam-macam motif batik. Tujuannya untuk mempengaruhi pengguna instagram agar tertarik dan mengetahui Batik Bogor dengan segala motif nya yang diambil dari ikon kota Bogor. dampak publikasi yang dilakukan melalui media sosial instagram yaitu pengguna instagram yang mengikuti (follow) akun @parbudkotaBogor dan juga @batikpancawati.id menjadi lebih informative mengenai Batik Bogor. Dinas Pariwisata dan Kebudayaan Kota Bogor mengadakan sebuah pameran tentunya pameran tersebut melakukan publikasi juga melalui media online juga media cetak seperti poster, fyler, undangan dll. Push Strategy p Acara atau kegiatan untuk menarik media untuk dieksplorasi oleh media massa yang perhatiannya memberikan arti penting. Untuk media event Dinas Pariwisata dan Kebudayaan Kota Bogor mengadakan Bogor batik run yang akan dilaksanakan dibotani square yang nantinya akan menarik perhatian media dan itu akan berdampak baik bagi Batik Bogor agar lebih dikenal dari informasi yang didapat dari media setelah media menyaksikan event yang diselenggarakan. Selain itu kegiatan yang dilakukan adalah mencanting dengan 100 anak pada saat hari batik juga ada kegiatan mencanting dengan 100 mahasiswa dari Jepang, Nigeria, juga Thailand. Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan upaya untuk mendorong promosi. Dalam mempromosikan Batik Bogor Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan promosi secara face to face, mengadakan event seperti pameran dan event Batik Bogor lainnya, lalu melalui media sosial instagram. Pelaksanaan promosi yang dilakukan meggunakan media online melalui media sosial instagram dengan mengupload foto Batik Bogor dengan motif yang berbeda, dan tentunya batik dari kampung batik Cibuluh. Selain mengupload foto di feeds instagram, promosi ini juga dilakukan dengan membuat instagram story, dengan begitu promosi melalui media online instagram akan muncul dicari para pengguna instagram. Kegiatan promosi melalui media sosial instagram mengenai Batik Bogor oleh Disparbud Kota Bogor bukan suatu hal yang asing lagi, karena semakin hari pengguna internet tentunya pengguna media sosial instagram terus bertambah. Media sosial sebagai medium yang dianggap relatif berbiaya rendah telah menjadi pilihan medium komunikasi pemerintah setempat dibandingkan menggunakan media promosi konvensional Siregar, M., & Hendri, E. (2019). Maka promosi melalui media sosial ini cukup Salah satu cara menarik perhatian masyarakat yang efektif dalam memperkenalkan suatu produk ke pasar adalah dengan mengikuti atau mengadakan pameran. Dinas Pariwisata dan Kebudayaan Kota Bogor membuat sebuah acara fashion show Batik Bogor yang diadakan di Lippo Plaza Keboen Raya Bogor yang mengundang sejumlah pejabat pemerintah Kota. Di saat acara tersebut yang menjadi model peragaan untuk Batik Bogor itu sendiri dari istri wali kota dan wakil wali Kota Bogor juga ambassador Batik Bogor Isabella Tan. Selain itu Batik Bogor pernah diikutsertakan dalam 15 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Strategi yang digunakan oleh Dinas Pariwisata dan Kebudayaan Kota Bogor pass strategy yaitu strategi mempengaruhi. Strategi ini untuk menjangkau masyarakat/konsumen yang makin tebal terhadap iklan. Push Strategy Maka informasi tentang pameran Batik Bogor tersebut akan tersampaikan kepada masyarakat. Tetapi untuk publikasi melalui media cetak tidak semua orang dapat menerima nya karena kesadaran masyarakat yang kurang maka dari itu publikasi yang efektif yaitu publikasi online. Faktor pe penghambat Terdapat faktor pendukung dan faktor penghambat dalam memperkenalkan Batik Pass Strategy Pass Strategy 16 Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Jurnal Penelitian Sosial Ilmu Komunikasi Bogor. Faktor pendukung dari segi sumber daya manusia sudah banyak perajin batik yang kompeten dan sudah bagus dalam mendesain Batik Bogor. Pemerintah bekerjasama dengan Baznas untuk membentuk kampung batik, disediakan sarana tempat yang didukung oleh pemerintah seperti dinas-dinas terkait untuk mereka (perajin batik) agar terus berkarya dengan memproduksi Batik Bogor. Faktor penghambat yang terdapat dalam penelitian ini dari pemasaran nya, karena pasar nya agak susah karena produksi nya mahal jadi mempengaruhi dalam penjualan nya. Juga hambatan dalam publikasi karena tidak semua orang bisa melihat publikasi yang di informasikan secara langsung, maupun melalui media sosial atau melalui media cetak seperti poster, flyer, dan koran. dapat menarik semua kalangan melalui media sosial instagram, poster, flyer dan melalui media cetak seperti koran. Pass Strategy, Strategi ini pun dilakukan dengan mengadakan program peduli yang diadakan oleh komunitas batik yaitu mencanting gratis dengan anak-anak jalanan di kampung mongol, dilapas, dan di bapas. Dinas Pariwisata dan Kebudayaan Kota Bogor menfasilitasi kegiatan yang akan dilakukan oleh perajin batik di kampung batik Cibuluh, lalu sponsorship yang dilakukan pada saat acara Mojang Jajaka Kota Bogor dan setiap dilangsungkannya kegiatan oleh Komunitas Cinta Berkain (KCBI). 2. ( ) Faktor pendukung dan faktor penghambat dalam memperkenalkan Batik Bogor. Faktor pendukung dari segi sumber daya manusia sudah banyak perajin batik yang kompeten dan sudah bagus dalam mendesain Batik Bogor. Pemerintah bekerjasama dengan Baznas untuk membentuk kampung batik, disediakan sarana tempat yang didukung oleh pemerintah seperti dinas-dinas terkait untuk mereka (perajin batik) agar terus berkarya dengan memproduksi Batik Bogor. Faktor penghambat yang terdapat dalam penelitian ini dari pemasaran nya, karena pasar nya agak susah karena produksi nya mahal jadi mempengaruhi dalam penjualan nya. Juga hambatan dalam publikasi karena tidak semua orang bisa melihat publikasi yang di informasikan secara langsung, maupun melalui media sosial atau melalui media cetak seperti poster, flyer, dan koran. Simpulan dan Saran Berdasarkan hasil penelitian dan keabsahan data yang di dapatkan penulis mengenai Analisis Strategi Marketing Public Relations Dinas Pariwisata dan Kebudayaan Kota Bogor Dalam Memperkenalkan Batik Bogor, maka dapat disimpulkan : disimpulkan : 1. Push strategy pada penelitian ini dalam mempromosikan Batik Bogor Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan promosi secara online melalui media sosial seperti instagram. Pull Strategy, strategi menarik perhatian konsumen atau masyarakat, mengadakan event Bogor batik run itu cukup menarik perhatian media juga setiap ada hari besar seperti hari jadi Bogor kegiatan lain yang dilakukan yaitu mencanting dengan 100 anak saat hari batik dan juga mencanting dengan 100 mahasiswa dari Jepang, Nigeria dan Thailand. Selain itu pameran yang dilakukan pernah ikut di Inakraf, saat Gebyar Ekonomi Kreatif Lomba Desain Batik Tulis Bogor juga ada fashion show Batik Bogor. Adapun publikasi yang dilakukan dengan membuat konten mengenai Batik Bogor yang p 1. Push strategy pada penelitian ini dalam mempromosikan Batik Bogor Dinas Pariwisata dan Kebudayaan Kota Bogor melakukan promosi secara online melalui media sosial seperti instagram. Pull Strategy, strategi menarik perhatian konsumen atau masyarakat, mengadakan event Bogor batik run itu cukup menarik perhatian media juga setiap ada hari besar seperti hari jadi Bogor kegiatan lain yang dilakukan yaitu mencanting dengan 100 anak saat hari batik dan juga mencanting dengan 100 mahasiswa dari Jepang, Nigeria dan Thailand. Selain itu pameran yang dilakukan pernah ikut di Inakraf, saat Gebyar Ekonomi Kreatif Lomba Desain Batik Tulis Bogor juga ada fashion show Batik Bogor. Adapun publikasi yang dilakukan dengan membuat konten mengenai Batik Bogor yang Adapun beberapa saran yang dapat penulis temukan untuk menjadikan pertimbangan bagi instansi, sebagai berikut: 1. Kegiatan marketing public relations salah satu nya mengadakan event atau pameran, diusahakan untuk 17 Jurnal Penelitian Sosial Ilmu Komunikasi https://journal.unpak.ac.id/index.php/apik Volume 5, nomor 1, April 2021, hlm. 11 - 18 e-ISSN: 2656-8306 mengadakan nya di outdoor, agar wisatawan yang datang ke Bogor dan masyarakat Bogor itu sendiri mengetahui bahwa ada event atau pameran Batik Bogor. p g 2. Humas Dinas Pariwisata dan Kebudayaan Kota Bogor dalam melakukan publikasi mengenai Batik Bogor ini harus lebih aktif memperkenalkan dan mempromosikan Batik Bogor melalui media yang disesuaikan dengan era atau perkembangan zaman. Referensi Bungin, Burhan. 2007. Penelitian Kualitatif. Jakarta : Kencana. Duncan, Tom. 2010. Integrated Marketing Communication: Using Advertising and Promotions and Build Brands. New York : Mc Graw Hill Hermawan. 2013. Komunikasi Pemasaran. Jakarta: Erlangga. Kasali, R. (2003). Manajemen Public Relations: Konsep dan Aplikasinya di Indonesia. Jakarta: Grafiti. Kotler. (2010). Manajemen Pemasaran. Jakarta: Erlangga. Kotler. (2010). Manajemen Pemasaran. Jakarta: Erlangga. Moleong, Lexy J. (2007). Metodologi Penelitian Kualitatif Edisi Revisi. Bandung: PT. Remaja Rosdakarya. Moleong, L. J. (2011). Metode Penelitian Kualitatif Edisi Revisi. Bandung: PT. Remaja Rosdakarya. Siregar, M., & Hendri, E. (2019). Komunikasi Primer dan Sekunder City Branding. Jurnal Sosial Humaniora, 10(1), 11-18. 18
https://openalex.org/W2086853203	https://europepmc.org/articles/pmc3835481?pdf=render	English	null	Irisin, Two Years Later	International journal of endocrinology	2,013	cc-by	7,343	Marta G. Novelle,1,2 Cristina Contreras,1,2 Amparo Romero-Picó,1,2 Miguel López,1,2 and Carlos Diéguez1,2 1 Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigaci´on Sanitaria (I 15782 Santiago de Compostela, Spain 2 CIBER Fisiopatolog´ıa de la Obesidad y Nutrici´on (CIBERobn), 15706 Santiago de Compostela, Spain Correspondence should be addressed to Marta G. Novelle; marta.garrido@usc.es and Carlos Di´eguez; carlos.dieguez@usc.es Correspondence should be addressed to Marta G. Novelle; marta.garrido@usc.es and Carlos Di´eguez; carlos.d Received 28 June 2013; Accepted 1 October 2013 Academic Editor: Paolo de Girolamo Copyright © 2013 Marta G. Novelle 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. In January 2012, Bostr¨om and colleagues identified a new muscle tissue secreted peptide, which they named irisin, to highlight its role as a messenger that comes from skeletal muscle to other parts of the body. Irisin is a cleaved and secreted fragment of FNDC5 (also known as FRCP2 and PeP), a member of fibronectin type III repeat containing gene family. Major interest in this protein arose because of its great therapeutic potential in diabetes and perhaps also therapy for obesity. Here we review the most important aspects of irisin’s action and discuss its involvement in energy and metabolic homeostasis and whether the beneficial effects of exercise in these disease states could be mediated by this protein. In addition the effects of irisin at the central nervous system (CNS) are highlighted. It is concluded that although current and upcoming research on irisin is very promising it is still necessary to deepen in several aspects in order to clarify its full potential as a meaningful drug target in human disease states. Hindawi Publishing Corporation International Journal of Endocrinology Volume 2013, Article ID 746281, 8 pages http://dx.doi.org/10.1155/2013/746281 Hindawi Publishing Corporation International Journal of Endocrinology Volume 2013, Article ID 746281, 8 pages http://dx.doi.org/10.1155/2013/746281 Review Article Irisin, Two Years Later 2. Irisin, A Bridge between Exercise and Thermogenesis 2.1. First Experimental Studies. Recently, Spielgman’s group described that transgenic PGC-1𝛼mice had greater levels of fibronectin type III domain containing (FNDC5) than wild- type mice [20]. FNDC5 (also known FRCP2 and PeP) is a type of transmembrane protein cloned by two groups in 2002. It has a signal peptide, two fibronectin domains and one hydrophobic domain inserted in the cell membrane [22, 23]. In fact, at present some authors question if FNDC5 might be a transmembrane receptor [24]. FNDC5 is proteolytically cleaved and secreted. Western blot of media fractions of cells overexpressing FNDC5 with antibodies against wild- type Fndc5 identified multiple bands; from 32 kDa to 20 kDa [20]. However, several aspects regarding proteolysis of this protein were not fully clarified yet. So, it seems that these possible discrepancies in molecular weight might be due to glycosylation in the culture media, while this is not observed in plasma mice. Moreover, the theoretically soluble secreted form, named irisin, would have a molecular weight of 12 kDa [20, 25] (Figure 1). A remarkable aspect about irisin is that the amino acid sequence is 100% identical among most mammalian species, which suggests a highly conserved function [20, 26]. p y Other studies have tried to elucidate the irisin role and other myokines in different physiological conditions. When male rats were subjected to calorie restriction (CR; ∼60% ad libitum) there were no significant diet-related differences in plasma levels of myonectin, myostatin, or irisin, although there were significant changes in fat and lean mass, and also in insulin resistance [31]. These results may indicate that alterations in plasma concentration of these proteins are not essential for the CR-related improvement in insulin sensitivity in rats; however, it does not rule out that these plasma proteins may be relevant for some of caloric restric- tion’s metabolic effects. On the other hand, S´anchez and collaborators have studied the possible effects of free fatty acids (FFA) alone and combined with adrenaline and AICAR (an activator of AMPK that acts as an exercise mimetic precursor) in the production of the myokines IL6, IL15, and irisin in mouse muscle cells in vitro [32]. They observed that FFA, adrenaline, and AICAR have a great influence in the IL6 expression and secretion, a little inhibitory effect on IL15 expression and almost no effect on the expression of FNDC5. International Journal of Endocrinology There is an extensive literature about different exercise-related signals that can reg- ulate the expression and/or secretion of the diverse myokines [4, 5, 27, 28]. In this context, it has recently been published that there is a close interaction between irisin and myo- statin. Myostatin, besides being a critical autocrine/paracrine inhibitor of skeletal muscle growth, has been shown to play an important role in metabolism [29]. In fact, it has been described as myostatin-knockout mice (Mstn−/−) that show an increase muscle mass and a concomitant reduction of fat mass. Moreover, these mice show WAT with characteristics of BAT, an effect mediated by the AMPK-PGC1𝛼-FNDC5 pathway in muscle [30]. 2.2. Interplay with Other Myokines. There is an extensive literature about different exercise-related signals that can reg- ulate the expression and/or secretion of the diverse myokines [4, 5, 27, 28]. In this context, it has recently been published that there is a close interaction between irisin and myo- statin. Myostatin, besides being a critical autocrine/paracrine inhibitor of skeletal muscle growth, has been shown to play an important role in metabolism [29]. In fact, it has been described as myostatin-knockout mice (Mstn−/−) that show an increase muscle mass and a concomitant reduction of fat mass. Moreover, these mice show WAT with characteristics of BAT, an effect mediated by the AMPK-PGC1𝛼-FNDC5 pathway in muscle [30]. International Journal of Endocrinology International Journal of Endocrinology is mainly expressed in BAT, it is also expressed at higher levels in red, oxidative muscle. In fact, its expression is increased by exercise in mice, in rats, and in human beings [15]. Exercise rapidly and robustly increases the expres- sion of PGC1𝛼, but this effect is transient as both mRNA and protein levels of PGC1𝛼quickly revert to preexercise values [16]. Exercise also activates AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism. AMPK directly phosphorylates PGC1𝛼, which is required for PGC1𝛼-dependent induction of the PGC1𝛼promoter [17]. While brief training produces only a transient rise in PGC1𝛼, endurance training results in persistent PGC1𝛼elevation [18]. Moreover, mice with transgenically increased PGC-1𝛼in muscle showed improved metabolic responses as age related obesity and insulin insensitivity [19]. When the adipose tissue of these transgenic mice was analysed, it was observed that subcutaneous fat inguinal had significantly increased thermogenic gene program. These “brite” (brown-in-white) adipocytes display several classical brown adipocyte charac- teristics, as elevated levels of UCP1 mRNA and protein [20]. Further, other reports showed that exercise also enhances certain brown adipocyte-specific gene expression in the BAT, as well as white adipose tissue (WAT), suggesting that exercise training may induce important alteration in BAT and/or BAT-like phenotypic changes in WAT [21]. In this context it has been proposed that irisin, a recently discovered myokine, may be the molecule that links exercise with increased thermogenesis. In fact, irisin is named for Iris, the Greek goddess who served as courier among the Gods [20]. FNDC5 to primary subcutaneous white adipocytes during differentiation a great increase in oxygen consumption was observed which suggests higher energy expenditure. More- over, the increase in uncoupled respiration was accompanied by an important induction of UCP1 mRNA and other known brown fat genes. However, genes characteristic to WAT were downregulated. Surprisingly, FNDC5 showed almost no effects on the classical brown fat cells isolated from the interscapular depot [20].h FNDC5 to primary subcutaneous white adipocytes during differentiation a great increase in oxygen consumption was observed which suggests higher energy expenditure. More- over, the increase in uncoupled respiration was accompanied by an important induction of UCP1 mRNA and other known brown fat genes. However, genes characteristic to WAT were downregulated. 1. Introduction Since human brown adipose tissue (BAT), especially in adults, was rediscovered several years ago by using positron emission tomography (PET) [6–9], it has been postulated as a major candidate for the treatment of obesity. This is based on the fact that brown adipose cells can dissipate energy in the form of heat leading to weight loss. This process takes place through a specialized mitochondrial protein called uncoupling protein 1 (UCP1). The uncoupling activity of UCP1 is explained by its ability to transport protons across the inner mitochondrial membrane, avoiding ATP synthesis and dissipating energy as heat [10]. Regulation of UCP1 is mainly at transcriptional level, where peroxisome proliferator-activated receptor Υ coactivator 1𝛼(PGC1𝛼) plays a key role [11]. Obesity is at present the most common nutritional disease in industrialized countries, constituting a priority health problem. It is associated with the development of cardiovas- cular disease, diabetes mellitus type II, increased incidence of certain forms of cancer, and respiratory complications from other diseases, which leads to higher rates of mortality and morbidity, reducing directly or indirectly the quality and life expectancy of sufferers [1, 2]. Lifestyle modification, specifically changes in diet, physical activity, and exercise, currently continues to be the best option for treatment of obesity. In this sense, the benefits of exercise have been extensively documented [3]. Moreover, it has recently been reported that especially during or immediately after physi- cal activity, skeletal muscle releases into circulation several hormones. These hormones, named myokines, can influence metabolism and modify cytokine production in different tissues and organs. On the basis of this, the concept of skeletal muscle must be reconsidered and being truly considered as an endocrine organ [4, 5]. Studies in immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC1𝛼corroborated their importance in thermogenesis [12]. Another important char- acteristic is its role in mitochondrial biogenesis; in fact the increased expression of PGC1𝛼is parallel to increased mitochondrial DNA and gene expression of OXPHOS system (oxidative phosphorylation) in BAT [13, 14]. Although PGC1𝛼 2 2 International Journal of Endocrinology Surprisingly, FNDC5 showed almost no effects on the classical brown fat cells isolated from the interscapular depot [20].h is mainly expressed in BAT, it is also expressed at higher levels in red, oxidative muscle. In fact, its expression is increased by exercise in mice, in rats, and in human beings [15]. Exercise rapidly and robustly increases the expres- sion of PGC1𝛼, but this effect is transient as both mRNA and protein levels of PGC1𝛼quickly revert to preexercise values [16]. Exercise also activates AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism. AMPK directly phosphorylates PGC1𝛼, which is required for PGC1𝛼-dependent induction of the PGC1𝛼promoter [17]. While brief training produces only a transient rise in PGC1𝛼, endurance training results in persistent PGC1𝛼elevation [18]. Moreover, mice with transgenically increased PGC-1𝛼in muscle showed improved metabolic responses as age related obesity and insulin insensitivity [19]. When the adipose tissue of these transgenic mice was analysed, it was observed that subcutaneous fat inguinal had significantly increased thermogenic gene program. These “brite” (brown-in-white) adipocytes display several classical brown adipocyte charac- teristics, as elevated levels of UCP1 mRNA and protein [20]. Further, other reports showed that exercise also enhances certain brown adipocyte-specific gene expression in the BAT, as well as white adipose tissue (WAT), suggesting that exercise training may induce important alteration in BAT and/or BAT-like phenotypic changes in WAT [21]. In this context it has been proposed that irisin, a recently discovered myokine, may be the molecule that links exercise with increased thermogenesis. In fact, irisin is named for Iris, the Greek goddess who served as courier among the Gods [20]. p p This evidence opened up some questions about the physiological role of irisin. In the same study, in vivo, it was demonstrated that injection intravenously of adenoviral vectors expressing full-length Fndc5 resulted in Ucp1 mRNA increased in the subcutaneous depot. Moreover, a moderate increase irisin blood levels caused a significant improvement in energy expenditure, body weight, and insulin resistance in mice that were fed a high fat diet. Finally, it was demonstrated that irisin was required for the effect of exercise in the brown- ing of subcutaneous white fat and concluded that the rise in irisin is mediated by augmented concentrations of PGC1𝛼 in muscle, while PPAR-𝛼(peroxisome proliferator-activated receptor-𝛼) acts as downstream target of this hormone [20]. 2.2. Interplay with Other Myokines. 2. Irisin, A Bridge between Exercise and Thermogenesis Thus, it would be possible that more signals may be required in vivo for inducing FNDC5 expression. In this sense, recent evidence using human rhabdomyosarcoma cells showed that treatment for 24 and 48 hours with omega 3 fatty acids significantly induced irisin expression [33]. Finally, it has also been found that just as FNDC5, heart-derived natri- uretic peptides activate white adipose thermogenic programs [34]. Taken together, these results may suggest that tissues such as skeletal and heart muscle, involved in high energy- expending activity, send signals to adipose tissue [35, 36]. obese rats with no functional leptin receptor showed signif- icantly diminished levels, while DIO (diet induce obesity) rats showed a significant increase. Ultimately, all these results suggest an interactions between muscle and adipose tissue interaction a regulatory feedback mechanism. In this same context, Roberts et al. showed that obese/diabetic prone Otsuka Long-Evans Tokushima Fatty (OLETF) rats have more muscle expression FNDC5 than lean Long Evans Tokushima Otsuka (LETO); however, LETO rats have higher circulating irisin levels. The authors also observed that triceps FNDC5 mRNA expression was corre- lated with fat mass and with plasma leptin; however, in vitro leptin treatment had no effect on FNDC5 mRNA expression in myotubes [37]. Given that the effect of leptin treatment depends on endogenous levels of this hormone and on the physiological state [38], many studies are still needed to determinate a possible interaction between leptin and irisin in the so-called muscle-adipose tissue axis. 2.3. Irisin Is Also an Adipokine. Current data by Roca-Rivada and coworkers proposed that irisin is not only secreted by muscle tissue. In fact, they demonstrated that irisin is a new adipokine with an important autocrine and endocrine function. Moreover, they showed that FNDC5/irisin has a different pattern of secretion depending on the anatomical location of adipose tissue. Thus, subcutaneous adipose tissue secretes more much FNDC5/irisin than visceral adipose tissue, reflecting one more time that visceral fat is more implicated in metabolic complications, while subcutaneous fat has a possible beneficial role. They also showed that short- term periods of exercise training induced FNDC5 secretion by WAT, that this secretion was significantly reduced in fasting animals, and that WAT of obese animals had an increase secretion of this hormone suggesting a type of resistance [25]. Another interesting feature, also reported by those authors, indicates that FNDC5/irisin has a secretion profile similar to other adipokines like leptin. 2. Irisin, A Bridge between Exercise and Thermogenesis In fact, the authors only found that FNDC5 had a tendency to be reduced with FFA and AICAR at isolated specific time Bostr¨om and colleagues demonstrated that irisin has potent effects on the browning of certain white adipose tissues, both in culture and in vivo. So, when they applied 3 International Journal of Endocrinology 3 domain C N domain C N Intracellular Extracellular Signal peptide C N FNDC5 IRISIN Fibronectin III domain Fibronectin III domain Proteolytic cleaved 12kDa Hydrophobic Hydrophobic Figure 1: Expression of FNDC5 (fibronectin type III domain containing 5), also known as FRCP2 and PeP, is stimulated in muscle by PGC1-𝛼 in response to exercise. It is a signal peptide with two fibronectin domains in its amino (N)-terminal part and a hydrophobic domain inserted in the lipidic bilayer at carboxy (C)-terminal domain. The first 29 aa of the mouse FNDC5 are a signal peptide, followed immediately by the single FNIII domain of 94 aa. The next 28 aa are of unknown structure and function and contain the putative cleavage site for irisin. This is followed by a 19 aa transmembrane domain and a 39 aa cytoplasmic domain. FNDC5 is thus a type I transmembrane protein with its FNIII domain extracellular, similar to some cytokine receptors. This structure is synthesized as a type I membrane protein and followed by proteolytic cleavage realising amino (N)-terminal part of the protein into the extracellular to circulation. Intracellular Extracellular FNDC5 Proteolytic cleaved domain C N Hydrophobic IRISIN N Figure 1: Expression of FNDC5 (fibronectin type III domain containing 5), also known as FRCP2 and PeP, is stimulated in muscle by PGC1-𝛼 in response to exercise. It is a signal peptide with two fibronectin domains in its amino (N)-terminal part and a hydrophobic domain inserted in the lipidic bilayer at carboxy (C)-terminal domain. The first 29 aa of the mouse FNDC5 are a signal peptide, followed immediately by the single FNIII domain of 94 aa. The next 28 aa are of unknown structure and function and contain the putative cleavage site for irisin. This is followed by a 19 aa transmembrane domain and a 39 aa cytoplasmic domain. FNDC5 is thus a type I transmembrane protein with its FNIII domain extracellular, similar to some cytokine receptors. This structure is synthesized as a type I membrane protein and followed by proteolytic cleavage realising amino (N)-terminal part of the protein into the extracellular to circulation. points. 2. Irisin, A Bridge between Exercise and Thermogenesis Moreover, it is suggested that this hormone might be implicated in the regulation of circulating FNDC5/irisin levels. In fact, Zucker International Journal of Endocrinology 4 International Journal of Endocrinology adipocyte metabolism via several intermediary synapses in the medulla and spinal cord, an interesting idea that still requires to be confirmed. increase in irisin levels occurs in states where more energy is needed, such as untrained individuals, while among trained individuals it is not necessary [47]. In the same direction, a recent study confirms that neither longer-term nor single exercise markedly increases skeletal muscle FNDC5 expres- sion or serum irisin [50].f i Supporting the role of FNDC5/irisin in the nervous system, it should be noted another study where it is demon- strated that FNDC5 is required for the adequate neural differentiation of mouse embryonic stem cells (mESCs) [43]. The authors observed that both Fndc5 knockdowns in mESCs during their differentiation after postneuronal progenitor formation and the neuronal differentiation were reduced. Finally, Moon et al. showed that hippocampal neurogenesis is regulated by irisin in a dose-dependent manner. So, while physiological concentrations of irisin (5–10 nmol/L) had no effect on mouse H19-7 hippocampal neuronal cells pro- liferation, pharmacological concentrations (50–100 nmol/L) increased proliferation when they were compared to control. This increase seems to occur trough signal transducer and activator of transcription (STAT)3 but not AMPK and/or extracellular signal-regulated kinase (ERK) signalling path- ways [44]. It seems, hence, that exercise might have an effect on irisin levels depending on physiological condition. In this sense, a new study describes that patients subjected to hemodialysis seem to have lower plasma irisin than healthy subjects and also show exercise training resistance; so despite increasing muscle mass, they not have higher irisin levels [51]. 4.2. Metabolic Diseases. When analyzing the correlation between body max index (BMI) and irisin levels, differences were also found. Some studies observed a positive correlation with BMI [47, 52, 53], while other reported null [49] or even a negative correlation [48]. It would be necessary a deeper investigation in this field, and a possible resistance to this protein should be characterized, as animal studies suggest [25]. In addition, bariatric surgery-induced weight loss has been reported to decrease irisin levels, independently of BMI [47]. However the functional significance of this finding needs further exploring. 4. Irisin, Studies in Humans 4.1. Human Exercise Gene. As stated above, irisin has a highly conserved function, and as in rodents, in humans this hormone is also predominantly expressed in muscle [47]. While, available data indicates that this is the main source of production, it was also found that both subcutaneous and visceral adipose tissue expressed and secreted FNDC5/irisin [25, 48]. On other hand, circulating irisin was detected in the serum or plasma of all subjects studied, whereas circulating FNDC5 was detected in only a minority of the subjects [47], which could be explained by a different processing in a minority group of humans.h p g In this same context, another controversy has been reported. The study of single nucleotide polymorphisms (SNPs) in the human Fndc5 locus, encoding the irisin precursor, showed that a common genetic variation in this locus determines insulin sensitivity [56]. Moreover, data from human myotubes revealed a negative association between FNDC5 expression and in vivo measures of insulin sensitivity. This result appears conflicting with the mouse data from Bostr¨om et al. who reported reduced insulin resistance in high fat-fed mice after adenoviral Fndc5 overexpression [20]. Considering the association of DMT2 and cardiovascular disease, a role for irisin is also tempting to be speculated. In this sense the FNDC5 expression in a skeletal muscle biopsy from heart failure (HF) patients, it was observed that this expression relates to functional capacity in a human HF and that a decrease in FNDC5 expression might reduce aerobic performance in HF patients [57]. Throughout the past two years, several studies in humans have tried to clarify the role of FNDC5/irisin in physiological conditions and in disease states. Spielgman’s group showed that endurance exercise training for 10 weeks in healthy adult humans increased plasma irisin levels compared to the baseline state [20]; however, there are some discrepancies about this. While Huh et al. also observed that circulating irisin levels were significantly upregulated 30 min after acute exercise [47], another study have questioned those results. So, other study has not been able to reproduce FNDC5 gene activation by aerobic exercise in younger subjects or in a resistance training study in 20–80 year olds [49]. These authors question therefore whether irisin is a human exercise gene. International Journal of Endocrinology Overall this evidence suggests a central role for irisin, In this regard, considering that the hippocampus is one of the principal regions affected by Alzheimer’s disease and that exercise causes neurogenesis in humans reducing risk of Alzheimer’s [45], Parkinson’s, and some other neurode- generative diseases [26, 46], irisin might be the link between exercise and healthy brain. Another interesting question that needs to be addressed is whether irisin may be expressed and play a role in other brain areas involved in the regulation of energy balance, such as the hypothalamus and the brainstem. Similarly, it has been established by some groups a relation between diabetes mellitus type 2 (DMT2) and irisin levels, although it is also reported that irisin expression is not related to diabetes status in humans [49]. Most studies show lower irisin levels in patients with DMT2 [48, 54, 55]. Fernandez-Real’s group suggests that a lesser production of irisin in muscle/adipose tissue in obese and patients with DMT2 could be responsible of the obesity-associated lower brown or beige adipocytes in human adipose tissue. So, they consider increasing irisin levels and browning adipose tissue as a potential target for metabolic diseases’ treatment [48]. 3. Irisin, Potential Roles in the Central Nervous System Besides interaction between skeletal muscle and adipose tis- sue, it has been described that FNDC5/irisin might have a role in the central nervous system. In fact, it has already described previously that PGC1-𝛼, an upstream of FNDC5, benefits tissues that do not have a primary metabolic function, such as the brain [39–41]. In this context, immunohistochemical studies have recently revealed that rat and mice cerebellar Purkinje cells expressed irisin and also FNDC5 [42]. Fur- thermore, the same authors hypothesize about a novel neural pathway, where irisin produced in cerebellum might regulate 4. Irisin, Studies in Humans Thermogenesis Heat P changes BAT-like phenotypes Figure 2: Skeletal muscle releases to circulation several hormones denomined myokines acting as endocrine organ. Thus during exercise PGC1𝛼is activated inducing FNDC5 release which is cleaved to irisin. Irisin can act on different tissues, thereby brown adipose tissue activates UCP1 in mitochondria triggering transport protons chain in the mitochondrial membrane, resulting ATP increased and dissipating energy in form of heat. This process increases energy expenditure, reduces body weight, and improves metabolic parameters such as insulin sensitivity. Irisin on white adipose tissue stimulates BAT-like phenotypes changes, increasing PGC1𝛼expression and thereby UCP1 and oxygen consumption while decreases WAT genes, process in which WAT stops behaving as energy reservoir for to use fat as energy source as in BAT, process named browning. For all of this, irisin has been proposed as a possible novel treatment in diabetes and obesity. Other target of irisin is nervous system where preliminary studies suggest that it could act on adipocyte metabolism through a novel neural pathway and on the other hand irisin induces neural proliferation and adequate neural differentiation, so it could also be a therapeutic target for neurodegenerative diseases such as Alzheimer or Parkinson. Another disease with altered energy expenditure and with high prevalence of metabolic imbalance and abnormal energy homeostasis is also chronic kidney disease (CKD). It was observed that patients with CKD have lower irisin levels at rest, independently of high-density lipoprotein cholesterol levels. The mechanism underlying the decrease in irisin in CKD is unknown, even though it seems that indoxyl sulphate, which is a protein-bound uremic toxin, decreases FNDC5 expression in skeletal muscle cells and irisin level in the cell culture medium [58]. Authors consider that these results show good evidence on how uremia may affect irisin levels. Although this study has some limitations, it is suggested that irisin may be a novel therapeutic agent for treating metabolic diseases in CKD patients. suggests that in a population of postmenopausal women with BMI between 24 and 45, irisin levels do not correlate with 24 h energy expenditure (EE); however, for a subpopulation with EE greater than predicted, irisin levels and EE are highly correlative [60]. Similar to physical activity, drugs might also increase irisin levels and thus affect lipid metabolism and improve risk among dyslipidemic and/or obesity individuals. 4. Irisin, Studies in Humans Given recent data, everything seems to indicate that between these drugs, statins could have an important role in this sense [61]. In this context, recently, Gouni-Berthold and collaborators have described that simvastatin, a hypolipidemic drug member of the statins, increases irisin concentrations both in vivo as in vitro [62]. Although it could be postulated that this increase could be beneficial, for example, by influencing adipose tissue metabolism and insulin resistance, it will be necessary to determine if irisin levels are result of myocyte damage or/and a mechanism of statin-induced cellular stress protection [62]. 4. Irisin, Studies in Humans These discrepancies might be explained as that an Circulating irisin has been also found to be directly asso- ciated with muscle mass and estradiol levels and inversely associated with age in middle-aged women. Also it is negatively correlated with age, insulin, cholesterol, and adiponectin levels [47, 52, 58], as well as intrahepatic triglyc- eride contents in obese adults [59]. While, another paper International Journal of Endocrinology 5 Physical activity BAT Thermogenesis Heat WAT Irisin Cleaved Browning Thermogenesis Heat changes Nervious system Skeletal muscle Mitochondria Novel neural Irisin as treatment of neurodegenerative diseases such as ∙Adequate neural differentiation ∙↑Proliferation pathway??? BAT-like ↑UCP1 ↓WAT genes UCP1 Transport protons chain ↑Energy expenditure ↑Insulin sensitivity ↓Body weight Irisin as treatment of diabetes and obesity? PGC1𝛼 FNDC5 ↑ATP PGCC1𝛼 ↑PGCC1𝛼 Alzheimer or Parkinson? phenotypes ↑Oxygen consumption Figure 2: Skeletal muscle releases to circulation several hormones denomined myokines acting as endocrine organ. Thus during exercise PGC1𝛼is activated inducing FNDC5 release which is cleaved to irisin. Irisin can act on different tissues, thereby brown adipose tissue activates UCP1 in mitochondria triggering transport protons chain in the mitochondrial membrane, resulting ATP increased and dissipating energy in form of heat. This process increases energy expenditure, reduces body weight, and improves metabolic parameters such as insulin sensitivity. Irisin on white adipose tissue stimulates BAT-like phenotypes changes, increasing PGC1𝛼expression and thereby UCP1 and oxygen consumption while decreases WAT genes, process in which WAT stops behaving as energy reservoir for to use fat as energy source as in BAT, process named browning. For all of this, irisin has been proposed as a possible novel treatment in diabetes and obesity. Other target of irisin is nervous system where preliminary studies suggest that it could act on adipocyte metabolism through a novel neural pathway and on the other hand irisin induces neural proliferation and adequate neural differentiation, so it could also be a therapeutic target for neurodegenerative diseases such as Alzheimer or Parkinson. Physical activity BAT Thermogenesis Heat WAT Irisin Cleaved Browning Thermogenesis Heat changes Nervious system Skeletal muscle Mitochondria Novel neural Irisin as treatment of neurodegenerative diseases such as ∙Adequate neural differentiation ∙↑Proliferation pathway??? BAT-like ↑UCP1 ↓WAT genes UCP1 Transport protons chain ↑Energy expenditure ↑Insulin sensitivity ↓Body weight Irisin as treatment of PGC1𝛼 FNDC5 ↑ATP PGCC1𝛼 ↑PGCC1𝛼 Alzheimer or Parkinson? phenotypes ↑Oxygen consumption Skeletal muscle Nervious system ∙Adequate neural differentiation ∙↑Proliferation Cleaved Novel neural pathway??? International Journal of Endocrinology 6 6 possible treatment for diabetes and perhaps also therapy for obesity. Moreover, it also was considered a possibility to treat patients with Alzheimer’s, Parkinson’s, and some other neurodegenerative diseases. However, new studies have started to question the initial expectations [24, 63]. So, while clear-cut data have been reported in rodents, the thermogenic effect of irisin in humans remains controversial, and it is not clear if exercise has an impact on irisin levels [49]. In fact, recently, Raschke and coworkers described that neither FNDC5 gene is activated by contraction in humans nor has effect on “brite” differentiation of human preadipocytes [63]; even they propose that irisin function for mice is lost in humans. Thus, it seems obvious that further studies are needed to elucidate, in depth, this field. [2] N. S. Mitchell, V. A. Catenacci, H. R. Wyatt, and J. O. Hill, “Obesity: overview of an epidemic,” Psychiatric Clinics of North America, vol. 34, no. 4, pp. 717–732, 2011. [3] B. Strasser, “Physical activity in obesity and metabolic syn- drome,” Annals of the New York Academy of Sciences, vol. 1281, pp. 141–159, 2013. [4] B. K. Pedersen, T. C. A. ˚Akerstr¨om, A. R. Nielsen, and C. P. Fischer, “Role of myokines in exercise and metabolism,” Journal of Applied Physiology, vol. 103, no. 3, pp. 1093–1098, 2007. [5] B. K. Pedersen and M. A. Febbraio, “Muscles, exercise and obes- ity: skeletal muscle as a secretory organ,” Nature Reviews Endocrinology, vol. 8, pp. 457–465, 2012. [6] A. M. Cypess, S. Lehman, G. Williams et al., “Identification and importance of brown adipose tissue in adult humans,” New England Journal of Medicine, vol. 360, no. 15, pp. 1509–1517, 2009. i First more studies would be necessary to determinate what the precise role of different forms of FNDC5/irisin is and if there is a different mechanism of proteolysis as it already was suggested [47]. On the other hand, it is absolutely necessary to characterize the receptor and the signalling pathway, which will allow a better understanding of irisin function. Just as with other hormones it seems to be a tolerance or resistance mechanism to irisin [25, 60]. So, the factors that contribute to irisin tolerance and/or resistance also would be defined. Similarly more extensive studies, with different cohorts, assessing genetic variations in the irisin gene and its relationships to obesity and associated comorbidities across life span are eagerly awaited. [1] P. G. Kopelman, “Obesity as a medical problem,” Nature, vol. 404, no. 6778, pp. 635–643, 2000. Acknowledgments [14] Z. Wu, P. Puigserver, U. Andersson et al., “Mechanisms controll- ing mitochondrial biogenesis and respiration through the ther- mogenic coactivator PGC-1,” Cell, vol. 98, no. 1, pp. 115–124, 1999. The research leading to these results has received fund- ing from the European Community’s Seventh Frame- work Programme (FP7/2007–2013) under Grant agreement no. 281854—the ObERStress European Research Council Project(ML) and no. 245009—the Neurofast project (ML and CD), and Xunta de Galicia (ML: 10PXIB208164PR and 2012-CP070), Instituto de Salud Carlos III (ISCIII) (ML: PI12/01814), MINECO were co-funded by the FEDER Pro- gram of EU (CD:BFU2011-29102). 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International Journal of Endocrinology Another important aspect that we need to consider is that human BAT is closely related to rodent beige fat, rather than classical BAT; so if we want to study the irisin effect in human BAT, a rodent model with beige fat would be necessary [26]. Intensive research efforts are needed to use BAT as a target organ for treatment of metabolic diseases. [7] J. Nedergaard, T. Bengtsson, and B. Cannon, “Unexpected evi- dence for active brown adipose tissue in adult humans,” Ameri- can Journal of Physiology, vol. 293, no. 2, pp. E444–E452, 2007. [8] W. D. van Marken Lichtenbelt, J. W. Vanhommerig, N. M. Smul- ders et al., “Cold-activated brown adipose tissue in healthy men,” New England Journal of Medicine, vol. 360, no. 15, pp. 1500–1508, 2009. [9] K. A. Virtanen, M. E. Lidell, J. Orava et al., “Functional brown adipose tissue in healthy adults,” New England Journal of Medicine, vol. 360, no. 15, pp. 1518–1525, 2009. [10] B. Cannon and J. 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https://openalex.org/W4322621526	https://ojs.uma.ac.id/index.php/biolink/article/download/7918/4767	English	null	ANALYSIS OF NEW PRIMER PAIR CANDIDATES OF rbcL GENE FOR IDENTIFICATION OF MICROALGAE SCENEDESMACEAE	Deleted Journal	2,023	cc-by	4,105	Department of Biology Faculty of Science and Technology, Universitas Islam Negeri Syarif Hidayatullah Jakarta, Indonesia Submited : 22-08-2022; Reviewed : 09-01-2023; Accepted : 19-01-2023 Corresponding author: E-mail : meggapikoli@uinjkt.ac.id How to Cite: Pikoli, M.R. (2023). Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae, BioLink: Jurnal Biologi Lingkungan, Industri dan Kesehatan, (9) (2): 163- 173 Keywords: rbcL gene; Scenesdesmaceae; Primer design; Molecular identification Megga Ratnasari Pikoli Department of Biology Faculty of Science and Technology, Universitas Islam Negeri Syarif Hidayatullah Jakarta, Indonesia ANALYSIS OF NEW PRIMER PAIR CANDIDATES OF rbcL GENE FOR IDENTIFICATION OF MICROALGAE SCENEDESMACEAE Megga Ratnasari Pikoli* BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023) ISSN: 2356- 458X (print) ISSN: 2597-5269 (online) DOI: 10.31289/biolink.v9i1.7918 BioLink Jurnal Biologi Lingkungan, Industri, Kesehatan Available online http://ojs.uma.ac.id/index.php/biolink BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023) ISSN: 2356- 458X (print) ISSN: 2597-5269 (online) DOI: 10.31289/biolink.v9i1.7918 BioLink Jurnal Biologi Lingkungan, Industri, Kesehatan Available online http://ojs.uma.ac.id/index.php/biolink BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023) ISSN: 2356- 458X (print) ISSN: 2597-5269 (online) DOI: 10.31289/biolink.v9i1.7918 Abstract Scenedesmaceae is one of the microalgae groups that has been widely studied as promising biodiesel feedstock. Its morphological identification is often confused by environmental changes, so it requires molecular identification as well. The current study aimed to obtain primer pair candidates that identify the Scenedesmaceae based on the rbcL gene. The research used bioinformatics tools, which harvested rbcL protein sequence data, performed multiple sequence alignments, and designed primers based on conserved and less-conserved regions. The best left and right primers selected based on sequence length, melting temperature, 3' end stability, number of hairpins, and self-dimers, were paired, and three candidates were obtained. The three pairs were examined based on melting temperature difference, number of hetero- dimers, length of amplified nucleotide product, number of hits, and number of genera captured from the GenBank. Sce-16 (F, 5'-TGGTCGTGCTGTTTATGAATGT-3' and 1_RL, 5'-TGCCAAACATGAATACCACCA-3'), which is back-translated according to Hariotina sp. (AOY36008.1), is the most preferred candidate compared to the other two pairs after discussing their advantages and disadvantages. In the future, the proposed primer candidate needs to be validated through in vitro amplification with some optimizations to eliminate potential weaknesses. Keywords: rbcL gene; Scenesdesmaceae; Primer design; Molecular identification 163 Pikoli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae INTRODUCTION The amino acid sequences were back-translated to nucleotide sequences (codons) according to the instruction in NCBI (Table 11 Bacteria, Archaea, Chloroplast) /transl_table=11), using https://www.ebi.ac.uk/Tools/st/emboss _backtranseq/ by selecting Anabaena sp. as codon usage table. The translation results were fed to Primer3Plus to obtain possible primers. The back-translation results were re-selected by using Primer3Plus. The default setting of %GC and melting temperature (Tm) was set to narrow down the results. The selection was carried out in stages and explained in the discussion. Each pair of primer candidates was analyzed for %GC, Tm, hairpin potential, self-dimer, heterodimer, etc., using Primer3Plus and OligoAnalyzer https://sg.idtdna.com/calc/analyzer. The Thus, the available primers from existing publications have not been specifically used for Scenedesmaceae. Therefore, by designing primers based on the alignment of conserved regions of available rbcL gene sequences from Scenedesmaceae members, the current study aimed to obtain suitable primer pairs to identify as many genera as possible that belong to the family. recorded. The sequence number for the species having the longest sequence was opened in the NCBI protein. The amino acid sequences were back-translated to nucleotide sequences (codons) according to the instruction in NCBI (Table 11 Bacteria, Archaea, Chloroplast) /transl_table=11), using https://www.ebi.ac.uk/Tools/st/emboss _backtranseq/ by selecting Anabaena sp. as codon usage table. The translation results were fed to Primer3Plus to obtain possible primers. The back-translation results were re-selected by using Primer3Plus. The default setting of %GC and melting temperature (Tm) was set to narrow down the results. The selection was carried out in stages and explained in the discussion. Each pair of primer candidates was analyzed for %GC, Tm, hairpin potential, self-dimer, heterodimer, etc., using Primer3Plus and OligoAnalyzer INTRODUCTION proteins, and other substances (Giraldo- Zuluaga et al., 2018). However, the variable morphology makes it difficult to identify the species. Microalgae is a source of biofuel that attracts global attention because it is a renewable fuel source with high-yield prospects. Its production can save land use, and coupling biofuel production with carbondioxide mitigation strategies and wastewater treatment (Shuba & Kifle, 2018; Rukminasari et al, 2021). Scenedesmaceae are among the microalgae with high potential as a source of lipids for biofuels. Scenedesmus is one member that contains up to 55% (w/v) lipid (Mata et al., 2010) and produces up to 39 mg L-1 d-1 (Mandotra et al., 2016). Molecular identification becomes a complement to morphological identification because it is not related to phenotypic changes by environmental conditions. Polymerase chain reaction (PCR) is one of the most useful molecular methods for the identification of microalgae on a small scale (Ebenezer et al., 2012). By PCR-amplifying particular genes, a microalgae can be identified by its genus or species. rbcL (ribulose-15- bisphosphate carboxylase/oxygenase large) is a gene in the chloroplast that is often used as a marker in the identification of algae and plants. In addition to producing a perfect match with a sequence from reference libraries associated with a single species (Manoylov, 2014), this gene is deposited a lot (>360,000) in GenBank NCBI so that it can be used as a comparison reference. The microalgae diversity is estimated at 200,000-800,000, which has not been fully described (Cheng & Ogden, 2011). Identification of organisms, including microlgae, is needed to determine their properties based on literature. Identification of microalgae is generally done by observing their cell morphology, and molecular method by comparing their genes. Microalgae morphology is partly determined by environmental conditions. Scenedesmus can build coenobia consisting of 1, 2, 4, and 8 cells as a form of adaptation to environmental changes. This polymorphism can be a marker of other properties, such as the number of lipids, PCR requires a pair of primers that start the process of amplifying DNA fragments from the left and right of the fragment. The rbcL sequence data which includes non-microalgae may also cause a lack of specificity in detecting this group, especially those from Scenedesmaceae. 164 BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023): Page: 163-173 recorded. The sequence number for the species having the longest sequence was opened in the NCBI protein. Materials The material used in this research is some tools for bioinformatics, namely MEGA11, GeneDoc, Primer3Plus, and OligoAnalyzer. The sequence used was obtained from GenBank, The National Center for Biotechnology Information (NCBI). Protein Scenedesmaceae rbcL were harvested in NCBI, and their amino acids were aligned in MEGA11. The alignment result was displayed in GeneDoc, then conserved regions (black to dark gray) and less conserved (light gray), both on the left end (for forward primers) and right end (for reverse primers) were observed. The sequences with a minimum of 6 consecutive amino acids were taken and https://sg.idtdna.com/calc/analyzer. The new primer candidates were also examined in Primer-BLAST NCBI. In analyzing data, the new primer candidates were compared with each other to select the best primer pair based on the smallest potential weakness and the ability to capture the most Scenedesmaceae. 165 Pikoli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae RESULTS AND DISCUSSION Results on Retrieving of the Scenedesmaceae rbcL Protein Sequence Protein NCBI as of 9 Agustus 2022 yielded 261 data. About 72 selected sequences were harvested, representing 16 genera (Table1). 15-bisphosphate carboxylase/oxygenase large) subunit partial (chloroplast) in NCBI as of August 9, 2022 No. Genera with available rbcL data Number of classified species No. Genera with unavailable rbcL data Number of classified species 1. Acutodesmus 1 1. Coelastrela 13 2. Asterarcys 2 2. Coelastropsis 1 3. Chodatodesmus 2 3. Coelastrum 8 4. Comasiella 1 4. Coronastrum 1 5. Crucigenia 4 5. Didymocystis 1 6. Desmodesmus 47 6. Dimorphococcus 1 7. Enallax 2 7. Hylodesmus 1 8. Flechtneria 1 8. Pectodictyon 1 9. Halochlorella 1 9. Pediludiella 1 10. Hariotina 6 10. Pseudodidymocystis 2 11. Komarekia 2 11. Pseudospongiococcum 1 12. Neodesmus 3 12. Tetranephris 1 13. Pectinodesmus 3 13. Verrucodesmus 2 14. Scenedesmus 23 15. Tetradesmus 14 16. Westella 1 Total species 113 Total species 34 rbcL data are not available. Those numbers do not include unclassified species. In addition, 16 genera or 113 species harvested by rbcL data represented 37% of genera or 33% of species belonging to Scenedesmaceae recorded in AlgaeBase. According to AlgaeBase as of August 9, 2022, the recorded Scenedesmaceae consisted of 349 species spread across at least 43 genera. Therefore, the harvested data are considered to be fairly Meanwhile, a search with Taxonomy NCBI showed that at least 29 genera were recorded as members of the Scenedesmaceae. Sixteen genera described 55% of the Scenedesmaceae members with known rbcL protein sequences, while the other 13 genera (45%) have no available information. However, genera for which protein sequences comprising 113 species are considered representative of other genera, compared to 34 species for which 166 BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023): Page: 163-173 representative of the unavailable rbcL sequences from other genera of Scenedesmaceae. These search result confirm that rbcL is a promising marker, which has been revealed by several researchers that it can be a barcode for green algae (Hadi et al., 2016). The availability of sequence libraries is used to determine the conserved and variable regions to design universal primers (Manoylov, 2014). of universal primers, as in the history of SSU rDNA sequences, is facilitated by the presence of conserved and variable regions that allow phylogenetic studies (Manoylov, 2014). ; Roslim et al., 2020; Agustina & Roslim, 2021). RESULTS AND DISCUSSION Results on Retrieving of the Scenedesmaceae rbcL Protein Sequence The center region of the protein is of concern to obtaining candidates for the primary pair. The concentration of the most conserved regions in the middle, not at the left nor right ends of the rbcL protein. The length of the PCR product that can be produced is about 600 bases, predicting that the DNA sequence encoding the rbcL would be produced was incomplete. But it is not considered as a big problem for identification purposes, as long as the amplified region is targeted, i.e. it can distinguish between species as accurately as possible. The length of this PCR product resembles that of amplification by other rbcL primer pair used in other Chlorophyte (Fitriyah et al., 2021; Hadi et al., 2016; Yanuhar et al., 2019), although the complete length of the gene is 1400 bp. The center region of the protein is of concern to obtaining candidates for the primary pair. The concentration of the most conserved regions in the middle, not at the left nor right ends of the rbcL protein. Results on Alignment of the Scenedesmaceae rbcL Protein Sequence The alignment results showed that the rbcL protein sequence data of Scenedesmaceae deposited in NCBI were not the same among the species. However, the alignment results showed that the most conserved region was mostly in the middle region marked by dark gray color at amino acid positions number 170s-370s shown using GeneDoc (Figure 1). No regions are completely conserved (dark or black colors are found). However, several variable regions (light gray) were distributed among the conserved regions. The design 167 Pikoli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae Figure 1. GeneDoc display of the alignment results of Scenedesmaceae rbcL protein sequences showing conserved (darker gray, red box) and variable (lighter gray, blue box) regions Figure 1. GeneDoc display of the alignment results of Scenedesmaceae rbcL protein sequences showing conserved (darker gray, red box) and variable (lighter gray, blue box) regions Table 2. Amino acid sequences of rbcL protein of Scenedesmaceae and their translated results targeted for searching candidates of primer pairs Table 2. Amino acid sequences of rbcL protein of Scenedesmaceae and their back- translated results targeted for searching candidates of primer pairs Region Amino acid sequence Sequence number* Back-translated sequences** Conserved region Left LGCTIKPKLGLSAKNY GRAVYECLRGGL 170-198 TTAGGTTGTACTATTAAACCAAAATTAGG TTTATCTGCTAAAAATTATGGTCGTGCTG TTTATGAATGTTTACGTGGTGGTTTA Right EKDRSRGIYFTGDW 355-369 GAAAAAGATCGTTCTCGTGGTATTTATTT TACTGGTGATTGG Less conserved region Left ICVERDKLNKYGRG 155-169 ATTCAAGTTGAACGTGATAAATTAAATAA ATATGGTCGTGGT Right MEVASGGIHVWHMP AIVEIFGDDA 375-399 ATTCAAGTTGAACGTGATAAATTAAATAA ATATGGTCGTGGT according to Hariotina sp. MMOGRB0030F (AOY36008.1) according to Anabaena codon (Table 2). The amino acid numbers follow the longest (most complete) sequence of the aligned species, namely AOY36008.1 ribulose-15-bisphosphate carboxylase/ oxygenase large subunit (chloroplast) Hariotina sp. MMOGRB0030F. The rbcL protein has been knew tend to be more variable, but still has the advantage of avoiding contamination by microorganisms that do not have chloroplasts (Krienitz & Bock, 2012). The alignment results also showed that there were less conserved regions at the left (no.160s) and right (no.390s) ends (Figure 1, signed by the blue box). As a precaution not to waste more region, the regions were also targeted to obtain candidates for primer pair. Therefore, there are two regions of concern as candidates, the conserved region and the non-conserved region so that the amino acid sequences were retrieved from them 168 BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023): Page: 163-173 Analysis Result of the Primer Pairs Candidates G (kcal.mole-1) for duplex disruption for the five 3' bases as calculated using the nearest-neighbor parameter values by Primer3Plus. In the ten primers, the number of GC bases at the 3' end does not exceed 3-5 bases (Sasmito et al., 2014; Utomo et al., 2019), so they are still good candidates for primers. Selecting primers using Primer3Plus on the back-translated sequences resulted in several candidate primers. By setting %GC min.40 and Tm min.55C, 9 left and 11 right primer candidates were obtained from conserved and less-conserved regions. No right primers were generated from the less- conserved regions with such settings, because the back-translated sequences are mostly AT bases (Table 2). And then to save on selection, the best five primers of each right and left, were taken based on the penalty values. Primer3Plus calculates the penalty value based on the percentage of max.%GC, min. Tm, possible product length, and non-redundancy. The primers were then sorted by penalties, with the lowest penalty defining the best primer (Table 3). However, the results of primary selection based on %GC and Tm contain weaknesses in the form of hairpin and self-dimer formation, which are a consequence of variations in the sequence of nitrogen bases (Table 3). Hairpins and self-dimers are difficult to avoid, so the primer selection is taken with the least risk. All of the ten primers had potency in forming hairpins mostly by G<2 kcal.mole-1, which is within tolerable values (Sasmito et al., 2014). Similarly, in the number of self-dimers, mostly only 2 base pairs occurs, although some of them are 4 base pairs. The potential for hairpin and self-dimer must be minimized through PCR mixture as often suggested by various PCR protocols. The 3’ stability is the maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The value is the maximum 169 koli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae Table 3. Analysis result of the primer pairs candidates by Primer3Plus (set: %GC min.40; Tm min.55) and IDT OligoAnalyzer*. Only the best five primers of each left and right are displayed. Left (F) and right (R) primers Sequence (5’ to 3’) Length (nt) Tm (C) GC (%)* 3’ stability, G (kcal.mole-1)* Penalty* No. of hairpins No. Analysis Result of the Primer Pairs Candidates Primer Pair Candidates with Tm difference and Hetero-dimer Analyzed by IDT OligoAnalyzer Name of primer pair Left (F) and right (R) primers Tm difference Hetero-dimer (no., max. G) Sce-2 1_F and R 0.3 19, -8 Sce-7 1_F and 2_R 0.4 19, -8 Sce-16 F and 1_RL 0.5 25, -7 Analysis Result of the Primer Pairs Candidates of self- dimers F TGGTCG TGCTGT TTATGA ATGT 22 58.6 40.9 2.7 3.408 8, mostly G<2 kcal.mole-1 8; 2 bp, G<- 4 kcal.mole-1 1_F TGGTCG TGCTGT TTATGA ATG 21 56.9 42.9 2.7 4.101 8, mostly G<2 kcal.mole-1 8; 2 bp, G<- 4 kcal.mole-1 2_F GGTCGT GCTGTT TATGAA TGT 21 56.9 42.9 2.7 4.103 8, mostly G<2 kcal.mole-1 8; 2 bp, G<- 4 kcal.mole-1 3_F GGTCGT GCTGTT TATGAA TGTT 22 57.6 40.9 2.7 4.420 8, mostly G<2 kcal.mole-1 9; 2 bp, G<- 4 kcal.mole-1 4_F TGAATG TTTACG TGGTGG TT 20 55.5 40.0 3.7 4.500 7, with G<2 kcal.mole-1 8; 1 of them 4 bp, G=-6 kcal.mole-1 R ACCACG AGAACG ATCTTT TTC 21 56.6 42.9 2.3 4.421 1, G= -1.8 kcal.mole-1 9; 1 of them 4 bp, G=-4 kcal.mole-1 2_R TACCAC GAGAAC GATCTT TTTC 22 56.5 40.9 2.3 5.503 1, G= -1.8 kcal.mole-1 10; 1 of them 4 bp, G=-4 kcal.mole-1 RL TGCCAA ACATGA ATACCA CCAG 22 59.2 45.5 4.0 2.825 5, with G<2 kcal.mole-1 11; 1 of them 4 bp, G=-5 kcal.mole-1 1_RL TGCCAA ACATGA ATACCA CCA 21 58.1 42.9 4.2 2.882 5, with G<2 kcal.mole-1 11; 1 of them 4 bp, G=-5 kcal.mole-1 2_RL GCCAAA CATGAA TACCAC CAGA 22 58.9 45.5 3.9 3.089 3, with G<2 kcal.mole-1 8; 1 of them 4 bp, G=-5 kcal.mole-1 The pairing of the tenth primer produced 25 primer pairs named Sce-1— Sce-25 which had been analyzed for the Tm difference and the number of hetero- dimers that might occur. However, the Tm difference between the left and right primers varied from 0.3—3.7C, so they have been selected again by 0.3—0.5C of Tm difference. The selection presented 3 candidates of primers with lower 170 Pikoli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae primer 1_F (Sce-2) is G, not T as in 2_F (Sce-3) (Table 3). The same reason applies to the selected Sce-7 instead of Sce-8. The nucleotide base at the 3' end of the primer should be G or C to avoid a mismatch of A or T (Utomo et al., 2019). differences between primers after excluding those having more heterodimers for the same Tm difference (Table 4). Sce-2 and Sce-3 have the same Tm difference and hetero-dimer, but Sce- 2 was chosen because the 3' end of the Table 4. REFERENCES Agustina, P., & Roslim, D. I. (2021). Analysis Of DNA Sequence Encoding Glyceraldehyde- 3-Phosphate Dehydrogenase (Gapdh) On Cocor Bebek (Kalanchoe X Laetivirens). BIOLINK (Jurnal Biologi Lingkungan Industri Kesehatan), 8(1), 72- 82. Results of Scenedesmaceae Trapped Using the Primer Candidates primers was not the genus used to design the primer. The genera not trapped by the three primers were Chodatodesmus, Halochlorella, and Neodesmus. It was presumably because their rbcL gene sequences deposited were too short and did not include the conserved regions being observed. The three candidates of primer pairs captures 14 genera (Table 5). Thirteen (81.3%) or most of them were part of the 16 genera possessing the rbcL gene (Table 1) that were retrieved for primer design. One other genus, Coelastrela, which was captured by the Table 5. Scenedesmaceae trapped using the primer candidates by using Primer-BLAST Name of primer pair Product length (bp) Number of hits trapped Number of genera Sce-2 529 109 14 Sce-7 529 109 14 Sce-16 603 67 14 higher %GC and less self-dimer, so in this context, Sce-7 is preferred over Sce-2 (Table 3). Meanwhile, Sce-16 produced a longer product (603 bp) than that of Sce- 2 and Sce-7, by the length of the partial rbcL gene containing conserved regions. Sce-2 and Sce-7, resulted in the same length of PCR product, number of hits, and genera (Table 5), indicating that the addition of base T at the 5' end of the primer 2_R did not affect the yield. However, primer 2_R (in Sce-7) has a 171 BioLink : Jurnal Biologi Lingkungan, Industri dan Kesehatan, Vol. 9 (2) Feb (2023): Page: 163-173 The lower number of hits from Sce-16 does not reduce the quality of gain because the number of genera remains the same as that of the others. The potential for hetero-dimers between the constituent primers of Sce-16 was greater than that of the others (Table 4). However, most of the pairing that occurs is 2 base pairs, and only one cross-dimer occurs at the 3' by G= -1.34 kcal.mole-1. The cross-dimer at the 3' end withG= -5 kcal.mole-1 was tolerated (Sasmito et al., 2014). Regardless, in applying the primers PCR reaction needs special modification of conditions and mixtures to eliminate the hetero-dimers. sequencing. The resulting primer pairs were selected according to the conserved and less-conserved regions of Scenedesmaceae, not yet taken from the non-Scenedesmaceae rbcL gene. Therefore, in the future in order to obtain primers that are specific for capturing only Scenedesmaceae rbcL and as few as possible of non-Scenedesmaceae, it is necessary to align them with broader representatives of rbcL at least in the Chlorophyta group. CONCLUSION The candidate of primer pairs proposed to capture Scenedesmaceae based on the conserved and less- conserved region of rbcL genes is Sce-16 (F, 5'-TGGTCGTGCTGTTTATGAATGT-3' and 1_RL, 5'- The candidate of primer pairs proposed to capture Scenedesmaceae based on the conserved and less- conserved region of rbcL genes is Sce-16 (F, 5'-TGGTCGTGCTGTTTATGAATGT-3' and 1_RL, 5'- TGCCAAACATGAATACCACCA-3'). The use of the primers has to be accompanied by observations of cell morphology which led to the initial assumption that the identified specimen belongs to the Scenedesmaceae group. In addition, both require validation in real PCR reactions, by the right conditions and mixtures, as well as product verification by Cheng, K. C., & Ogden, K. L. (2011). Algal biofuels: The research. Chemical Engineering Progress, 107(3), 42–47. Ebenezer, V., Medlin, L. K., & Ki, J. S. (2012). Molecular detection, quantification, and diversity evaluation of microalgae. Marine Biotechnology, 14(2), 129–142. https://doi.org/10.1007/s10126-011-9427-y Fitriyah, F., Faramitha, Y., Sari, D. A., Kresnawaty, I., Panji, T., & Santoso, D. (2021). Molecular identification and phylogenetic analysis of Chlorella isolates from Indonesia using the rbcL gene. E-Journal Menara Perkebunan, 89(1), 17–25. https://doi.org/10.22302/iribb.jur.mp.v89i1 .408 TGCCAAACATGAATACCACCA-3'). The use of the primers has to be accompanied by observations of cell morphology which led to the initial assumption that the identified specimen belongs to the Scenedesmaceae group. In addition, both require validation in real PCR reactions, by the right conditions and mixtures, as well as product verification by Giraldo-Zuluaga, J. H., Salazar, A., Diez, G., Gomez, A., Martínez, T., Vargas, J. F., & Peñuela, M. (2018). Automatic identification of Scenedesmus polymorphic microalgae from microscopic 172 Pikoli, M.R. Analysis Of New Primer Pair Candidates Of rbcl Gene For Identification Of Microalgae Scenedesmaceae images. Pattern Analysis and Applications, 21(2), 601–612. https://doi.org/10.1007/s10044-017-0662-3 Roslim, D. I., Hairima, H., Herman, H., & Lestari, W. (2020). The DNA Sequence Encoding Glyceraldehyde 3-Phosphate Dehydrogenase (Gapc) Enzyme On Tuntun Angin Plant (Elaeocarpus Floribundus BI). BIOLINK (Jurnal Biologi Lingkungan Industri Kesehatan), 7(1), 16- 22. Hadi, S. I. I. A., Santana, H., Brunale, P. P. M., Gomes, T. G., Oliveira, M. D., Matthiensen, A., Oliveira, M. E. C., Silva, F. C. P., & Brasil, B. S. A. F. (2016). DNA barcoding green microalgae isolated from neotropical inland waters. PLoS ONE, 11(2). https://doi.org/10.1371/journal.pone.01492 84 Rukminasari, N., Omar, S. A., & Lukman, M. (2021). Temperature and nitrate concentration effect on the abundance and growth rate of melosira sp. BIOLINK (Jurnal Biologi Lingkungan Industri Kesehatan), 7(2), 185-194. CONCLUSION Krienitz, L., & Bock, C. (2012). The present state of the systematics of planktonic coccoid green algae of inland waters. Hydrobiologia, 698, 295–326. https://doi.org/10.1007/s10750-012-1079-z Sasmito, D. E. K., Kurniawan, R., & Muhimmah, I. (2014). Karakteristik primer pada polymerase chain reaction(PCR) untuk sekuensing DNA: Mini review. Seminar Informatika Medis 2014, 93–102. http://snimed.fit.uii.ac.id/ Mandotra, S. K., Kumar, P., Suseela, M. R., Nayaka, S., & Ramteke, P. W. (2016). Evaluation of fatty acid profile and biodiesel properties of microalga Scenedesmus abundance under the influence of phosphorus, pH, and light intensities. Bioresource Technology, 201(November), 222–229. https://doi.org/10.1016/j.biortech.2015.11.0 42 Shuba, E. S., & Kifle, D. (2018). Microalgae to biofuels: ‘Promising’ alternative and renewable energy, review. Renewable and Sustainable Energy Reviews, 81(May 2017), 743–755. https://doi.org/10.1016/j.rser.2017.08.042 Manoylov, K. M. (2014). Taxonomic identification of algae (morphological and molecular): species concepts, methodologies, and their implications for ecological bioassessment. Journal of Phycology, 50(3), 409–424. https://doi.org/10.1111/jpy.12183 Utomo, D. H., Ichsan, M., & Puti, J. F. (2019). Prinsip dasar desain primer dengan bioinformatika (Tim Inbio Indonesia (ed.)). Global Science. Yanuhar, U., Caesar, N. R., & Musa, M. (2019). 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https://openalex.org/W4294325988	https://pure.tue.nl/ws/files/301659006/TCHES2022_4_19.pdf	English	null	ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge	IACR transactions on cryptographic hardware and embedded systems	2,022	cc-by	14,727	Document status and date: Published: 31/08/2022 Document status and date: Published: 31/08/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Document license: CC BY Document license: CC BY DOI: 10.46586/tches.v2022.i4.527-552 Document status and date: Published: 31/08/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There ca important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visi DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication DOI: 10.46586/tches.v2022.i4.527-552 ECDSA White-Box Implementations Citation for published version (APA): Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Citation for published version (APA): Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Download date: 24. Oct. 2024 www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 IACR Transactions on Cryptographic Hardware and Embedded Systems ISSN 2569-2925, Vol. 2022, No. 4, pp. 527–552. DOI:10.46586/tches.v2022.i4.527-552 Guillaume Barbu1 , Ward Beullens2, Emmanuelle Dottax1 , Christophe Giraud1 , Agathe Houzelot1,3 , Chaoyun Li4 , Mohammad Mahzoun5, Adrián Ranea4 and Jianrui Xie4 1 IDEMIA, Cryptography & Security Labs, Pessac, France firstname.lastname@idemia.com 2 IBM Research, Zurich, Switzerland wbe@zurich.ibm.com 3 LaBRI, CNRS, Université de Bordeaux, Bordeaux, France 4 imec-COSIC, KU Leuven, Leuven, Belgium firstname.lastname@esat.kuleuven.be 5 Eindhoven University of Technology, Eindhoven, Netherlands m.mahzoun@tue.nl Abstract. Despite the growing demand for software implementations of ECDSA secure against attackers with full control of the execution environment, scientiﬁc literature on ECDSA white-box design is scarce. The CHES 2021 WhibOx contest was thus held to assess the state-of-the-art and encourage relevant practical research, inviting developers to submit ECDSA white-box implementations and attackers to break the corresponding submissions. In this work, attackers (team TheRealIdeﬁx) and designers (team zerokey) join to describe several attack techniques and designs used during this contest. We explain the methods used by the team TheRealIdeﬁx, which broke the most challenges, and we show the eﬃciency of each of these methods against all the submitted implementations. Moreover, we describe the designs of the two winning challenges submitted by the team zerokey; these designs represent the ECDSA signature algorithm by a sequence of systems of low-degree equations, which are obfuscated with aﬃne encodings and extra random variables and equations. The WhibOx contest has shown that securing ECDSA in the white-box model is an open and challenging problem, as no implementation survived more than two days. In this context, our designs provide a starting methodology for further research, and our attacks highlight the weak points future work should address. Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 1 Introduction Cryptographic techniques are primarily designed to be secure in a context where the conﬁdentiality of secret keys is ensured with black-box access to the algorithm – only inputs and outputs are available to the attacker. Conﬁdence in security is built from detailed studies, carefully deﬁned security notions, and security proofs. Such a strong level of conﬁdence is now a standard expectation. However, real-life scenarios for implementations might jeopardize initial assumptions, where attackers have access to additional information via side channels (e.g., timing or power consumption) or can modify the algorithm execution Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 Published: 2022-08-31 Published: 2022-08-31 528 Attacks and Designs from CHES 2021 Challenge and exploit faulty results. This is called the grey-box model. Developers have to put countermeasures in place to reach the originally expected security level. and exploit faulty results. This is called the grey-box model. Developers have to put countermeasures in place to reach the originally expected security level. In the context of mobile applications – contactless payments, cryptocurrency wallets, streaming services – or connected objects, devices often lack secure storage to protect secret keys, and their generally open execution environment exposes a large attack surface. This hostile environment is captured by the white-box model, which assumes an attacker having control of every aspect of the implementation: execution ﬂow, memory content and addresses. The ﬁrst white-box implementations were proposed in the early 2000s by Chow et al. [CEJvO02,CEJv03], and the ﬁeld has continuously developed since then, with design proposals [BG03,BCD06,XL09,Kar11,DFLM18,RW19,SEL21,BCC21], attacks [BGEC04,GMQ07,WMGP07,MGH09,DWP10,DRP13,LRD+14,AMR19,GRW20] and eﬀorts to deﬁne security notions [SWP09,DLPR14,AABM20]. The industry shows a growing interest in white-box cryptography owing to the widespread usage of security-related applications on connected devices. The WhibOx contest, attached to the CHES conference, has been held biennially since 2017 to encourage practical experiments both from the designer and attacker perspectives. It lasts several months, inviting coders to post white-box implementations and attackers to break them. Participants can remain anonymous and silent about any detail on their work. The ﬁrst two editions in 2017 and 2019 focused on white-box implementations of AES and exhibited the community’s strong interest in this subject. Some candidates survived all attacks in the second edition in 2019, showing a certain maturity for this algorithm. 1Guillaume Barbu, Emmanuelle Dottax, Christophe Giraud and Agathe Houzelot are part of the team TheRealIdeﬁx; Ward Beullens, Chaoyun Li, Mohammad Mahzoun, Adrián Ranea and Jianrui Xie compose the team zerokey. 2 Rules of the WhibOx 2021 Contest Designers were required to post challenges computing ECDSA signatures on the NIST P-256 curve under a hard-coded, freely chosen key, and accepting as input any 256-bit message digest e = H(m). Notice that the cryptographic hash function H is excluded from the intended white-box implementation of ECDSA and the message m is also not provided. At the same time, attackers were encouraged to extract the private keys. In addition, acceptance of submitted implementations was conditioned on some requirements: • the public key corresponding to the embedded private key, as well as a proof of knowledge of the private key, had to be provided, • submissions had to be source code in portable C, • linking to external libraries was forbidden, except for the GNU Multi Precision library [Gt20], • the signature algorithm had to be deterministic, • the execution time was limited to 3 seconds, the program size to 20 MB, and the RAM usage to 20 MB as well. • the execution time was limited to 3 seconds, the program size to 20 MB, and the RAM usage to 20 MB as well. There was an elaborate system with scoreboards to reward designers and attackers. A challenge gains strawberries as time goes by till broken. Challenges with a higher performance score (measured in terms of execution time, code size, and RAM usage) gain strawberries faster. Eventually, the challenge with the highest number of strawberries wins the competition. Accordingly, when submitting a matching private key to the system, attackers receive bananas, the number of which is determined by the number of strawberries of the challenge at the time of the break. More detailed information can be found on the contest website [CHE]. 1 Introduction Section 5 discloses the designs of Challenges 226 and 227 proposed by the team zerokey, and Section 6 concludes this paper. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx 2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding white-box implementations. Section 4 presents the diﬀerent methods that have been used by the team TheRealIdeﬁx to break various implementations and some statistics regarding the success rate of these methods. Section 5 discloses the designs of Challenges 226 and 227 proposed by the team zerokey, and Section 6 concludes this paper. 1 Introduction In 2021, organisers changed the target and decided to consider the ECDSA signature algorithm, whose white-box implementation is of substantial interest to the industry but virtually lacks scientiﬁc literature. From May 17th to August 22nd 2021, 97 candidate implementations were submitted for scrutiny by 37 (teams of) attackers. All challenges were broken within 35 hours, suggesting the diﬃculty of achieving a secure white-box implementation of ECDSA. Thus, studying the attacks would help to discern weak points inside the implementations. Besides, the analysis of the design of the most resistant challenges, which successfully defeated most attackers, would also give directions for future designs. Contributions. In this paper, teams TheRealIdeﬁx — who broke the most challenges — and zerokey — who proposed the two winning challenges — join to present how they proceeded during the contest1. On the attack side, we describe a strategy to achieve eﬃcient attacks. As reverse engineering is a time-consuming task, automated attacks are desirable. We consider diﬀerent attack paths against ECDSA white-boxes: the ones inherited from traditional cryptanalysis, the extensions of attacks in the grey-box model, and the logical attacks of the software. We discuss the feasibility of automating each attack path and provide detailed information regarding which attacks succeeded (or failed) on each candidate. Our results show that, with few exceptions, it was suﬃcient to fully recover the secret value by these automated attacks. On the design side, we describe the methodology we used to build the two winning challenges, Challenges 226 and 227. It includes modifying the implicit framework [RVP22] originally proposed for block ciphers, applying techniques from multivariate public-key cryptosystems, and obfuscating the resulting C code with a C obfuscator. Our design thus turns the ECDSA signature algorithm into a sequence of systems of low-degree equations which are obfuscated with large aﬃne encodings and additional variables and equations. Finally, we show how to break Challenge 226 with automated attacks and how to break Challenge 227 once reverse-engineered. 529 Barbu et al. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx 2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding white-box implementations. Section 4 presents the diﬀerent methods that have been used by the team TheRealIdeﬁx to break various implementations and some statistics regarding the success rate of these methods. 3.1 ECDSA In 1992, Vanstone introduced a variant of DSA based on elliptic curves. The result- ing public-key signature algorithm is called Elliptic Curve Digital Signature Algorithm (ECDSA) [Van92]. Its parameters are an elliptic curve E over a ﬁeld Fq, a point G of prime order n, and a cryptographic hash function H. The private key d is randomly drawn from J1, n −1K, and the public key consists of the point Q = [d]G where [d]G corresponds to the scalar multiplication of the point G by the scalar d. The ECDSA signature is described in Algorithm 1, where Rx and Ry denote the coordinates of the point R. Note that the key d is not the only sensitive value in that scheme. Indeed, the recovery of the nonce k allows the computation of d from the signature (r, s) and the message m: d = (ks −H(m))r−1 mod n . (1) d = (ks −H(m))r−1 mod n . (1) Attacks and Designs from CHES 2021 Challenge 530 Algorithm 1: ECDSA signature Input : the message m Output : the signature (r, s) 1 e ←H(m) 2 k $←−J1, n −1K 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) 1 e ←H(m) 2 k $←−J1, n −1K 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) The nonce must not only remain secret but also diﬀer for each execution of the algorithm. Indeed, an eﬃcient way to recover its value is to ﬁnd another signature (r′, s′) of a diﬀerent message m′ ̸= m using the same nonce, that is with k′ = k. In that case, we also have r′ = r, so the adversary may compute k = (H(m′) −H(m))(s′ −s)−1 mod n . (2) (2) In the black-box model, the security of ECDSA is based on the diﬃculty of the Elliptic Curve Discrete Logarithm Problem (ECDLP), i.e., on the diﬃculty of computing the scalar k (resp. d) from the points G and R = [k]G (resp. Q = [d]G). 3.1 ECDSA To ensure that this problem is diﬃcult to solve, there are several standards to deﬁne elliptic curves, e.g. [Loc10,Sta10,JOR11,FIP13]. However, there is a gap between the security of ECDSA in theory and that of ECDSA implementations. Many grey-box attacks have been described in the literature (see for example [FV12]). Some of them directly target the key d while others aim at recovering some information on the nonce k. As explained previously, the knowledge of the nonce allows an adversary to compute the secret key. Recovering a few bits of the nonces associated to diﬀerent signatures may be enough for an attacker. Indeed, this allows the construction of a system of equations that can be solved using lattice-based algorithms [BH19,JSSS20] or Bleichenbacher’s FFT-based approach [ANT+20]. These bits could, for example, be recovered via side-channel analysis if the implementation is not protected or simply guessed if the nonce is not drawn uniformly at random. These attacks show that it is already complicated to achieve a secure implementation of ECDSA in the grey-box model, and of course, things get worse in the white-box context. 4 Breaking the Challenges White-box implementations usually rely on encodings and other theoretically sound approaches to protect the secret values and their manipulations. It is also very often the case that code obfuscation techniques are used to make understanding the design a time-consuming and challenging task. Extensive use of such obfuscation techniques in the submitted source ﬁles causes independently reverse-engineering each challenge to be overwhelming in time. We thus focused on designing attack methods that could be eﬃcient and easily automated. This section looks at the diﬀerent attacks that can be automated in a white-box context and gives the rationale for using and discarding them. We then present the results of applying the selected methods to the whole set of submissions. 3.2 White-box Implementation of ECDSA The white-box model assumes that the attacker has total access to the executable: he can read and modify it at will. He also has access to all the memory used during execution, so a white-box designer does not only have to protect his implementation against grey-box attacks but also against an adversary who can dump the memory and search for sensitive values such as k or d. The ﬁrst technique to prevent secret data from appearing in plain was introduced by Chow et al. in [CEJv03]. Their idea is to embed the key into the algorithm, and each operation is performed with the help of look-up tables protected by carefully crafted encodings. Informally, the algorithm is split into low-level operations, and each operation op is replaced by f −1 ◦op ◦f ′, where f and f ′ are bijections called respectively input and output encodings. The drawback of this technique is that the required memory drastically increases with the algorithm’s complexity. Using it to secure operations as 531 Barbu et al. complex as scalar multiplications or inversions while remaining eﬃcient is thus a real challenge. complex as scalar multiplications or inversions while remaining eﬃcient is thus a real challenge. Another challenge in white-box cryptography is the impossibility of relying on any external source of randomness. An attacker could simply disable such a source and ﬁx its output to a constant value. For example, in the context of AES, this renders some countermeasures against side-channel or fault attacks based on randomization techniques completely ineﬃcient. When one considers ECDSA signatures, disabling the source of randomness yields multiple uses of the same nonce and, thus, easy recovery of the private key, as seen in Sect. 3.1. The solution is to compute k as a function of the only source of randomness available, the input message: k = f(m). In order to maintain the security of the signature scheme, this mapping must be computationally indistinguishable from what a randomly and uniformly chosen function would return. We will see in the next section that many challenges of the WhibOx competition did not fulﬁll this requirement. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 4.1.1 Hooking Shared Libraries The contest rules were a clear incentive for developers to use the GMP library for big number arithmetic operations. A ﬁrst attempt to break the submitted challenges was then to search if sensitive values were manipulated in clear by the GMP library. In order to perform this automatically, our approach has been to hook the calls to GMP functions thanks to the so-called LD_PRELOAD trick. Pre-loading is a feature of the dynamic linker on UNIX systems that allows loading a speciﬁc shared library before all other libraries linked to a given executable binary2. In our speciﬁc case, we built a shared library deﬁning the same function as the GMP library (e.g. mpz_mul, mpz_mod or mpz_invert). Each of these functions simply updates a log of the given parameters before calling the real GMP function, explicitly using the dynamic linker (thanks to the <dlfcn.h> module) to ensure the correct execution of the white-box implementation. It is then only necessary to add our shared library to the LD_PRELOAD environment variable of the dynamic linker on our system before calling the ECDSA binary to have our custom functions called in place of the genuine GMP ones. The corresponding log is analysed in a second step to eventually reveal the secret key if d, k or related values such as r · d or e + r · d are found in the log. Such an approach allowed us to break 32% of the challenges. As a side note, this technique also jeopardizes implementations relying on system- dependent random generators such as srand or mpz_XrandomX functions, or on other sources such as time. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 532 Attacks and Designs from CHES 2021 Challenge Attacks and Designs from CHES 2021 Challenge 4.1.2 Biased Nonces To sum up, the relations we used for our lattice attacks are the following (with ei ranging from 0 to 999): • assuming l = 6 known most- or least-signiﬁcant bits of the ephemeral key: kmsb2L + klsb = s−1(e + rd) mod n , (3) (3) with L = 256 −l for the MSB case and L = l for the LSB case (we considered both cases where the known value is 0 or 63 = 26 −1), with L = 256 −l for the MSB case and L = l for the LSB case (we considered both cases where the known value is 0 or 63 = 26 −1), • assuming the ephemeral keys are ki = tκi: t = κ−1 0 (e0 + r0d) mod n, κi = t−1(s−1 i ei −s−1 i rir−1 0 e0) + κ0(s−1 i rir−1 0 s0) mod n, (4) (4) with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. Such an approach allowed us to break 72% of the challenges. 4.1.2 Biased Nonces As explained in Sect. 3.2, white-box designers usually generate the nonce k from the input. In the case of the WhibOx contest, the nonce is thus computed as a function of the hash, i.e. k = f(e). However, if the function f is not carefully selected, it could happen that the ki’s generated from diﬀerent ei’s are not uniformly random. In the worst case, we have collisions such that diﬀerent hash values e0 and e1 produce the same nonce (k0 = k1). If such a collision occurs, one can recover the private key d as explained in Sect. 3.1. Furthermore, collisions can be eﬃciently detected by looking at the r part of the signature. To eﬃciently browse a subset of hash values in search of such collisions, we limited ourselves to hash values with a Hamming weight equal to 1 or 2. We thus considered 32 896 hash values and were able to break 60% of the challenges with this technique. In those cases where we did not ﬁnd any collision, we looked for biases in the nonce generation. We used well-known lattice attacks derived from [NS03] and [FGR13] to exploit such a potential weakness. Such attacks can recover an ECDSA private key only with the knowledge of a few bits of the ephemeral keys of several signatures. A concrete example showing why such techniques can succeed in our context consists in considering f = Id. Then ki = ei and with providing ei ranging from 0 to 99 we obtained 100 signatures for which the 249 most-signiﬁcant bits of the nonces are 0. This bias is more than enough for a lattice attack to recover the private key d. Lattice-based attacks can also be applied when the ephemeral key is the product of a small random κ by another (large) constant scalar t. Such a design allows to eﬃciently perform the scalar multiplication as R = [κ]T = [k]G, with T = [t]G a precomputed value. The point is that the small size of κ reduces the cost of the scalar multiplication. 4.1.4 Fault Injections Another attack method is to disturb the algorithm execution and exploit the resulting faulty output. In the white-box context, faults can be easily induced since the attacker can modify the binary or use debugging tools to stop the execution and, for example, skip an instruction or modify the value of a particular register. Again, this attack can be automated and does not require an earlier reverse engineering step. All the fault attacks that can be performed in the grey-box context are obviously also a potential threat in the white-box context. In the case of ECDSA, diﬀerent faults can be induced on diﬀerent variables to give an exploitable result. The most obvious attack is to force the use of a weak elliptic curve during the scalar multiplication by disturbing the curve parameters [BMM00] in order to solve the discrete logarithm problem easily. The attacker can also force the use of biased nonces, for instance, by sticking a 32-bit word of k at zero during several executions. The corresponding signatures can then be used to obtain information on the key using lattice-based algorithms. Finally, modifying one byte of d during the computation of rd may allow one to recover information on the key, as shown in [GK04]. In addition, the white-box model oﬀers new possibilities [PSS+18,ABF+18,DGH21]. They arise from the fact that deterministic versions of the scheme have to be implemented due to the impossibility of relying on a source of randomness in this context. When the algorithm is used twice on the same message, the same nonce k is derived. The attacker may thus obtain a correct signature for a given digest e, and an erroneous one by modifying a second execution of the same signature. To break the challenges of the WhibOx contest, we mainly disturbed the computation of the ﬁrst part of the signature r, obtaining faulty results ˜r and ˜s = k−1(e + ˜rd) mod n. Some secret information can be deduced from the correct and faulty signatures: (r −˜r)(s −˜s)−1 ≡(r −˜r)(k−1d(r −˜r))−1 ≡kd−1 mod n . (5) (5) Let α = kd−1 mod n. The adversary can then compute the private key: Let α = kd−1 mod n. The adversary can then compute the private key: d = e(αs −r)−1 mod n . (6) (6) It is also possible to disturb other variables, but still, the faulty value must be known to exploit the result. 4.1.3 DCA In 2016, Bos et al. showed that although ﬁrstly described for the grey-box context, the well-known side-channel attacks could be very well adapted to the white-box model. The resulting attack [BHMT16] is called Diﬀerential Computational Analysis (DCA). The principle is very similar to classical side-channel attacks: secret values are extracted from leakage traces obtained during several executions of a cryptographic algorithm with the help of statistical tools. The only diﬀerence relies upon the nature of the traces. Whereas in the grey-box context, one can record the power consumption of the device in which the algorithm is implemented, a white-box attacker can simply use software execution traces. By instrumenting the binary, he can record completely noiseless traces of all accessed addresses and data over time, leading to much more eﬃcient attacks. 533 Barbu et al. In theory, this attack is particularly devastating since it can be fully automated and does not require any earlier reverse engineering step. In practice, it is quite diﬃcult to apply because of the size of the traces, in particular for cryptosystems such as ECDSA that have relatively long execution time. Indeed, if the whole white-box execution were to be recorded, each trace would easily reach several gigabytes. For instance, tracing n 64-bit registers on a 3GHz machine during 3 seconds would lead to a single trace of 9 ∗8 ∗n Gb. Therefore, iterating over dozens of traces for a CPA would be overwhelming in time and memory. A time-consuming step of reverse engineering allowing to select a smaller window of the implementation before the attack is thus required, which is why we did not use this technique to break the challenges of the WhibOx contest. 4.1.4 Fault Injections Interestingly, when one modiﬁes the ﬁrst part of the signature, if no countermeasure is implemented, the faulty value is just given to the attacker as part of the output. Furthermore, the attack surface is huge: the fault may happen anywhere during the scalar multiplication. This is why we considered only this perturbation in the context of this competition. This approach is the most successful one, allowing us to break 75% of the challenges. 534 Attacks and Designs from CHES 2021 Challenge 4.2 Attacks Results When applying the various attack methods described above, we obtain the results presented in Table 1. We observe that lattice and fault attacks are very eﬃcient. Collision attacks also give good results. Table 1: Success rate of each attack on the 97 challenges. Attack type Percentage of broken challenges Hooking 32% Bad nonce - Collision 60% - Lattice 72% Fault Injection 75% We give in Appendix A the speciﬁc vulnerabilities of each of the 97 submitted challenges as well as the corresponding private key. However, we noticed that many challenges had a low level of security, some of which were even plain implementations. We thus excluded 30 challenges3 where the nonce and/or the private key were manipulated in plain. Table 2 illustrates the eﬃciency of the attacks presented in Sect. 4.1 on the remaining 67 challenges. We observe that hooking gives no signiﬁcant result anymore, collision and lattice attacks become less eﬃcient, and fault injection seems the most powerful attack. Table 2: Success rate of each attack on the 67 strongest challenges. Table 2: Success rate of each attack on the 67 strongest challenges. Attack type Percentage of broken challenges Hooking 1% Bad nonce - Collision 49% - Lattice 61% Fault Injection 69% Among the 67 strongest challenges, Challenges 226 and 227 are the winning ones. In the next section, we present the design of these two white-box implementations. 3The challenges 3, 4, 8, 10, 11, 32, 45, 54, 55, 57, 85, 97, 114, 135, 136, 139, 153, 157, 174, 185, 187, 231, 235, 267, 274, 299, 307, 320, 321, and 323 are considered as weak. 5.1 Implicit White-box Implementations The implicit framework is a method to obtain a white-box implementation of a block cipher. Its main idea is to represent the round functions of the cipher by implicit functions of low degree and to protect these implicit functions with large aﬃne encodings. Before introducing implicit white-box implementations, we need to introduce the notions of encoding, encoded implementation, and quasilinear implicit functions. While these notions are originally deﬁned in [RVP22] for vectorial functions over the binary ﬁeld, we extend these notions for an arbitrary ﬁnite ﬁeld. Let Fq be the ﬁnite ﬁeld with q elements. A vectorial function F from the vector space (Fq)l to (Fq)l′ is called a (l, l′) function over Fq, and its l′ component functions are denoted by (F1, F2, . . . , Fl′). The degree of an (l, l′) function F denotes the maximum polynomial degree of the l′ multivariate polynomials uniquely representing the component functions of F. Deﬁnition 1. Let F be an (l, l′) function over Fq, A be an (l, l) permutation over Fq and B be an (l′, l′) permutation over Fq. The function F = B ◦F ◦A is called an encoded function of F, and A and B are called the input and output encodings respectively. Deﬁnition 2. Let F = F (t) ◦F (t−1) ◦· · ·◦F (1) be a vectorial function over Fq. An encoded implementation of F, denoted by F, is an encoded function of F composed of encoded functions of F (i), that is, F = F (t) ◦· · · ◦F (1) = (B(t) ◦F (t) ◦A(t)) ◦· · · ◦(B(1) ◦F (1) ◦A(1)) , where the input and output encodings (A(i), B(i)) are permutations over Fq such that A(r+1) = B(r)−1. The ﬁrst and last encodings (A(1), B(t)) are called the external encodings. where the input and output encodings (A(i), B(i)) are permutations over Fq such that A(r+1) = B(r)−1. The ﬁrst and last encodings (A(1), B(t)) are called the external encodings. Deﬁnition 3. Let F be an (l, l′) function over Fq. A (l + l′, l′′) function T is called an implicit function of F if it satisﬁes T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 ⇐⇒F(u1, u2, . . . , ul) = v1, v2, . . . , vl′ . 5 Design of the Winning Challenges In this section, we describe the designs of the two winning challenges of the WhibOx contest: Challenges 226 and 227. The designs of both challenges were inspired from the white-box implicit framework [RVP22], which allows encoding the whole state with large aﬃne permutations eﬃciently. We implemented both challenges with the same methodology; they only diﬀer in some additional countermeasures used. As mentioned in Sect. 2, in the WhibOx contest, a challenge gains strawberries quadrat- ically with time before being broken. The rule is that challenges that are either smaller, faster, or less memory-consuming gain strawberries faster. As a result, we strategically 535 Barbu et al. posted two challenges with diﬀerent trade-oﬀs between security level and implementa- tion cost. Challenge 227, our lightweight variant, was the winning implementation of the contest, obtaining the highest number of strawberries (20.39). On the other hand, Challenge 226, our hardened but heavier variant, achieved second place in the contest with the second-highest number of strawberries (11.19). However, it stood unbroken for the longest time (35 hours). Note that these challenges were speciﬁcally built for the WhibOx contest, where attackers did not know the design. Against an attacker who knows the design details, these challenges are easy to break once reverse-engineered. This section ﬁrst introduces the implicit framework, then describes the shared design approach of both challenges, and ﬁnally explains the additional countermeasures used in each challenge. For access to the underlying software used to build these challenges, please contact the authors from the team zerokey. 5.1 Implicit White-box Implementations An implicit implementation of F with underlying encoded implementation F is a set of quasilinear implicit functions {T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). 5.1 Implicit White-box Implementations In this case, T is said to be quasilinear if for any (u1, u2, . . . , ul) ∈(Fq)l, the function (v1, v2, . . . , vl′) 7→T(u1, u2, . . . , ul, v1, v2, . . . , vl′) is aﬃne over Fq. The following lemma from [RVP22] describes how the composition of aﬃne permutations translates to implicit functions. 536 Attacks and Designs from CHES 2021 Challenge Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′) function of F. Let A be an aﬃne (l, l) permutation over Fq, B be an aﬃne (l′, l′) permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦ (A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′) function of F. Let A be an aﬃne (l, l) permutation over Fq, B be an aﬃne (l′, l′) permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦ (A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. The quasilinear property allows the implicit evaluation of F in a point (u1, u2, . . . , ul) by solving the aﬃne system T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 for the variables v1, v2, . . . , vl′. We are ready to present the deﬁnition of an implicit implementation. Deﬁnition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. An implicit implementation of F with underlying encoded implementation F is a set of quasilinear implicit functions {T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). Deﬁnition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. 5.2 White-boxing ECDSA Signature Algorithm Using the Implicit Frame work In the WhibOx contest, designers submitted white-box implementations of the ECDSA signature algorithm on the NIST P256 curve. As opposed to the standard ECDSA algorithm (cf. Algorithm 1), the algorithm for the WhibOx contest (hereafter denoted by E) takes as input the 256-bit message digest. The private key is not an input of the algorithm; it is freely chosen by the designer, but it is ﬁxed (hard-coded) in the implementation. Algorithm 2 depicts a high-level overview of this deterministic variant of ECDSA, where the deterministic nonce derivation mechanism is chosen freely by the designer. Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest Input : 256-bit message digest e Output : the signature (r, s) 1 state ←e 2 k, state ←NonceDerivation(state) 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest Input : 256-bit message digest e Output : the signature (r, s) 1 state ←e 2 k, state ←NonceDerivation(state) 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Ret ( ) The main steps of E can be represented by the functions E(1) and E(2). The Fp-function E(1) is given by E(1)(e) = (Rx, k, e) , (7) (7) which takes as input e ∈Fp and computes the scalar multiplication R = [k]G over Fp. On the other hand, the Fn-function E(2) can be written as E(2)(R′ x, k′, e′) = (r, s) , (8) (8) which takes as input (R′ x, k′, e′) = (Rx mod n, k mod n, e mod n) and computes (r, s) = (R′ x, k−1(e + rd)) over Fn. Inspired from the implicit framework, we built the white-box implementations of Challenges 226 and 227 by encoding E(1) and E(2) with aﬃne permutations and obtaining low-degree implicit round functions of E(1) and E(2), the encoded functions of E(1) and E(2). We will ﬁrst describe the implicit implementation of E(1) and then that of E(2). 537 Barbu et al. 5.2.1 White-boxing the Scalar Multiplication To build an implicit implementation of E(1), we need ﬁrst to decompose E(1) as the composition of Fp-functions that we call round functions. Then we explain how to encode these round functions and how to obtain low-degree quasilinear implicit functions of the encoded round functions. Decomposing E(1) into round functions. The function E(1)(e) = (Rx, k, e), mainly consists of the scalar multiplication r = [k]G of the nonce k and the point G. For the scalar multiplication, we perform the following subroutine. First, we precompute and store a list of t random point pairs on the curve, i.e., (Gi,0 = [ki,0]G, Gi,1 = [ki,1]G) for 1 ≤i ≤t . Then, for each pair we select one of the two points together with its logarithm, denoted as (Gi,bi, ki,bi), where bi ∈{0, 1} and 1 ≤i ≤t . We add the selected points and the selected logarithms, obtaining the scalar multiplication G1,b1 + · · · + Gt,bt = [k1,b1 + · · · + kt,bt]G = [k]G , (9) (9) where k = k1,b1 + · · · + kt,bt. This selection is done in a deterministic way depending on the bits (e1, e2, . . . , e256) of the hash e, the only source of entropy in the algorithm. Moreover, the selection is done with Fp-arithmetic operations rather than with conditional instructions, so that each iteration only performs Fp operations. The subroutine is given in Algorithm 3. It is worth pointing out that the values ki,j are chosen such that the sum of max(ki,0, ki,1) for all i is always smaller than n. That is, we have k < n. Hence, r and s are never 0. In this way, we avoid the trivial case, i.e., avoid going to Step 7 in Algorithm 2. Algorithm 3: Round-based scalar multiplication used in E(1) Input : the bits (e1, e2, . . . 5.2.1 White-boxing the Scalar Multiplication , e256) of the hash e (little-endian order) Output : x-coordinate of [k]G and the message-dependent scalar k /* Round 1: input e1, k1,0, k1,1, G1,0, G1,1 embedded values / 1 R ←[1 −e1]G1,0 + [e1]G1,1 2 k ←(1 −e1)k1,0 + e1k1,1 / Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values / 3 for 2 ≤i ≤t do 4 R ←R + [1 −ei]Gi,0 + [ei]Gi,1 5 k ←k + (1 −ei)ki,0 + eiki,1 6 end 7 return Rx, k // (Rx, Ry) = R = [k]G / Round 1: input e1, k1,0, k1,1, G1,0, G1,1 embedded values 1 R ←[1 −e1]G1,0 + [e1]G1,1 k ←(1 )k + k [ ] , [ ] , 2 k ←(1 −e1)k1,0 + e1k1,1 /* Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values 3 for 2 ≤i ≤t do /* Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values 3 for 2 ≤i ≤t do 4 R ←R + [1 −ei]Gi,0 + [ei]Gi,1 5 k ←k + (1 −ei)ki,0 + eiki,1 6 end // (Rx, Ry) = R = [k]G 7 return Rx, k By considering the precomputed points Gi,j and their logarithms ki,j as ﬁxed values and by representing the elliptic curve additions by operations over Fp, we can represent E(1) given by Algorithm 3 as an iterated function over Fp, that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) , (10) (10) where each (4, 4) round function F (i) is given by the following component functions F (i) 1,2(u1, u2, u3, u4) = (u1, u2) + [1 −ei]Gi,0 + [ei]Gi,1 F (i) 3 (u1, u2, u3, u4) = u3 + (1 −ei)ki,0 + eiki,1 , F (i) 4 (u1, u2, u3, u4) = u4 + ei2i . (11) (11) F (i) 4 (u1, u2, u3, u4) = u4 + ei2i . 538 Attacks and Designs from CHES 2021 Challenge Note that the input value of F (1) is (0, 0, 0, 0), and each round function F (i) takes the hash bit ei as an additional input value. The pair of component functions F (i) 1,2 return a point on the elliptic curve with F (i) 1 and F (i) 2 the x- and y- coordinates respectively. 5.2.1 White-boxing the Scalar Multiplication The component function F (i) 3 updates and returns the current nonce, while F (i) 4 updates the current hash. The hash e is recomputed so that E(1) can output the hash e and provide it in an encoded form to E(2) . Encoding the round functions. To protect the round functions, we encode each round with random Fp-aﬃne permutations A(i), obtaining the encoded round functions F (i) = A(i) ◦F (i) ◦(A(i−1))−1, 1 ≤i ≤t . (12) (12) In other words, the input and output encodings of F (i) are (A(i−1))−1, A(i) , and the composition of the round functions cancels all intermediate encodings except (A(0))−1 and A(t), that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity g to preserve the input-output behaviour of E. E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) is the number of rounds The input encoding (A(0))−1 of F (1) is set as the identity E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) (13) where t is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity mapping to preserve the input-output behaviour of E. Obtaining the implicit round functions. Now we proceed to obtain an implicit round function T (i) of each encoded round function F (i). To this end, we ﬁrst show how to derive an implicit function of the elliptic curve addition. Let ADD(Px, Py, Qx, Qy) = (Rx, Ry) be the vectorial Fp-function denoting the elliptic curve addition P + Q = R where P and Q are not the point at inﬁnity and where P and Q have diﬀerent x-coordinates4. In this case, R can be written as [KL14] Rx = (Qy −Py)2((Qx −Px)2)−1 −Px −Qx Ry = (Qy −Py)(Px −Rx)(Qx −Px)−1 −Py . (14) (14) From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j, and the probability that these additions involve points at inﬁnity or points with the same x-coordinate is negligible. 5.2.2 White-boxing the Computation of s Now we turn our attention to E(2), the second step of the signing algorithm, where we compute r = Rx mod n and s = k−1(e + dRx) mod n, and output the signature (r, s). As opposed to E(1), we do not decompose E(2) but build a single (vectorial) quasilinear implicit function of E(2) = E(2) ◦(A(t))−1, the encoded version of E(2). ( ) The vectorial Fn-function T (t+1) deﬁned as ( ) The vectorial Fn-function T (t+1) deﬁned as ( T (t+1) 1 (Rx, Ry, k, e; s, r) = ks −e −dRx T (t+1) 2 (Rx, Ry, k, e; s, r) = r −Rx (19) (19) is a quasilinear implicit function of E(2). In other words, the polynomial system T (t+1) = {T (t+1) 1 , T (t+1) 2 } implicitly deﬁnes E(2) because (s, r) = E(2)(Rx, Ry, k, e) if and only if T (t+1)(Rx, Ry, k, e; s, r) = 0. Moreover, the system is aﬃne in r and s, so after plugging in values for Rx, Ry, k and e, the system can be solved for r, s eﬃciently. The encoded version E(2) gets as input u = A(t)(Rx, Ry, k, e), where A(t) is the aﬃne function that protects the last round of E(1). By Lemma 1, we build the implicit round function of E(2) as T (t+1)(u; s, r) = M · T (t+1)((A(t))−1(u); s, r) , (20) (20) where (A(t))−1 is the inverse of A(t) mod n, and where M is a random invertible 2-by-2 matrix mod n. The function T (t+1) is quasilinear, and we can implicitly evaluate E(2) on input u = A(t)(Rx, Ry, k, e) by plugging u in the ﬁrst slot of T (t+1) and solving the remaining system (which is aﬃne) for r and s over Fn. However, the fact that E(1) works in Fp while E(2) works in Fn causes a problem. The input to E(2) is u = A(t)(Rx, Ry, k, e) reduced by mod p, so (A(t))−1(u) is in general not equal to (Rx, Ry, k, e) mod n if there are overﬂows in the computation of u. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. The white-box implementations of Challenges 226 and 227 contain this implicit imple- mentation of E(1), with underlying encoded implementation E(1), given by the t implicit round functions {T (1), . . . , T (t)} in Eq. (18). Moreover, E(1) is evaluated in our white-box implementations by implicitly evaluating the encoded round functions F (i). In other words, given the output u of the round i−1, the output v of the ith round is computed by ﬁnding the solution of the aﬃne system T (i)(u; v) = 0 for v. Barbu et al. is a quasilinea The white- mentation of E round function implementatio given the outp the solution of 5.2.2 White Now we turn compute r = As opposed to implicit functi The vector is a quasilinea {T (t+1) 1 , T (t+1) 2 T (t+1)(Rx, Ry in values for R The encode function that function of E( where (A(t))− matrix mod n on input u = remaining syst However, t input to E(2) equal to (Rx, R vector of overﬂ then (A(t))−1( aﬃne map A(t To deal wit vector o and s If the guess is r, s will be rec to get a candi output the ﬁrs need to protec If A(t) was would be very 539 Barbu et al. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. 5.2.1 White-boxing the Scalar Multiplication (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations (Px + Qx + Rx)(Qx −Px)2 = (Qy −Py)2 (15) (Ry + Py)(Qx −Px) = (Qy −Py)(Px −Rx) (16) (15) (16) (15) (16) (16) hold. Note that these relations have degree 3 (degree 1 over the variables Rx and Ry), while Eq. (14) has a high degree due to the inversion over Fp. g p Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) deﬁned by ( ) p Thus, the function IMP(Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) deﬁned by IMP0 = (Qy −Py)2 −(Px + Qx + Rx)(Qx −Px)2 IMP1 = (Qy −Py)(Px −Rx) −(Ry + Py)(Qx −Px) (17) (17) is a quasilinear implicit round function of ADD with degree 3, assuming none of the points is the point at inﬁnity and assuming the x-coordinates of the points are diﬀerent. From the above implicit function of the elliptic curve addition, it is easy to derive a quasilinear implicit function T (i) of each round function F (i). Then, we sample a linear permutation M (i) for each round i, and by Lemma 1 the function T (i) = M (i) ◦T (i) ◦ (A(i−1))−1, (A(i))−1 (18) (18) 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j, and the probability that these additions involve points at inﬁnity or points with the same x-coordinate is negligible. 5.2.2 White-boxing the Computation of s Let o be the vector of overﬂows mod p, such that u = A(t)(Rx, Ry, k, e) −po , (21) (21) then (A(t))−1(u) = (Rx, Ry, k, e) −pL−1 t (o) mod n, where Lt is the linear part of the aﬃne map A(t) (i.e., A(t)(x) = Lt(x) + c for some constant term c). To deal with this problem, we correct for the overﬂow mod p by guessing the overﬂow vector o and setting u′ = u + po before plugging u′ into T (t+1)(u; s, r) to solve for (r, s). If the guess is correct, then u′ is equal to A(t)(Rx, Ry, k, e) over the integers, so the correct r, s will be recovered. Therefore, we repeatedly run the last step with random guesses of o to get a candidate signature (r, s). Then we run the veriﬁcation algorithm on (r, s) and output the ﬁrst (r, s) for which the veriﬁcation algorithm succeeds. Note that we do not need to protect the veriﬁcation algorithm because it does not use secret information. If A(t) was a random aﬃne map with entries of size up to p, then guessing o correctly would be very unlikely. Therefore, we choose the aﬃne map A(t) with small entries. For Attacks and Designs from CHES 2021 Challenge 540 xample, we could use A(t)(Rx, Ry, k, e) =     1 0 1 2 1 1 2 0 0 1 2 1 1 2 0 1         Rx Ry k e    + c . (22) (22) With this choice, the weight of each row is four, so there are at most four overﬂows mod p in each entry of u, which means o can be guessed more easily. Not all guesses are equally likely, (e.g., o = [4, 4, 4, 4] only occurs if Rx, Ry, k, e are all quite big, which is unlikely). Rather than ineﬃciently guessing o ∈[0, 4]4 at random, we precompute a list of guesses L ordered from more likely to be correct to less likely, and we iterate through the list of guesses in that order. The white-box implementations of Challenges 226 and 227 contain the implicit function T (t+1), which allows the implicit evaluation of E(2), together with the correction for the overﬂow mod p described above and summarized in Algorithm 4. 5.2.2 White-boxing the Computation of s Note that the severe restriction on the size of the entries of A(t) makes the conversion from Fp to Fn one of the most vulnerable points in the white-box implementation. In particular, an attacker knowing the speciﬁcations of the design can easily recover A(t) by exhaustive search if no additional countermeasures are used. Algorithm 4: White-box implementation of ECDSA signature algorithm for winning challenges Input : 256-bit hashed message digest e Output : the signature (r, s) 1 e ←e mod p 2 (v1, v2, v3) ←E(1)(e) // implicit evaluation 3 for o in L do 4 (u1, u2, u3) ←(v1, v2, v3) + p · o 5 (r, s) ←E(2)(u1, u2, u3) // implicit evaluation 6 if VerifySignature(r, s, e) = valid then 7 return (r, s) 8 end 9 end Algorithm 4: White-box implementation of ECDSA signature algorithm for winning challenges // implicit evaluation 5https://tigress.wtf/transformations.html 6https://github.com/CryptoExperts/whibox_contest_submission_server 5.3 Additional Countermeasures The representation of the implicit round functions as systems of multivariate polynomials allows applying countermeasures from multivariate public-key cryptosystems. In fact, Challenges 227 and 226 only diﬀer in the additional countermeasures used. In particular, we considered two techniques. First, we obfuscated the components (seen as polynomials) of the implicit round functions T (i) by multiplying them with random polynomials in the input variables. Note that the multiplication of input variables preserves the quasilinear property. Moreover, the image of a random polynomial is non-zero with high probability, and multiplying an equation with a non-zero value does not change its solution set. In the unlikely case that one of the added polynomials vanishes, the output of the corresponding implicit function will be invalid, and no valid signature will be obtained. To prevent this extreme case, we made the ﬁrst implicit round function dependent on an initial value; if no valid signature is found, we simply repeated the whole process with a diﬀerent initial value. 541 Barbu et al. This ﬁrst technique increases the degree of the implicit round functions, signiﬁcantly increasing the implementation size. Thus, for the lightweight Challenge 227 we only applied this technique to raise the degree of the components to the total degree of the functions, but for Challenge 226 we multiplied with polynomials of higher degree to increase the total degrees of the implicit round functions. The ﬁnal degrees are listed in Tables 3 and 4. The second technique we used was adding additional variables and components to the implicit round functions but preserving the input-output behaviour of the underlying encoded round functions. In particular, to avoid the bias in the most signiﬁcant part of the nonce k due to the constraint k < n (see Section 5.2.1), we duplicated the nonce variable and its equations so that E(1) outputs an additional nonce variable k′ similarly to k, k′ = k′ 1,b1 + · · · + k′ t,bt, X i max(k′ i,0, k′ i,1) < n , and E(2) uses the sum of the nonce variables k + k′ as the ﬁnal nonce. On top of that, instead of e, the input L(e) is given to E(1) for some hard-coded low-degree encoding L, and its inverse L(−1) is composed to E(2) to recover e. 5.3 Additional Countermeasures Note that this is a minor trick since the encoding L is not merged or composed with other functions (as opposed to the other encodings A(i)), and the computation L(e) is done in clear. Since adding additional variables and equations also introduces signiﬁcant overhead in the implementation size, we only applied the second technique to Challenge 226. In particular, we added two variables and two equations in the implicit round functions of E(1), and two variables and one equation in those of E(2). We also used Tigress [Col] for both challenges to obfuscate the C source code. Tigress is an obfuscator for C language that protects programs against dynamic and static reverse engineering attacks. We used the transformations5 Flatten (ﬂattens the code to remove structured ﬂow), AntiTaintAnalysis (disrupts tools that make use of dynamic taint analysis), AddOpaque (adds opaque predicates), EncodeLiterals (replaces integers and strings with run-time expressions) and CleanUp (renames variables and functions). 5.4.2 Security Analysis Challenge 227 can be broken in several ways. Here, we explain how the attacks of Sect. 4 allow one to recover the secret key of Challenge 227 or why they do not work. Hooking shared libraries. During the implicit evaluation of E(2) for the valid input u = A(t)(Rx, Ry, k, e), the aﬃne system T (t+1)(u; s, r) is solved for r and s. By denoting the entries of M as M = m0 m1 m2 m3 , it is easy to see that this aﬃne system is given by the equations the equations ( c1s + c3r −c5 = 0 c2s + c4r −c6 = 0 (23) (23) where the coeﬃcients ci are given by c1 = m0k, c3 = m1, c5 = m0e −m0dRx −m1Rx c2 = m2k, c4 = m3, c6 = m2e −m2dRx −m3Rx . (24) (24) We stress that k, e, and Rx do not appear in the clear. They are expressed as linear combinations of the input u = A(t)(Rx, Ry, k, e) of E(2). Nevertheless, the coeﬃcients ci are operated in the clear during the Gaussian elimination, and the adversary can obtain their values. Some of these coeﬃcients are sensitive. In particular, if the attacker manages to ﬁnd c1 during the computation of two diﬀerent signatures (r, s) and (r′, s′), he may solve the following system of two equations with two unknowns (m0 and d) in Fn: ( m0(e + rd) = c1s m0(e′ + r′d) = c1s′ . (25) (25) Therefore, recovering the value c1 = m0k mod n for two diﬀerent signatures allows an attacker to compute the private key. Finding the interesting values inside the white-box may seem diﬃcult without a reverse engineering step. However, the attack turns out to be easily automated on challenges that use the GMP library, such as Challenge 227. Indeed, one of the coeﬃcients of s, say c1 = m0k, will be inverted modulo n during the resolution of the system, and ﬁnding this inversion is easy when one can simply trace the calls to the function mpz_invert(). The team TheRealIdeﬁx was thus able to eﬃciently apply this attack on Challenge 227 during the contest without any reverse engineering step. 5.4.1 Description Following the method described in Section 5.2, we built Challenge 227 (keen_ptolemy) as a lightweight white-box implementation. The only additional countermeasures from Section 5.3 included in Challenge 227 are the degree increase of each component to the total degree of the corresponding vectorial function and the code obfuscation by Tigress. Challenge 227 was the winning implementation of the WhibOx contest; it achieved the highest number of strawberries (20.39) and stood for 33 hours as the second-longest. ( ) Table 3 describes the memory complexity of Challenge 227 (after applying the additional countermeasures) by describing {T (1), . . . , T (t)} and T (t+1), the implicit round functions of E(1) and E(2) respectively. The number of coeﬃcients in Table 3 denotes the maximum number of non-zero coeﬃcients of a quasilinear vectorial function with a given number of input variables, components, and degrees. If each coeﬃcient is represented with 256 bits, {T (1), . . . , T (t)} and T (t+1) require in total roughly 4 MB. After obfuscating the code with Tigress, the size of the ﬁnal C source code of Challenge 227 is 4.4 MB. In a modern personal laptop with the environment6 provided by the competition, the size of the compiled binary is 4.42 MB, and the average running time and 542 Attacks and Designs from CHES 2021 Challenge Table 3: Information of the implicit round functions T (i) of Challenge 227. T (1) {T (2), . . . , T (t−1)} T (t) T (t+1) input variables 2+4 5+4 5+3 3+2 number of components 4 4 3 2 degree 3 3 4 2 number of coeﬃcients 27 × 4 130 × 4 255 × 3 18 × 2 Table 3: Information of the implicit round functions T (i) of Challenge 227. RAM consumed are 0.04 seconds and 6.14 MB respectively. The code obfuscation did not impact the running time but increase the binary size by 8% and the average RAM by 3%. 5.4.2 Security Analysis This attack may seem very speciﬁc, but multiplying the nonce with a constant may appear as an easy way to protect the inversion step, and could very well be used by designers. This attack shows it is not a robust countermeasure. 543 Barbu et al. Biased nonces. There exists a more generic way of breaking Challenge 227. Indeed, the way the ephemeral key is constructed (see Sect. 5.2.1) opens the way for an attack using lattice reduction techniques. Given that the ephemeral key k is obtained by summing 256 scalars ki,j according to each bit of the input, one can obtain the following signatures by selecting a couple of hashes (e0, ei), with e0 = 0 and ei = 2i :            s0 = 255 P j=0 kj,0 !−1 (e0 + r0d) mod n si = ki,1 + 255 P j=0,j̸=i kj,0 !−1 (ei + rid) mod n , (26) (26) which allows us to construct 256 equations involving only one of the ki,j: ki,1 + 255 P j=0,j̸=i kj,0 − 255 P j=0 kj,0 = s−1 i (ei + rid) −s−1 0 (e0 + r0d) mod n ki,1 −ki,0 = s−1 i ei −s−1 0 e0 + (s−1 i ri −s−1 0 r0)d mod n . (27) (27) Now, the additional constraint k < n lets us estimate that each ki,j is sampled from J0, ⌊n/256⌋K. Consequently, \|ki,1 −ki,0\|n = \|s−1 i ei −s−1 0 e0 + (s−1 i ri −s−1 0 r0)d\|n < j n 256 k , (28) (28) with \|y\|n := min a∈Z \|y −an\| to denote the distance of y ∈R to the closest integer multiple of n. with \|y\|n := min a∈Z \|y −an\| to denote the distance of y ∈R to the closest integer multiple of n We recognize in Eq. (28) an instance of the Hidden Number Problem (HNP) [BV96]. Indeed, we are given many HNP inequalities of the form: \|αti −ui\|n < j n 256 k , (29) (29) with ti = s−1 i ri −s−1 0 r0, ui = s−1 0 e0 −s−1 i ei and the hidden number α is the private key d. Solving HNP instances in the context of ECDSA given inequalities such as Eq. (29) has been described numerous times in the literature. We refer the reader to [JSSS20] for a more detailed description7. 7We also highlight that the authors of [JSSS20] made their code available at https://github.com/crocs- muni/minerva. 5.4.2 Security Analysis In particular, the authors detail the reduction of the HNP instance to a Closest Vector Problem instance in a speciﬁc lattice as well as the construction of this lattice. Finally, we use 75 relations such as Eq. (28) (out of the 255 we can establish) to build a lattice whose reduction allows us to recover the private key d. DCA. As explained in Sect. 4, we did not mount this side-channel attack during the contest. With the design of Challenge 227 in hand, we can see that it would have been unsuccessful, at least at the ﬁrst order, thanks to the linear masking scheme used to protect all the implementation. Fault injections. None of the faults injected on Challenge 227 during the contest were exploitable. This can be explained by the presence of the signature veriﬁcation that is used to check if the guess for the overﬂow between E1 and E2 is correct. If a fault is induced, the signature is rejected and recomputed. g Of course, a reverse engineering step could be performed to get rid of this veriﬁcation, but this would be quite time-consuming. Furthermore, even without this veriﬁcation, 544 Attacks and Designs from CHES 2021 Challenge the fault attack is still not trivial to perform because of the linear masking scheme. In particular, Rx is not manipulated directly in E2. It is expressed as a linear combination of the input A(t)(Rx, Ry, k, e), so modifying one of the shares would probably also fault e, k or Ry, making the resulting faulty signature unexploitable. 5.5.1 Description Challenge 226 (clever_kare) was the second white-box implementation that we built following the method described in Section 5.2 and including all the additional countermea- sures from Section 5.3. While this challenge stood for the longest (35 hours), Challenge 226 achieved the second-highest number of strawberries (11.19) due to its higher time and memory complexity than Challenge 227. Table 4 describes the memory complexity of {T (1), . . . , T (t)} and T (t+1) of Challenge 226 after applying the additional countermeasures. Given each coeﬃcient as a 256-bit value, {T (1), . . . , T (t)} and T (t+1) require in total roughly 15 MB. The impact of the additional countermeasures on the number of equations, the degree, and the number of variables can be seen by comparing this table with Table 3. Table 4: Information of the implicit round functions T (i) of Challenge 226. Table 4: Information of the implicit round functions T (i) of Challenge 226. T (1) {T (2), . . . , T (t−1)} T (t) T (t+1) input variables 2+6 7+6 7+5 5+2 number of components 6 6 5 2 degree 3 3 4 5 number of coeﬃcients 37 × 6 322 × 6 854 × 5 504 × 2 The size of the ﬁnal C source code of Challenge 226 is 17.54 MB, the size of the compiled binary is 15.44 MB, and the average running time and RAM consumed are 0.15 seconds and 17.27 MB, respectively. The code obfuscation did not signiﬁcantly impact the performance of Challenge 226; the running time, the binary size, and the average RAM increased by less than 1%. in d in order to obtain the secret key. in d in order to obtain the secret key. Therefore, this challenge can be easily broken once reverse-engineered. Nevertheless, such an attack is quite time-consuming, and resisting TheRealIdeﬁx’s automated attacks on ECDSA in the white-box contest is already an achievement considering that only 5 challenges resisted these attacks during the contest. 5.5.2 Security Analysis During the WhibOx contest, the team theRealIdeﬁx did not manage to break Challenge 226 with any of the automated attacks presented in Sect. 4.1. DCA and fault injection, which fail to break Challenge 227, are also not applicable to Challenge 226 since it is designed to be more secure. Moreover, the two attacks presented in Sect. 5.4.2 also fail to recover any secret information. Hooking shared libraries. As mentioned in Sect. 5.3, Challenge 226 implements an additional countermeasure which consists in multiplying the components of the implicit round functions T (i) with random polynomials in the input variables. Hence, the coeﬃcients of s in the system T (t+1)(u; s, r) for the valid input u are no longer ﬁxed multiples of k, and the attack cannot be mounted anymore. Biased nonces. Likewise, the additional countermeasures implemented in Challenge 226 makes the lattice attack described in Sect. 5.4.2 fail. The additional variable k′ alone would only reduce by 1 bit the bias observed in Eq. 29 and the attack would still be 545 Barbu et al. practical. However, without the knowledge of the encoding L introduced in this challenge it is impossible to exhibit such a bias leading to key recovery. practical. However, without the knowledge of the encoding L introduced in this challenge it is impossible to exhibit such a bias leading to key recovery. As explained, none of the automated attacks that the TheRealIdeﬁx team used during the contest were successful on Challenge 226. Nevertheless, with the design in hand, one could easily break this challenge. Indeed, knowing that the matrix M of the last aﬃne encoding A(t) contains small entries, the attacker could, for example: • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note that v1 −v2 contains the nonce diﬀerence κ = k1 −k2. • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note that v1 −v2 contains the nonce diﬀerence κ = k1 −k2. • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 = (M ′)−1(u1 −u2) and check if one of its entries κ satisﬁes (κG)x = r1 −r2. 5.5.2 Security Analysis • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 = (M ′)−1(u1 −u2) and check if one of its entries κ satisﬁes (κG)x = r1 −r2. • Solve the equation s−1(e1 + r1d) −s−2(e2 + r2d) = κ (30) (30) s−1(e1 + r1d) −s−2(e2 + r2d) = κ 6 Conclusion This work describes several attack techniques and designs used in the WhibOx 2021 contest. We explained the attack methods used by the team TheRealIdeﬁx, which broke the most challenges, and we showed the success of each method against all the implementations in the contest. Fault attacks were the most eﬃcient and eﬀective ones; collision and lattice attacks were slightly less eﬃcient, and hooking succeeded against weak implementations only. Among the white-box implementations that resisted these attacks, the one with the highest score was Challenge 226 (clever_kare). This challenge, together with Challenge 227 (keen_ptolemy), was submitted by the team zerokey, and they obtained the second- highest and the highest score in the contest, respectively. In this work, we described the design methodology of these two challenges, which was inspired by the implicit white-box framework. The large number of implementations broken by our automated attacks and the fact that no challenge survived more than two days show that securing ECDSA in the white-box model is a challenging problem. White-box attacks beneﬁt from the huge progress in side-channel and fault attacks against ECDSA implementations, but not much research has been done on the design part. To this end, our designs provide insightful examples for future works, and our attacks highlight the weak points future research should address. One of the main challenges speciﬁc to white-boxing ECDSA is the conversion from Fp to Fn. While grey-box countermeasures can protect this step (e.g., with Arithmetic-to-Boolean and Boolean-to-Arithmetic mask conversions), these techniques rely on randomness, which is ineﬀective in white-box implementations. In particular, the conversion from Fp to Fn is one of the weakest points in our designs, and further research in white-boxing the ﬁeld conversion is needed. 546 Attacks and Designs from CHES 2021 Challenge Acknowledgment The authors would like to thank the other members of the TheRealIdeﬁx team: Yannick Bequer, Luk Bettale, Laurent Castelnovi, Thomas Chabrier, Nicolas Debande, Roch Lescuyer, Sarah Lopez and Nathan Reboud. Adrián Ranea is supported by a PhD Fellowship from the Research Foundation – Flanders (FWO) under grant No. 11E1921N. Chaoyun Li is an FWO post-doctoral fellow under grant No. 1283121N. Ward Beullens is an FWO post-doctoral fellow under grant No. 1S95620N. 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A Attacks Summary Table g Challenge Hooking Collision Fault Lattice Key 3 ✓ ✓ ✓ ✓ 45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE 4 ✓ ✓ ✓ ✓ 22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D 8 ✓ ✓ ✓ ✓ F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE 10 ✓ ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC 11 ✓ ✓ ✓ ✓ E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38 12 ✓ ✓ 773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED 13 ✓ ✓ 034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE 15 ✓ ✓ 3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7 16 ✓ 23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A 32 ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 33 ✓ CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242 34 ✓ ✓ ✓ 70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257 36 ✓ 10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A 38 ✓ 70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE 42 ✓ 1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65 44 ✓ B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C 45 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 50 ✓ 7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15 54 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790 55 ✓ ✓ ✓ ✓ 00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790 57 ✓ ✓ ✓ ✓ 7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083 58 ✓ ✓ ✓ BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255 61 ✓ ✓ ✓ F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95 62 ✓ ✓ ✓ 0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED 66 ✓ ✓ ✓ 8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC 70 ✓ ✓ ✓ 21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C 71 ✓ ✓ ✓ 588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF 72 ✓ ✓ ✓ B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF 73 ✓ ✓ ✓ 4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F 74 ✓ ✓ ✓ 12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816 76 ✓ ✓ ✓ F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318 77 ✓ ✓ ✓ A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93 78 ✓ ✓ ✓ 511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F 79 ✓ ✓ ✓ 595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686 80 ✓ ✓ ✓ 8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52 81 ✓ ✓ ✓ F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE 84 ✓ ✓ ✓ 36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7 85 ✓ ✓ ✓ ✓ AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8 87 ✓ ✓ ✓ 9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088 89 ✓ ✓ C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482 94 ✓ ✓ A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90 96 ✓ ✓ AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0 97 ✓ ✓ ✓ ✓ 0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829 100 ✓ 7F58EDB783C1F3FA7FF424CF7F5DF6D4BCDCF18D8A98CE4559EC22EB17030578 101 ✓ 25D31D3AFF5773799ECF43DEC1882B8F05D9231697BDDA5482DE05B14FB8A63B 103 ✓ ✓ CC977E0748722D615B845C1B10EA554B69DFCA640440CA5C468BBEF84B8C0442 104 ✓ ✓ ✓ 638C9DFBF9F376CBB3E3B01DF27960EC53A689D2FF4DFF23D97EE5351ED4A3D0 105 ✓ ✓ ✓ D29E9D130016D930BF830BCAD071BC6503F877FB207922A9E495CF71A79631FE Key C H C Fa La K 3 ✓ ✓ ✓ ✓ 45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE 4 ✓ ✓ ✓ ✓ 22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D 8 ✓ ✓ ✓ ✓ F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE 10 ✓ ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC 11 ✓ ✓ ✓ ✓ E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38 12 ✓ ✓ 773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED 13 ✓ ✓ 034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE 15 ✓ ✓ 3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7 16 ✓ 23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A 32 ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 33 ✓ CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242 34 ✓ ✓ ✓ 70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257 36 ✓ 10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A 38 ✓ 70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE 42 ✓ 1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65 44 ✓ B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C 45 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 50 ✓ 7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15 54 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790 55 ✓ ✓ ✓ ✓ 00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790 57 ✓ ✓ ✓ ✓ 7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083 58 ✓ ✓ ✓ BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255 61 ✓ ✓ ✓ F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95 62 ✓ ✓ ✓ 0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED 66 ✓ ✓ ✓ 8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC 70 ✓ ✓ ✓ 21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C 71 ✓ ✓ ✓ 588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF 72 ✓ ✓ ✓ B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF 73 ✓ ✓ ✓ 4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F 74 ✓ ✓ ✓ 12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816 76 ✓ ✓ ✓ F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318 77 ✓ ✓ ✓ A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93 78 ✓ ✓ ✓ 511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F 79 ✓ ✓ ✓ 595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686 80 ✓ ✓ ✓ 8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52 81 ✓ ✓ ✓ F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE 84 ✓ ✓ ✓ 36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7 85 ✓ ✓ ✓ ✓ AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8 87 ✓ ✓ ✓ 9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088 89 ✓ ✓ C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482 94 ✓ ✓ A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90 96 ✓ ✓ AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0 97 ✓ ✓ ✓ ✓ 0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829 100 ✓ 7F58EDB783C1F3FA7FF424CF7F5DF6D4BCDCF18D8A98CE4559EC22EB17030578 101 ✓ 25D31D3AFF5773799ECF43DEC1882B8F05D9231697BDDA5482DE05B14FB8A63B 103 ✓ ✓ CC977E0748722D615B845C1B10EA554B69DFCA640440CA5C468BBEF84B8C0442 104 ✓ ✓ ✓ 638C9DFBF9F376CBB3E3B01DF27960EC53A689D2FF4DFF23D97EE5351ED4A3D0 105 ✓ ✓ ✓ D29E9D130016D930BF830BCAD071BC6503F877FB207922A9E495CF71A79631FE 551 Barbu et al. 9Challenges 54, 55, 57, 58, 61, 62, 66, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 84, 87, 89, 94, 96, 103, 104, 105, 107, 136 and 139 are not deterministic. A Attacks Summary Table Challenge Hooking Collision Fault Lattice Key 107 ✓ ✓ ✓ 4E420B6AA9E9F07F19CF7ED97497871C1223BC2A68E83716575C235DE6D63E17 108 ✓ 60609404F0B9086D3A995AF0680D048724CF2B1AF2B33CEA8DD4AF4B62A5DDBB 114 ✓ ✓ ✓ 0000000000000000000000000000000000000000000000000000000000000005 127 ✓ 1144D82B9568581405D10CF8B219FF7E94E4559E0832B06056F1F87D43C75777 135 ✓ ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33576B3123CECBB6406837BF51F5 136 ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33476B3123CECBB6406837BF51F4 139 ✓ ✓ ✓ 000000000000000000000000000000004319055358E8617B0C46353D039CDAA9 153 ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875C3 157 ✓ ✓ ✓ ✓ F04DBFD1147F9D43747538C1C9256DD2BC20562F9D92B83E9AFA751299B160A4 165 ✓ ✓ ✓ 84DAF8B6620FC6669BF1EE264D1B214A4FBECACEADDFDC0DCBC89CF4B6E3232B 166 ✓ ✓ C746740A4A6BCBD462D9041023A0FEF5CCF0328FF80D9C50132682030D77D33C 172 ✓ ✓ ✓ 285E57F7BDDAAA6201D8870A0B9B168C7A5D8200085F62504EE3EBFCC11EF150 174 ✓ ✓ ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 185 ✓ ✓ ✓ ✓ 7729EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 187 ✓ ✓ ✓ ✓ 7779EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 192 ✓ 09302BDFA5313312B9A665316F7E9365DCC57DA7E21FD8612CDCD553BABB51FE 193 ✓ E0FE06BE0684455EDD2F5134A3AE8B9F6852561C821672FA16606986233BF811 209 ✓ 6E3A09F8EC613B8A524F7608CB80B2D3C510E27506AD84FA14C3B6D018E659F7 212 ✓ D663E156F036F11D4E73CC0EC09A952DEAED316947DF73EB28467EC623C5740D 2268 6F1D9093F3D5AE7C5F133659295914C9AF22E54B4ADE38CA421CA9BBD3D48A50 227 ✓ ✓ ADA6C6A1049825989811C9495D83681A68C67AB5E8EBDDC126CEE77056A7BB27 228 ✓ EA7BA345EB9D99F54261D01AE6319B184769E5745621706D77018E0DB46DDAFA 231 ✓ ✓ ✓ ✓ 8ADE24EE6413C6E408784DBB4D81D04F33238AB503CBE35C77400517EE5ABC96 235 ✓ ✓ ✓ ✓ 000000000000000000000000000000000000000000000000D0FACADE0DEFACED 251 ✓ ✓ ✓ DDE098A74086ECBB4DBA1848511BEA924145D1A9ED2EC9E64E0C5934BAAC97AE 253 ✓ ✓ B22DB44C9E66D567B3B2CBB3C720309D1EEAD38717017F5E79F05274F289A52C 256 ✓ F1662664E7E303740C0CA3927F9870A789978DAE95892302E73C85E3993B4CC9 261 ✓ ✓ 3266C9F6379DFDAE4AA763E8E6BA94526504CA364C482306829D4BF1E97BFF92 262 ✓ A0F00DCAA5DAB169FD4DFE2186BCBCBD22631AB68BFEFF1FC19306174EAF8970 264 ✓ D0EE17829A397C18074EA3888057AE815B5336773F9668E6CE4464D4B2B05F1F 267 ✓ ✓ ✓ ✓ C17536B60BCF94326A9C8CA17E0FC4EDBD76822532B350E8237CA2D8CF9C74B0 274 ✓ ✓ ✓ ✓ 0080ECD2A00080ECD2A00080ECD2A00080ECD2A00080ECD2A00080ECD2A00080 283 ✓ 79FE8D884DC2F7440824DE79C9F7C513C2B4549631D343523C73CB8F85983A4F 299 ✓ ✓ ✓ ✓ 3A0F803A874CD5B826023F2073FF200371D399E76E66B05E1241AA787B0564D6 304 ✓ EE8942A527CA1A58B8A8EA369441CB8518836DDB98F6380B8008B6053BC8182C 305 ✓ 311EA92FBCDD3C6A29D269589A9E71F13A231FFEC85FF36B398967EC9934805E 307 ✓ ✓ FA3FCDE70679E7E44391F7157E2B5822F5B9B9C93ADD95C2BA90FF4B95C8A6BB 308 ✓ ✓ 84CCCAA904CB397F41A36FF9E05D4EB6C58B8E203E02373C465B6C3F03280C82 314 ✓ 7E045DB89DD77BD6B2EAF23172A89A656B5084748642DB82BBAE931E737560C2 320 ✓ ✓ ✓ D235C2B1D089F158A0AE4E7799C2DCA9985E3D44C8F243BAD8B5E1A4EB647E1B 321 ✓ ✓ ✓ ✓ BA15757E1B0DB122F349C0C50C97071A4CFFF4FD2875B4A092FBDD985E8595DE 323 ✓ ✓ ✓ ✓ C7491BBC530FFA9DDCF3E7D732536FACF04239693D549C50DDAD41931A6244C2 325 ✓ ✓ 6902CD65AE124A45B9DD16BAEFD26D9CFFB5C291DC1E256D9CCE17BE3CF11775 327 ✓ 2BC6F2467C7F8DFA164EDC68DDCF65E795B8A2153182565481D8D6878D80EA81 328 ✓ 37170CF851A89AAFD3511234BE2B96C89B783A44D7A6C22E9A150872809F7CDF 335 ✓ EC90CC12DC70E3C5A7D47B6083A988F3F6C6B2B63EB0D8991F84B19E21ACC061 3368 D2E2AE325946DDADC9A67A2DFE8EE74065D8D39968707F5D818D7B62910894EA 345 ✓ 3266C9F6378DFDAE4AA763E9166B131E6514CA364C482306829D4BF1E97BFF92 346 ✓ 5EE43950837D0ABA419FE5B586D1A7AA44DDAAC6327DADC3133F18A850211B9F Challenge Hooking Collision Fault Lattice Key 107 ✓ ✓ ✓ 4E420B6AA9E9F07F19CF7ED97497871C1223BC2A68E83716575C235DE6D63E17 108 ✓ 60609404F0B9086D3A995AF0680D048724CF2B1AF2B33CEA8DD4AF4B62A5DDBB 114 ✓ ✓ ✓ 0000000000000000000000000000000000000000000000000000000000000005 127 ✓ 1144D82B9568581405D10CF8B219FF7E94E4559E0832B06056F1F87D43C75777 135 ✓ ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33576B3123CECBB6406837BF51F5 136 ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33476B3123CECBB6406837BF51F4 139 ✓ ✓ ✓ 000000000000000000000000000000004319055358E8617B0C46353D039CDAA9 153 ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875C3 157 ✓ ✓ ✓ ✓ F04DBFD1147F9D43747538C1C9256DD2BC20562F9D92B83E9AFA751299B160A4 165 ✓ ✓ ✓ 84DAF8B6620FC6669BF1EE264D1B214A4FBECACEADDFDC0DCBC89CF4B6E3232B 166 ✓ ✓ C746740A4A6BCBD462D9041023A0FEF5CCF0328FF80D9C50132682030D77D33C 172 ✓ ✓ ✓ 285E57F7BDDAAA6201D8870A0B9B168C7A5D8200085F62504EE3EBFCC11EF150 174 ✓ ✓ ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 185 ✓ ✓ ✓ ✓ 7729EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 187 ✓ ✓ ✓ ✓ 7779EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 192 ✓ 09302BDFA5313312B9A665316F7E9365DCC57DA7E21FD8612CDCD553BABB51FE 193 ✓ E0FE06BE0684455EDD2F5134A3AE8B9F6852561C821672FA16606986233BF811 209 ✓ 6E3A09F8EC613B8A524F7608CB80B2D3C510E27506AD84FA14C3B6D018E659F7 212 ✓ D663E156F036F11D4E73CC0EC09A952DEAED316947DF73EB28467EC623C5740D 2268 6F1D9093F3D5AE7C5F133659295914C9AF22E54B4ADE38CA421CA9BBD3D48A50 227 ✓ ✓ ADA6C6A1049825989811C9495D83681A68C67AB5E8EBDDC126CEE77056A7BB27 228 ✓ EA7BA345EB9D99F54261D01AE6319B184769E5745621706D77018E0DB46DDAFA 231 ✓ ✓ ✓ ✓ 8ADE24EE6413C6E408784DBB4D81D04F33238AB503CBE35C77400517EE5ABC96 235 ✓ ✓ ✓ ✓ 000000000000000000000000000000000000000000000000D0FACADE0DEFACED 251 ✓ ✓ ✓ DDE098A74086ECBB4DBA1848511BEA924145D1A9ED2EC9E64E0C5934BAAC97AE 253 ✓ ✓ B22DB44C9E66D567B3B2CBB3C720309D1EEAD38717017F5E79F05274F289A52C 256 ✓ F1662664E7E303740C0CA3927F9870A789978DAE95892302E73C85E3993B4CC9 261 ✓ ✓ 3266C9F6379DFDAE4AA763E8E6BA94526504CA364C482306829D4BF1E97BFF92 262 ✓ 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B Some Remarks on the Challenges Among the various submissions, we notice the following facts: • Challenges 15 and 16 have a very small code size, only 194 bytes! To obtain such tiny implementations, the designers use a ﬁxed nonce k = 1 (i.e. r = Gx) and a private key d such that dr ≡2i mod n. In such a case, the signature of a hash e is equal to (Gx, e + 2i). • Challenge 114 uses a very small private key, indeed d114 = 5 • Some designer teams modify a few bits only of the private key in several challenges (cf. Challenges 174, 185 and 187 for instance). In such a case, if one implementation is broken, then the private keys of the other challenges of the same team could be recovered by brute force search. • Despite what is indicated in the rules (cf. Sect. 2), some challenges are not deter- ministic9, i.e. the two signatures of the same message could be diﬀerent. All these challenges use the time() function to obtain some randomness. However, it is easy to hook such calls and return a constant value.
https://openalex.org/W2101652307	https://europepmc.org/articles/pmc544864?pdf=render	English	null	Desmoplastic Infantile Ganglioglioma: cytologic findings and differential diagnosis on aspiration material.	Cyto journal/CytoJournal	2,005	cc-by	4,391	Address: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA Address: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA Email: Oluwole Fadare* - oluwole.fadare@yale.edu; M Rajan Mariappan - rajm1@stanford.edu; Denise Hileeto - denise.hileeto@yale.edu; Arthur W Zieske - ArtZ@BaylorHealth.edu; Jung H Kim - jung.kim@yale.edu; Idris Tolgay Ocal - idris.ocal@yale.edu * C di h * Corresponding author Published: 11 January 2005 CytoJournal 2005, 2:1 doi:10.1186/1742-6413-2-1 Received: 27 November 2004 Accepted: 11 January 2005 Received: 27 November 2004 Accepted: 11 January 2005 © 2005 Fadare et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BioMed Central Abstract Background: Desmoplastic infantile ganglioglioma (DIG) is a rare WHO Grade I tumor of infancy that is characterized by large volume, superficial location, invariable supratentoriality, fronto- parietal lobe predilection and morphologically, by an admixture of astroglial and neuroepithelial elements in a desmoplastic milieu. With over 50 cases described, the histologic and radiographic spectrum of DIG has been well-characterized. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology; however, the cytologic features of this rare tumor have only been reported once previously. Case Presentation: We present herein cytomorphologic findings from the intraoperative aspiration of a typical case of DIG diagnosed in a 1-year-old male. As evaluated on a single liquid- based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells with eccentrically located hyperchromatic nuclei (which were occasionally binucleated) and abundant unipolar cytoplasm. Rare smaller astroglial cells were intermixed. Despite the tumor's characteristic desmoplastic histologic appearance, no stromal fragments were identified on the aspiration material. Conclusions: A differential diagnosis is presented and analyzed in detail and it is concluded that when these large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities. described, such that the clinical, radiologic and his- topathologic features of this tumor are now well-defined. An uncommon tumor that constituted less than 0.04% of all central nervous system (CNS) tumors in one series [2], DIG is classified as a Grade 1 tumor in the World Health Organization (WHO) classification of CNS tumors [3]. They most commonly occur in children less than 18 CytoJournal Open Access Page 1 of 6 (page number not for citation purposes) Background g The clinicopathologic features of 11 examples of a distinc- tive pediatric tumor designated desmoplastic supratentorial neuroepithelial tumors of infancy (also known as desmoplas- tic infantile ganglioglioma, [DIG]) were originally described by Vandenberg et al in 1987 [1]. Since that sem- inal report, at least 40 additional cases have been Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) http://www.cytojournal.com/content/2/1/1 CytoJournal 2005, 2:1 idase complex with antibodies ki-67 (dilution 1:320, DakoCytomation Corp, Carpinteria, CA), synaptophysin (dilution 1:600, DakoCytomation) and glial fibrillary acid protein [GFAP] (dilution 1:10, DakoCytomation). months of age [1] who typically present with symptoms related to an intracranial mass effect [3]. DIGs are gener- ally of large size, are solid to cystic, show a predilection for the frontal and parietal cerebral lobes, and are typically superficially located with at least focal attachment to the overlying dura [1-3]. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology. However, there is a dearth of information on the cytomorphologic features of these tumors [4]. To contribute information of possible utility in their pre-operative or intra-operative assessment, we report herein cytomorphologic features associated with a typical case of DIG. Case Presentation A one-year-old boy was noted to have a striking increase in head circumference as compared to a previous meas- urement. Neurological examination and developmental status were normal at that point. Within 2 weeks, the patient deteriorated rapidly, with poor mobilization, feeding and verbalization. He was brought to the emer- gency room where an emergent computed tomographic scan showed a large left hemispheric cerebral mass (Figure 1-1) with an underlying cystic component and a more superficial area of bright enhancement; the rest of the brain showed massive edema. He was emergently admit- ted and within 24 hours, a gross resection of the tumor was carried out. At surgery, following a parietal craniot- omy, 50–60 cc of straw colored fluid was aspirated from the cystic component through a taut dura. After the exci- sion of the dura, the bright area of enhancement previ- ously noted was an area of tumor attachment to the dura in the parietal region. Otherwise, the tumor showed a well-demarcated interface with the subjacent normal brain parenchyma and a complete gross resection was achieved. A follow-up magnetic resonance image at 12 months post-surgery showed no evidence of tumor recur- rence. Functionally, the patient was felt to have a mild right hemiparesis and some probable language delay, but otherwise showed no neurological deficits. Vascular structures or stroma were not present. Histologi- cally, the tumor was partially attached to the dura and was present in the subarachnoid space. The bulk of the speci- men was a variably cellular desmoplastic component whose predominant constituent cells were elongated spindle cells arranged in a reticulin-rich, storiform pattern (figure 1-5). At higher magnification, ganglion-type cells (Nissl stain positive) with 1–4 round nuclei, prominent nucleoli, and abundant unipolar cytoplasm were present (Figure 1-5, inset). Immature or abortive ganglion cells with enlarged single nuclei and markedly irregular nuclear membranes were rare but identifiable morphologically. Less "differentiated" aggregates of cells with hyperchro- matic nuclei and minimal cytoplasm, as has been well- described in DIGs [1-3], were present. Mitotic figures were rare and small foci of necrosis were limited to the less dif- ferentiated component. Immunohistochemical stains for synaptophysin was positive in the ganglion cells only. Pathologic findings g f g As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised pre- dominantly of a population of dispersed (occasionally clustered) large neuronal cells (~70 µm diameter each) with round eccentrically placed uniform hyperchromatic nuclei, undulating and slightly convoluted nuclear mem- branes, and abundant unipolar granular cytoplasm (gan- glion cells) (figures 1-2 and 1-3). Occasional cells were binucleated (figure 1-3a). A spectrum in the degree of nuclear membrane irregularities was noted, with most cells displaying irregular features as described above, while other cell showed bland nuclear features (figure 1- 3b). However, all displayed nuclear polarity to the cyto- plasm. Rare smaller cells interpreted as astroglial cells were interspersed between the larger cells. The latter cells showed nuclear hyperchromasia, more prominent irregu- larities in their nuclear membranes and a smaller cyto- plasmic rim. (figure 1-4). Overall, significant proportions of both cellular populations showed varying degrees of degenerative changes manifested as lack of clear delinea- tion of nuclear and cytoplasmic borders and a loss of nuclear detail. Several clusters were composed of large cells, and in these clusters, constituent large cells showed less cytoplasm but retained a unipolarity in relation to the nuclei and their nuclear features were identical to those of the more predominant population of large cells. Addi- tionally, scattered foamy histiocytes were present. A finely granular background material consistent with necrosis was present, but there was no distinct neurofibrillary material. Page 2 of 6 (page number not for citation purposes) Materials and Methods For cytology, a slide was prepared from 50–60 cc of straw- colored fluid utilizing the ThinPrep® 2000 Automated Slide Processor (Cytyc, Boxborough, MA) according to the manufacturer's instructions. For the tumor specimen, approximately 10 × 6.5 cm of fragmented gray and white cerebral tissue was received and entirely processed rou- tinely: tissue sections were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned to 4 µ-thick sections and stained with hematoxylin and eosin, Nissl stain and reticulin. The immunohistochemical pro- file of the tumor was evaluated on 4 µ thick, formalin- fixed, deparaffinized sections using a DAKO Autostainer (Carpinteria, CA, USA) based on the avidin-biotin-perox- Page 2 of 6 (page number not for citation purposes) Page 2 of 6 (page number not for citation purposes) CytoJournal 2005, 2:1 http://www.cytojournal.com/content/2/1/1 Radiologic, cytologic and morphologic appearance of the tumor Figure 1 Radiologic, cytologic and morphologic appearance of the tumor. 1: This computed-tomographic scan of the patient's cerebr mass shows a large cystic mass with peripheral enhancement at the solid portion which attached to the overlying dura; 2: In ddition to scattered individual cells, variably sized clusters of neuronal cells were identified, all composed of cells with ecce rically located, occasionally binucleated hyperchromatic nuclei and abundant unipolar cytoplasm [original magnifications ×40 : Occasional neuronal cells were binucleated (3a) while others showed bland nuclear features (3b) [original magnifications 400]; 4: Scattered astroglial cells with more convoluted nuclear contours and less cytoplasm were also present. [original m ifications ×400]; 5: Typical histologic appearance of desmoplastic infantile ganglioglioma, showing scattered ganglion cells in esmoplastic and fibroblastic, vaguely storiform background (original magnification ×200, inset ×400) Radiologic, cytologic and morphologic appearance of the tumor igure 1 Radiologic, cytologic and morphologic appearance of the tumor. 1: This computed-tomographic scan of the patient's cerebr mass shows a large cystic mass with peripheral enhancement at the solid portion which attached to the overlying dura; 2: In ddition to scattered individual cells, variably sized clusters of neuronal cells were identified, all composed of cells with ecce rically located, occasionally binucleated hyperchromatic nuclei and abundant unipolar cytoplasm [original magnifications ×400 : Occasional neuronal cells were binucleated (3a) while others showed bland nuclear features (3b) [original magnifications 400]; 4: Scattered astroglial cells with more convoluted nuclear contours and less cytoplasm were also present. Discussion h l l The clinical presentation, radiographic appearance and histopathologic features of this case are entirely consistent with those described for desmoplastic infantile gangliog- lioma [1-3]. There has been a significant evolution in the understanding of this rare tumor since its original descrip- tion in 1987 [1]. DIGs are typically large supratentorial tumors that, at least as observed radiographically in one patient, are initially solid then become cystic [5]. Although this tumor is considered a grade 1 tumor based on the histopathologic features of cases described prior to the publication of the WHO monograph in 2000, at least one report has since documented anaplastic features in a case of DIG, which was ultimately fatal [6]. However, fol- low-up has generally been favorable following complete resection in the reported cases of DIG, with a median post-surgical interval of 8.7 years without metastases or recurrence in one series of 14 patients [2]. Additionally, in some cases, spontaneous regression of tumor following subtotal tumor resection has been documented [7,8]. The finding of large cells with eccentrically located nuclei and abundant unipolar cytoplasm in an aspiration speci- men of a cerebral mass occurring in a young person should generate a differential diagnosis that includes DIG, atypical teratoid/rhabdoid tumor (AT/RT), dysembroplas- tic neuroepithelial tumor (DNT), ganglioglioma, supratentorial primitive neuroectodermal tumour (PNET) with ganglionic differentiation (ganglioneuroblastoma), anaplastic large cell lymphoma and pleomorphic xan- thoastrocytoma. In our opinion, clinical features as well as immunohistochemical analysis can significantly help reduce the likelihood for most of the aforementioned entities. The distinction of DIG from AT/RT is probably of the greatest prognostic significance, since in contrast to DIG, AT/RT is a highly malignant tumor that is almost uniformly fatal [9]. Although AT/RT occurs in infants or young children, most cases occur in the posterior fossa, in contrast to DIGs, which are invariably supratentorial [1- 3]. Radiographically, AT/RT are typically not distinctly cystic, although necrosis may impart an irregularly cystic appearance. The distinctive clinical features of DIG, being a typically superficially located tumor occurring in young children (with potentially unclosed fontanelles), may render them particularly amenable to pre-operative assessment using aspiration cytology. Discussion h l l In addition, familiarization of practi- tioners with the cytopathologic features of DIG may be useful because 1) With the aforementioned cases of DIG regressing after subtotal resection [7,8], it might be unnec- essary to aggressively resect these tumors to negative mar- gins, and a preoperative aspiration diagnosis of DIG will be helpful in the neurosurgical planning and 2) In their intra-operative assessment, imprint cytopathology may potentially be more diagnostic than histopathology. However, to our knowledge, the cytologic features of DIG have been documented only once previously [4]. In that report, Hasegawa et al [4] reported aspiration and imprint cytology findings in two cases of DIG. Two distinct cellu- lar populations were identified, a predominant popula- tion of small to intermediate sized astroglial cells and "a few" large cells with round nuclei, prominent nucleoli and profuse cytoplasm that was unipolar to the nuclei in all their illustrations. In the current case, the reverse was found, with the predominant cells being an identical pop- ulation of large cells with round nuclei, prominent nucle- oli and abundant unipolar cytoplasm, and only rare unequivocal astroglial cells. Most of the analysis of the aforementioned report [4] was on the imprint smears, and although a mixture of small and large cells were also iden- tified on the aspiration smear, there was no stated assess- Immunohistochemically, the rhabdoid cells of AT/RT co- express vimentin and epithelial membrane antigen [10], in contrast to the ganglion cells of DIG. However, rhab- doid cells may rarely express neurofilament, an immu- nophenotypic overlap with DIG. Morphologically, the distinct cytoplasmic borders, "inclu- sion-like" cytoplasmic globule and overall dense eosi- nophilia of the cytoplasm of rhabdoid cells, in conjunction with the aforementioned clinicopathologic parameters, may help in their distinction from ganglion cells of DIG [9,10]. The separation of DIG from other tumors containing true ganglion cells based on cytomor- phology alone would probably pose the greatest diffi- culty. These tumors include DNT, ganglioglioma, and supratentorial PNET with ganglionic differentiation; all have a predilection for, or at least may potentially occur in children. In addition to ganglion cells, cytomorphologic features of DNT include oligodendroglial-like cells arranged in lobules and neurons in abundant extracellular mucin or neurofibrillary material [11,12]; these findings were neither identified in the 2 cases of DIG reported by Hasegawa et al [4], nor the current case. Materials and Methods [original m ifications ×400]; 5: Typical histologic appearance of desmoplastic infantile ganglioglioma, showing scattered ganglion cells in esmoplastic and fibroblastic, vaguely storiform background (original magnification ×200, inset ×400) Radiologic Figure 1 Radiologic, cytologic and morphologic appearance of the tumor Figure 1 Radiologic, cytologic and morphologic appearance of the tumor. 1: This computed-tomographic scan of the patient's cerebral mass shows a large cystic mass with peripheral enhancement at the solid portion which attached to the overlying dura; 2: In addition to scattered individual cells, variably sized clusters of neuronal cells were identified, all composed of cells with eccen- trically located, occasionally binucleated hyperchromatic nuclei and abundant unipolar cytoplasm [original magnifications ×400]; 3: Occasional neuronal cells were binucleated (3a) while others showed bland nuclear features (3b) [original magnifications ×400]; 4: Scattered astroglial cells with more convoluted nuclear contours and less cytoplasm were also present. [original mag- nifications ×400]; 5: Typical histologic appearance of desmoplastic infantile ganglioglioma, showing scattered ganglion cells in a desmoplastic and fibroblastic, vaguely storiform background (original magnification ×200, inset ×400) Page 3 of 6 (page number not for citation purposes) CytoJournal 2005, 2:1 http://www.cytojournal.com/content/2/1/1 GFAP was positive in astroglial elements within the desmoplastic regions; the latter was negative for synapto- physin. The ki-67 labelling index was 11.3% (evaluated in the area of greatest density of positive staining cells). ment or low-power illustration of the relative ratio of small to large cells on the latter. In the current case, a cell- block for immunohistochemical confirmation of the nature of two-cell population was unavailable; however, the larger cells were positive for neurofilament (confirm- ing their neuronal nature) while the smaller cells were positive for GFAP (confirming their astroglial nature) in the report of Hasegawa et al [4]. References In World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System Edited by: Kleihues P Cavenee WK Lyon: IARC Press; 2000:96-98 1. VandenBerg SR, May EE, Rubinstein LJ, Herman MM, Perentes E, Vinores SA, Collins VP, Park TS: Desmoplastic supratentorial neuroepithelial tumors of infancy with divergent differentia- tion potential ("desmoplastic infantile gangliogliomas"). J Neurosurg 1987, 66:58-71. Other less likely differential considerations include pleo- morphic xanthoastrocytoma and anaplastic large cell lym- phoma (ALCL), both of which may contain large cells with eccentrically located nuclei and abundant cytoplasm. The temporal lobe predilection, lack of neuronal differen- tiation, presence of xanthomatous cells, smaller tumor size and older age of patients with pleomorphic xan- thoastrocytoma should permit an easy distinction of the large cells in this tumor from those of DIG. Primary brain ALCL is exceedingly rare and generally occurs in older individuals, with a mean age of 29 years in one series [16]. Immunoreactivity for CD30, ALK and CD45 in ALCL and absence of similar immunoreactivity in DIG should facil- itate a distinction in rare cases that occur in very young children. g 2. VandenBerg SR: Desmoplastic infantile ganglioglioma and desmoplastic infantile astrocytoma of infancy. Brain Pathol 1993, 3:275-281. 3. Taratuto AL, VandenBerg SR, Rorke LB: Desmoplastic infantile astrocytoma and ganglioglioma. In World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nerv- ous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:99-102. 4. Hasegawa Y, Hayabuchi Y, Namba I, Watanabe T, Kato K, Ijiri R, Tan- aka Y, Sekido K, Kigasawa H, Hara M: Cytologic features of desmoplastic infantile ganglioglioma: a report of two cases. Acta Cytol 2001, 45:1037-1042. y 5. Tseng JH, Tseng MY, Kuo MF, Tseng CL, Chang YL: Chronological changes on magnetic resonance images in a case of desmo- plastic infantile ganglioglioma. Pediatr Neurosurg 2002, 36:29-32. p g g g g 6. De Munnynck K, Van Gool S, Van Calenbergh F, Demaerel P, Uytte- broeck A, Buyse G, Sciot R: Desmoplastic infantile gangliogli- oma: a potentially malignant tumor? Am J Surg Pathol 2002, 26:1515-1522. 7. Takeshima H, Kawahara Y, Hirano H, Obara S, Niiro M, Kuratsu J: Postoperative regression of desmoplastic infantile gangliog- liomas: report of two cases. Neurosurgery 2003, 53:979-983. 8. Tamburrini G, Colosimo C Jr, Giangaspero F, Riccardi R, Di Rocco C: D l i i f il li li Child N S 2003 8. Authors' contributions alone may be impossible even in the presence of a signif- icant stromal component on the aspirate. Ganglioglio- mas, like DIG may be solid to cystic and show desmoplasia, although in contrast to DIG, they have a pre- dilection for the temporal lobe and most commonly occur in an age group slightly older than is typical for DIG [13]. However, it should be noted that conventional gangliog- liomas and DIG may exist on a morphologic spectrum, and a case with morphologic features of both entities has been described [14]. Supratentorial PNET with ganglionic differentiation (ganglioneuroblastomas) also show signif- icant clinicopathologic overlap with DIG, as they are supratentorial, occur in young children, may be cystic and may show desmoplasia [15]. Cerebral ganglioneuroblast- omas are extremely rare, and in the absence of treatment- related cytodifferentiation, will show a significant neuro- nal component of smaller cells. However, the cytomor- phologic features of pure ganglioneuroblastoma have not been well-characterized. All authors made substantial contributions to the intellec- tual content and/or presentation of the manuscript. ITO (cytologist), diagnosed the cytopathological aspects of the case and co-supervised the entire project. JHK (neu- ropathologist), diagnosed the histological aspects of the case and co-supervised the project. OF wrote the initial version of the manuscript. MRM, DH and AWZ collected pathological, clinical and/or photographical information and revised the manuscript. References When the cytomorphologic findings of DIG are described in more cases, it is likely that the cytologic spectrum will mirror the histologic heterogeneity of this tumor. For example, astroglial cells predominated in the two cases of Hasegawa et al [4] while the ganglion cells predominated in ours. In addition, it is conceivable that an aspirate would only capture the immature neuroepithelial cells which frequently characterizes DIG. Nonetheless, it is concluded that when the large neuronal cells are encoun- tered in an aspirate of a brain mass in a child, a combina- tion of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities. Desmoplastic infantile ganglioglioma. Childs Nerv Syst 2003, 19:292-97. 9. Burger PC, Yu IT, Tihan T, Friedman HS, Strother DR, Kepner JL, Duffner PK, Kun LE, Perlman EJ: Atypical teratoid/rhabdoid tumor of the central nervous system: a highly malignant tumor of infancy and childhood frequently mistaken for medulloblastoma: a pediatric Oncology Group Study. Am J Surg Pathol 1998, 22:1083-1092. g 10. Rorke LB, Biegel JA: Atypical teratoid/rhabdoid tumour. In World Health Organization Classification of Tumours: Pathology and Genet- ics of Tumours of the Nervous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:145-48. y 11. Bleggi-Torres LF, Netto MR, Gasparetto EL, Goncalves E, Silva A, Moro M: Dysembrioplastic neuroepithelial tumor: cytological diagnosis by intraoperative smear preparation. Diagn Cytopathol 2002, 26:92-4. y p 12. Park JY, Suh YL, Han J: Dysembryoplastic neuroepithelial tumor. Features distinguishing it from oligodendroglioma on cytologic squash preparations. Acta Cytol 2003, 47:624-9. Discussion h l l The distinction of gangliogliomas form DIG based on cytomorphology Page 4 of 6 (page number not for citation purposes) http://www.cytojournal.com/content/2/1/1 CytoJournal 2005, 2:1 Acknowledgement Due to the archival nature of the case as well as the absence of any poten- tially identifying patient information, the acquisition of patient consent to specifically report the case was deemed unnecessary. Page 5 of 6 (page number not for citation purposes) References References 1. VandenBerg SR, May EE, Rubinstein LJ, Herman MM, Perentes E, Vinores SA, Collins VP, Park TS: Desmoplastic supratentorial neuroepithelial tumors of infancy with divergent differentia- tion potential ("desmoplastic infantile gangliogliomas"). J Neurosurg 1987, 66:58-71. 2. VandenBerg SR: Desmoplastic infantile ganglioglioma and desmoplastic infantile astrocytoma of infancy. Brain Pathol 1993, 3:275-281. 3. Taratuto AL, VandenBerg SR, Rorke LB: Desmoplastic infantile astrocytoma and ganglioglioma. In World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nerv- ous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:99-102. 4. Hasegawa Y, Hayabuchi Y, Namba I, Watanabe T, Kato K, Ijiri R, Tan- aka Y, Sekido K, Kigasawa H, Hara M: Cytologic features of desmoplastic infantile ganglioglioma: a report of two cases. Acta Cytol 2001, 45:1037-1042. 5. Tseng JH, Tseng MY, Kuo MF, Tseng CL, Chang YL: Chronological changes on magnetic resonance images in a case of desmo- plastic infantile ganglioglioma. Pediatr Neurosurg 2002, 36:29-32. 6. De Munnynck K, Van Gool S, Van Calenbergh F, Demaerel P, Uytte- broeck A, Buyse G, Sciot R: Desmoplastic infantile gangliogli- oma: a potentially malignant tumor? Am J Surg Pathol 2002, 26:1515-1522. 7. Takeshima H, Kawahara Y, Hirano H, Obara S, Niiro M, Kuratsu J: Postoperative regression of desmoplastic infantile gangliog- liomas: report of two cases. Neurosurgery 2003, 53:979-983. 8. Tamburrini G, Colosimo C Jr, Giangaspero F, Riccardi R, Di Rocco C: Desmoplastic infantile ganglioglioma. Childs Nerv Syst 2003, 19:292-97. 9. Burger PC, Yu IT, Tihan T, Friedman HS, Strother DR, Kepner JL, Duffner PK, Kun LE, Perlman EJ: Atypical teratoid/rhabdoid tumor of the central nervous system: a highly malignant tumor of infancy and childhood frequently mistaken for medulloblastoma: a pediatric Oncology Group Study. Am J Surg Pathol 1998, 22:1083-1092. 10. Rorke LB, Biegel JA: Atypical teratoid/rhabdoid tumour. In World Health Organization Classification of Tumours: Pathology and Genet- ics of Tumours of the Nervous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:145-48. 11. Bleggi-Torres LF, Netto MR, Gasparetto EL, Goncalves E, Silva A, Moro M: Dysembrioplastic neuroepithelial tumor: cytological diagnosis by intraoperative smear preparation. Diagn Cytopathol 2002, 26:92-4. 12. Park JY, Suh YL, Han J: Dysembryoplastic neuroepithelial tumor. Features distinguishing it from oligodendroglioma on cytologic squash preparations. Acta Cytol 2003, 47:624-9. 13. Nelson JS, Bruner JM, Wiestler OD, VandenBerg SR: Ganglioglioma and gangliocytoma. 16. George DH, Scheithauer BW, Aker FV, Kurtin PJ, Burger PC, Cameselle-Teijeiro J, McLendon RE, Parisi JE, Paulus W, Roggendorf W, Sotelo C: Primary anaplastic large cell lymphoma of the central nervous system: prognostic effect of ALK-1 expression. Am J Surg Pathol 2003, 27:487-493. Competing interests y g q p p y 13. Nelson JS, Bruner JM, Wiestler OD, VandenBerg SR: Ganglioglioma and gangliocytoma. In World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:96-98. The author(s) declare that they have no competing interests. y y 14. Komori T, Scheithauer BW, Parisi JE, Watterson J, Priest JR: Mixed conventional and desmoplastic infantile ganglioglioma: an y y 14. Komori T, Scheithauer BW, Parisi JE, Watterson J, Priest JR: Mixed conventional and desmoplastic infantile ganglioglioma: an Page 5 of 6 (page number not for citation purposes) Page 5 of 6 (page number not for citation purposes) Page 5 of 6 (page number not for citation purposes) CytoJournal 2005, 2:1 http://www.cytojournal.com/content/2/1/1 autopsied case with 6-year follow-up. Mod Pathol 2001, 14:720-726. 15. Rorke LB, Hart MN, McLendon RE: Supratentorial primitive neu- roectodermal tumour (PNET). In World Health Organization Clas- sification of Tumours: Pathology and Genetics of Tumours of the Nervous System Edited by: Kleihues P, Cavenee WK. Lyon: IARC Press; 2000:141-144. 16. George DH, Scheithauer BW, Aker FV, Kurtin PJ, Burger PC, Cameselle-Teijeiro J, McLendon RE, Parisi JE, Paulus W, Roggendorf W, Sotelo C: Primary anaplastic large cell lymphoma of the central nervous system: prognostic effect of ALK-1 expression. Am J Surg Pathol 2003, 27:487-493. 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W42050145.txt	https://journals.calstate.edu/cjhp/article/download/1692/1534	en		Who Has Potatoes?	Californian journal of health promotion	2,003	cc-by	8,086	K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 Who Has Potatoes? Turning Points in Migratory Experience Keith V. Bletzer Arizona State University Abstract / Resumen Para el migrante, viajar en busca de trabajo es díficil, ya sea que trabaje en agricultura o en otras labores. Este ensayo examina ciertas etapas en la vida de un hombre (estudio de un solo caso) que examina los cambios que le han ocurrido durante un período en que él consumía grandes cantidades de alcohol en los estados y en su país, seguido por un período de sobriedad (no tomaba alcohol, no usaba drogas) en este país en que él comienza una etapa de rehabilitación. Migratory farm labor like other forms of migrant work both in and outside agriculture impedes on the opportunity to make choices. The following essay explores particular phases in the life of one man (a single case study) and examines how he considers turning points in his life that led to a long period of substance use, both as an immigrant in the country and as a working man in his home country, followed by a cessation of use and the beginning stages of recovery. © 2003 Californian Journal of Health Promotion. All rights reserved. Keywords: Migratory labor, recovery from substance use, immigration, and narrative analysis Works that cover multiple cases generally present extreme situations that may show an improvement or describe conditions that are sub-standard, such as discriminatory practices that lead to a poor working environment. Examples of this approach, for improvements in lifestyle, include strawberry growers who once were sharecroppers along the Central Coast of California (Wells, 1996), and, for the sub-standard, most of the 70 narratives presented by Rothenberg (1998), wherein farm labor hardships are presented first, before more upbeat narratives by children of migrant families in the final chapters. In this essay, I use the single case study approach to explore one man’s experiences, which represent both typical and other than typical events in the life of a migrant who traveled more extensively than prior single case studies in the literature. Conditions upon which migratory labor are based in the United States generate a life of transition that extends beyond that of other occupations and lifestyles. Migration by its very nature removes people from one place, compels them to seek work and/or residence in another, and may return them to the same place with new ways of looking at the world. Or it may push them not to return, once they experience another place or obtain something that they believe is better than a prior experience in work, residence and recreation. Most single case studies on farm labor in the social science literature seek to present a view that is considered “typical.” Instances of this approach are the unnamed labor camp in Illinois studied by Alicia Chavira-Prado (1992), the migrant family based in El Valle of South Texas accompanied by Isabel Valle (1994), a journalist, and the experience of Pedro, an older teen who came to California from a rural area of Guanajuato, Mexico, and worked in the Central Valley of California, as described by Juan Vincent Palerm (1992). Having worked with migrant and seasonal farm workers for nearly 15 years, primarily in relation to risk behaviors, I recently collected life stories from farm workers in three states of the Southeast for a study of initiation into 148 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 arrived, less known than maids, nicknamed gatas (“cats”), who make daily purchases for employers in apartments along the adjacent streets, and less known than poor women who walk from nearby colonias (“neighborhoods”) to make weekly purchases of vegetables and fruits, since bulk purchases are cost-reductive. drug and alcohol use. Although the main source of data was taped interviews, I incorporated techniques from my training in anthropology, wherein I spent time with those whom I interviewed. Worker-users in one state where I conducted fieldwork I came to know over time that I spent in a house rented by men in recovery, which I visited to collect life stories through the cooperation of a substance abuse program designed for farm workers. It is this data that I wish to explore in this essay, particularly my contact with one man over four interviews that became much like the ethnographic encounter that anthropologists cultivate (Marcus, 1998 [1997]). Life stories are a particularly good way to learn details of migrant life. This narrative excerpt from which the opening quotation is taken occurs in the second of four interviews I completed with D.N. over a seven-month period.1 I will return to this setting of "sidewalk selling" later in the essay. Despite coming to the states at the age of 17 and spending most of his adult life in cities and rural areas of this country, D.N. identifies with the time that he spent as a fruit-vegetable vendor in Mexico. Owing to time in the states and no house of his own, he lives in his parents’ home, when he returns. Unlike men and women from Mexico who come to the states to earn money, often with the goal of constructing a house in Mexico (Chavez, 1992; Davis, 1990; Rothenberg, 1998), and unlike D.N.’s hometown compatriots in a local branch of a national political party active in his hometown, he never sought title to land by "take-over," which entails replacement of farmlands with neighborhood housing and reimbursement of farmers by the municipal government for lands "taken" and "occupied." Like other urban areas in Mexico, the city is expanding to which he has returned four times since he first came to the states 18 years ago. D.N. talked of building a house in Mexico shortly after he left treatment, when he recontacted his family after 11 years of no communication. The evening that I transported Celso and he to an Alcoholics Anonymous meeting in a nearby town, he sought out Celso's advice, since Celso, nine years older than D.N. and free of drinking and drugs for some time, was able over the years to purchase a house in his native state for his family. "In his mother's house" is how D.N. describes his residence in Mexico, since it is the house where she lived with her parents, grandparents and parents' siblings while growing up. Coming from an adjacent province, his father has lived all his adult life My focus is the dynamics of transition, as they appear in narrative, focusing on the case of one man's recovery from a long period of substance use. I collected nearly one-fourth of my life stories from men in a residential treatment program for those who are or were tied to farmwork. Bi-monthly visits permitted me to find recruits for new interviews and reinterview participants still in treatment, as well as those who “graduated.” D.N. (fictitious initials) was among the first men whom I interviewed. He became a source of material on each visit through a combination of ethnographic encounters and audio-taped interviews. As Rosenwald (1992) reminds us: "When people tell life stories, they do so in accordance with models of intelligibility specific to the culture (p. 265) ... [T]he issue of human development is obscured if cultural forces are declared to be sovereign (p. 267)." Introduction to D.N. Je, quién tiene papas? ("Hey, who has potatoes?"), D.N. imitates the voice of a customer, as he explains his selling from a wheeled-platform in his hometown in a northcentral province of Mexico. Unlike his coworkers and he, who line the sidewalk in a heavily walked avenue of the capital city, the customer who wants "potatoes" is mobile. He drives to the area to transact business where these men work from wheeled-platform stands (called plataformas) and he leaves, as he 149 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 uxorilocally2 with his wife’s family. D.N. recently came to the idea of "having a house." Forty-five days into treatment, he told me: atypical is his engagement in illicit drug use (primarily use of marijuana). Seeking treatment for alcohol and drug addiction is uncommon among men and women who perform agricultural labor. The infrequency of treatment owes itself to a lack of appropriate services for men and women in farm labor rather than a lack of desire. For farm workers, D.N.’s choices typify common substances that farm workers may use (Weatherby et al., 1997) and/or initiate on both sides of the border. “I was living with my family at the time, since the idea I was going to build a house in Mexico never entered my mind [nunca se me sembró en la mente que voy a hacer una casa en México]. Though I used to accompany the town’s “land petitioners,” I myself never petitioned for land [nunca pedí terreno, sí los acompañaba].” D.N. recognizes that substance abuse thwarted any plans that he may have had for saving for himself or remitting money to his parents back home to Mexico. Later in the second interview, D.N. explained his drinking and drug use. New Developments Mi mamá me manda foto de la hija a los cinco años ("My mom is sending me a picture of my daughter at age five"), D.N. tells me as we select coffee and pastries from a self-serve coffee shop one block from the rented house where several "graduates" of the treatment program are living. Me dice que ahora tiene 16 años ("She says she's now 16"); he elaborates on news he received from his family after 11 years of neither phone calls nor letters, news that also is new to me, since my last visit was three weeks earlier. Boracho yo, ni sabía ("Drunk I was [when I knew her], I didn't even know what happened"), he says with measured words, then continues with his earlier pacing: Me dice que me quiere conocer ("She says she wants to get to know me"). By this time, I recognized that he is sharing something integral to his sense of identity: he is demonstrating an interest not only in his daughter but also her mother, the one woman among several for whom he continues to feel affection (owing to the young age at which he left Mexico, all the women he has known lived in the states). He is enthused, yet cautious, as he explains that his mother will first send a photo of his daughter at age five, before sending one at age 16 (it is possible that his mother has no recent photo at age 16). Both the girl and her mother live in central Texas, and the link between mother-daughter, D.N. 's mother and D.N., is his aunt, also living in central Texas. D.N. first came to the states with an older brother, and the two of them spent two years working on a farm along the border in Texas. When the brother was injured (cow-kicked), D.N. went with him on “In my mind I was carrying only thoughts of beer [En mi mente yo traía pura cerveza]. (pause) To think that I was going to eat, to think that I was going to work, it never entered my mind. So constant was the thought that it spilled out from my thoughts, (pause) and I had to bring along a bottle in my hand.” D.N. was twice interviewed during treatment for substance abuse (inhalants, alcohol, pills, marijuana, crack-cocaine). Thus, his narrative follows the process of recovery and provides an inside look at substance use by a man who worked inside and outside agriculture in the United States. Translocated by choice from Mexico to the states, before finding himself years later "ready for treatment," he presents a tale typical of farm workers in several respects (Chavez, 1992; Griffith & Kissam, 1995; Heppel & Amendola, 1992; HondagneuSotelo, 1994): variable rates of pay across employment (hourly wage, piece rate), shortterm work and brief periods of no work (never collecting unemployment compensation), work outside agriculture (golf course, house construction, restaurant, among others), residential and recreational experience in towns and cities (for example, 8 months in Chicago, 6 months in a southern town of 1,219 persons,3 among others). Where his tale is 150 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 Four months after their daughter was born 16 years ago, Guadalupe left him for another man. Despite D.N.’s transgressions, which included his bouts of anger and lack of demonstrative interest in the baby,4 D.N. and Guadalupe remained fond of each other. During the second interview, D.N. described the relationship after they separated. For analysis, I begin with a general translation of his narrative; later I examine in more detail the narrative devices that he uses. In the excerpt below, D.N. is describing how Guadalupe took up with another man (double parenthesis indicates notes on speech and non-verbal behavior, and a question mark at the end of a sentence indicates a rise in inflection). a bus to the border. Rather than return home with his brother, however, D.N. went to live with a maternal aunt and uncle in central Texas, where he shifted from farm labor to non-agricultural work. Working in a poultry plant, he met Guadalupe, the woman who gave birth to his daughter. At the time, she had friends talk to him of her interest in him. Given his experience with women (Chicanas and Anglo) and the fact that three women followed him to Mexico on separate occasions when he returned home, I suspect his mother is equally cautious and seeks to emphasize the tie to his daughter through a reference to a childhood unknown to him, as her father (perhaps age five was a happy time for D.N. as a child). I consider a word of support is appropriate and I tell him: Está haciéndolo con pasos despacios ("She's taking it one step at a time"). When I start to pay for my coffee, he tells the cashier that he is paying as he adds two cheese rolls for me to carry out. Like José Limón (1994, p. 168-186) and several other ethnographers, I recognize that his gesture of largesse is important for him, and I slowly return the money to my pocket as we return to the rented house. Later that evening, after transporting D.N. and Celso to the A.A. meeting in a nearby town, I place the unspent money in the offering basket. “So I continued, in the dance (el baile) ((he taps table)) and she, well, I would see the guy and her there. “Well,” I said, “That’s no way” (Ni modo). She loved him (ella quiso), right?” Since D.N. omits an object to quiso (“loved”), past tense of the verb querer, the meaning is ambiguous and could also mean, “She loved me.” He continues, “And she would continue dancing, but this made me get drunker (más borracho). I’d go and take her to dance, and she would go out with me (salía conmigo). And the guy she would leave sitting (lo dejaba sentado) ((rise in tone)). If I wanted to take her some place, I would take her some place (la quería llevar, yo me la llevaba). She’d return, two-three days later? And that’s the way that I had her (así la tuve yo?) ((rise in tone, emphatic)). And she would come to get me (ella me buscaba), cause her brothers gave her money and she bought a nice car. She’d go and find me (me buscaba) where I was with my friends. Like she still really loved me (sea que sí me quería?) ((rise in tone, more emphatic)).” D.N. 's disclosure on news of his daughter is my entry point for analysis. Since the last interview with D.N. was more open-ended than the previous three, I allowed him to provide direction. He chose to discuss the women he had known in the states, and the pain he recognized that he inflicted. Into his narrative, he interwove reflections on alcohol and drugs. To review the interviews, I started in reverse order, since the news from central Texas through his mother brought an unexpected revelation to his recovery process, and allowed me to explore how he presented his feelings on gender relations in earlier interviews as well as his feelings for Guadalupe, their now-grown daughter, and the other women whom he has known in the states. To describe their relationship, D.N. uses the dance as a place where men and women "achieve artful control" over their bodies, which are prone to a dominance pervasive in 151 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 “She used to come to get me (me buscaba). I don’t know why I did what I did. The beer, ((pause)) all that marijuana and all that. Did I ever take hold of them in a big way (los agarré muy muchomucho-mucho), right? I took hold of many resentments (agarré muchos resentimientos), she had left me-since she was going with another guy (me había dejada-sea que se iba juntada con otro).” other sectors of their lives (Limón, 1994, p.141-167). Taking her out on the dance floor is a metaphor for her "going out" with him (salía has both meanings here). There is an expression of disbelief in his narrative where he describes Guadalupe's behavior and a lack of response from the "other youth." D.N. is cognizant of male protectiveness over women in Mexican culture. He indicates surprise at three points in the narrative, each indicated by a rising tone (emphatic in the last two). Starting with the young man "left sitting there by himself" at the dance (by implication, at home, when Guadalupe spent time with D.N. for two-three days), D.N. follows with two clauses that suggest that Guadalupe was more interested in him than the youth. That she accepted his advances (spending two-three days with him) is further illustrated by her “seeking him out.” In this sense, "the dance" to which D.N. refers is a set of maneuvers offthe-dance-floor, in which the couple show a fondness for each other that cannot escape the watchful eyes of those around them. Whereas this behavior would be unacceptable, albeit tolerated, in Mexico (Gutmann, 1996, p. 111145; Limón, 1998, p. 179-185), it is not unknown for women on this side of the border to "assert" themselves for issues that matter to them, including their interest in a particular man. Instead of interpreting this behavior as a breakdown of cultural morés, I suggest that women's assertiveness sometimes is necessary, given distractions that compete for men's attention on this side of the border. Thus, women are following a strategy of corrective action for a "sadness" that confronts men and women, as Latinos, and in this case, as Mexicanos, who find themselves in a world with distinct cultural expectations (VelezIbañez 1997:182-206; also Gonzalez 1994). D.N. continues by illustrating a closeness he felt toward her family, and his feelings on the situation of Guadalupe and her new beau. One day I arrived to visit her brothers, and her mother came out, the mother of her brothers (salió su mamá, de ellos). “My son (Mi'jo), listen. You are the child’s father. We will never deny that you see her. Come inside to see her (pásele que la mire). The other guy is there [in the house]-the one who stay-the one with whom my daughter got together (allí está el otro muchacho que se quedó mi-se juntó mi hija con él). But, no matter, come inside.” “No, I may not be under control” (No, no tenga cuidado). I said, "No, señora, here I am okay." “Well, let me bring the child to you," and they graciously showed the child to me. He recognizes a tension in the situation he created, notwithstanding the willingness of Guadalupe to start living with another man, a strategy on her part to make D.N. jealous. Interestingly, the mother catches herself in D.N.’s narrative, saying that this other man “stayed” with her daughter, implying that it was D.N. who left their daughter. He blames Guadalupe’s breaking up for contributing to his drinking and use of marijuana. At this time, D.N. had tried all but one drug, crackcocaine, which he would briefly use ten years later. Since drinking alcohol and marijuana smoking represent his repertoire of use at this point in time, he signals the fullness of his use with "and all that." Use of pills and inhalants were things he used when he was living in Mexico (more on these substances later). He uses the pretense of visiting her brothers to come to her house (her brothers he met after he met her). Under the watchful eye of her Later in the narrative, it becomes clear that her parents supported Guadalupe, and hence support my interpretation of her assertive behavior, however dissonant it may appear on the surface. At the same time, they encouraged D.N. 's responsibility to the daughter. At this point, he acknowledges a recurring theme in his life, his diversion into heavy drinking. 152 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 There is a strong indication in this passage that for all the pain that D.N. felt after the relationship with Guadalupe, he retains a fondness for her. He wants to go back, and he becomes nostalgic in stating a desire to return to the dance, before he catches himself and recognizes the "dance" with Guadalupe has ended. Her attributes his pain to her taking up with another man, prefacing the passage with his recognition of vulnerability that suggests a continuing interest in Guadalupe. mother, he is welcomed and asked to come inside to see the child. Another point of interest is that D.N. identifies her as the mother of the brothers, rather than the mother of Guadalupe or grandmother of his child. He thus erases the real reason he came to visit. Whether his statement that he might "loose it" with the other youth is real or postured is not known; D.N. never described in any of his four interviews significant participation as an aggressor in fights with men. For example, for incidents in which he was shot while living with his aunt and uncle, before meeting Guadalupe, and almost attacked by a gang in Chicago, he never sought retaliation. It is women toward whom he showed abusive behavior (but never to an extent where he was jailed or reported to the police). All incidents with women occurred after he left central Texas and Guadalupe. Later during the second interview, D.N. acknowledges his womanizing at the same time that he characterizes such behavior as self-centered and, distancing himself from a sense of responsibility, a part of his youth: “I thought the world was mine.” I would take one woman (ya agarraba una), I would take another women (agarraba otra). Then, [there were] no more girlfriends (no más novias) to hug and kiss and dance and all that. I would have three, or four girlfriends ((rising tone)), wherever I was ((rising tone)). I used to say, "No, I'm too young to get married." D.N. expresses appreciation for an opportunity to see his daughter; the agency of Guadalupe's mother in arranging this is obscured by use of “they,” as an expression of gratitude toward her family and an acknowledgment of the importance of family in Mexican culture. The tension between vulnerability and pain for Guadalupe, and justification for womanizing, are replaced with later statements that shift the allocation of responsibility. This time D.N. uses a rise in tone over disbelief of his behavior. After six months of recovery, D.N. 's perspective balances more equitably contributions of she and he to the dissolution of their relationship. He views the symmetrical aspect of pain to each of them: “I had to leave there, cause at that time, I did not want to hurt her more nor hurt myself more, better to not see her again (no-no-no quería yo, sea, más lastimarla a ella ni yo mismo, mejor que, así, no mirarla más).” He remembered the pain he felt during these final weeks in central Texas. A few lines after the above excerpt, he attributes his pain to actions of Guadalupe in taking another lover: “We all have hearts ((forcefully)), that can be broken! Right? (TODOS TENEMOS CORAZON. Verdad?) I went thinking that, one day she might [experience this], also weighted with rage (coraje). Most likely, it seems, she many not suffer the pain she caused (a la mejor, pos, no sufra el dolor que hizo). After that, I left that place, Dallas. I left for good and never returned.” When I asked D.N. for clarification, if that was the last time, he tells me, “I've not gone back. I have a strong desire to return (ganas de volver), to go back there, and to the dance to see-I don’t know, more than likely they’re no longer there (ir allí al baile a ver-no sé, a la mejor ya ni están allí).” D.N. recognizes the pain experienced by he and Guadalupe, and he responds by leaving. Despite his departure, even before he received renewed communication from his mother on the daughter and Guadalupe in Texas that hinted at the possibility of a reconciliation, he recognized, through the medium of his 153 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 or relationship that could or would never be the same. These are turning points that Denzin (1989, p. 71-73) describes as "epiphanies," which are problematic and eruptive, but otherwise represent a crisis that is an important part of a person's life. interview, that he still cared for Guadalupe. Drinking and womanizing were disruptive to the relationship. It is the final interview a few months after sobriety in which D.N. first acknowledges the potential for children to be affected by adult drinking and drug use. Until this time, he had acknowledged a lateral effect within his generation, most acutely felt by him in the loss of women whom he had known in the states. Previously, he had not expressed concern for the vertical effect of substance use generated between adults and children: “I thought I was never going to pay for what I made my parents suffer (lo que hice sufrir a mis padres), and now I have to pay with my own suffering (tengo que pagar con el mio).” From central Texas, D.N. went to west Texas, where he became friends with the pastor of a small church. It was here that he lived with the preacher’s family and participated in church activities, when he wasn't working. These were things that D.N. was able to do, willingly, admirably, and free of a need for pastoral chastisement, at least for a short time. He describes another turning point that he experienced while living with the preacher. For this excerpt I follow narrative techniques outlined by Riessman (1993, p. 34-40), who follows Labov & others. Following Riessman, regular breath pauses are indicated with a comma, longer pauses are indicated by (.), and a hyphen indicates interrupted speech. For this excerpt, I present D.N.’s speech stanza by stanza. Turning Points As indicated, D.N. acknowledged conflicts in relationships with women, and he named one woman per locale to highlight the tenor of his experience: Guadalupe in central Texas, Lucy in west Texas, Vanessa in east Texas, Susie in the Middle South, Luz María in the Southwest and Deborah in the Lower South [fictitious names]. One characteristic of his narrative style is inclusion of internal dialogue, which he foregrounds with decir ("say,” “tell,” “reply"). D.N. is not alone in the use of internal dialogue; there are several interviews with English-speakers and Spanish-speakers who strategically use dialogue to heighten the complicating actions or "plot" (Reissman 1993) in their narrative. Some farm workers shift pitch when (re)producing the speech of others. Others "mark" the onset of dialogue with decir (Spanish) or "say" (English). So [there was] this one occasion, I arrived (yo llegué), I bathed (me bañé). I noticed he was looking rather serious (.) to one side there. Then (.) he said to me, "Brother, I want to speak with you." Cause he called me that, Brother. We went to church [together]. I say to him (le digo), "Yes, what do you want, Brother?" (.). "Like, tell me, s-straight up, the truth. (.) Do you know a (.) girl [who is] such-andsuch?" (.). And I told him (le dije), "Oh my! No, no." "Yes, you do know her." (.) "No." "Yes, do you recall that she works in the restaurant." (.) I told him (le dije), "Oh, yes, yes, yes, it's certain, yes I know her, but by name, (.) never, no-it's that I didn't remember, her name. K: /huh. G: /huh. [overlap due to suspense] But of course I know her-why, Bro'?" (.) K: /huh. G: /huh. [overlap due to suspense] “Know what, Brother? Well, you know I gave you my trust, I thought you would not go sinning (pecando) out there. (.) But I had a sinner here in my house." I tell him (le digo), "No, no, no." “Yes, (.) it seems that What caught my attention in the internal dialogue with D.N. 's narratives was a shift in tense to mark onset. Although events he narrates, and the talk he recalls, took place in the past, D.N. uses present tense to mark occasions in which his behavior pulled him into trouble or he acted in less than an admirable fashion. More than this, these occasions represent a time to which D.N. cannot return. At the historic moment when they occurred, the person with whom he was speaking was referring to change in a situation 154 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 of his inappropriate actions, D.N. validates his interpretation. Moreover, he inadvertently, or clumsily, depending on one's reading, permits a severance of their once-trusting relationship. Shortly after this incident, D.N. left this new town where he was working and residing. you went and you misused a position of trust with that girl (fué y se revolcó con esa muchacha). And now she’s pregnant, (.) ((softly)) and it's your child." "Brother, hey! How can you be sure?" He says (dice), “Yes, for sure.” [D.N. uses "No" transitionally to link prior and impending statements] He says (dice), "That girl is my niece," (.) “Say what?” ("Qué-qué?") “Yes, that girl is my niece." Onset and Dissolution of Substance Use There are other sections of his narrative where D.N. uses a shift in tenses to mark a turning point. There are two in particular that are related to the main point of this essay; this is a description of D.N. 's first use of substances and continuing use prior to the time he entered the residential treatment program. He equates both incidents with bringing death upon himself. D.N. uses two narrative devices to highlight the impact of confrontation with the preacher. He indicates his duplicity by pausing in his responses to the preacher (rendered as "normative" pauses), and marks the sureness and authority of the preacher as an immediate response, without pause, that counters his two attempts of denial. Second, he uses past tense ("I arrived, I bathed," and "I saw him") to set the stage for the discussion that took place between the preacher and he, which he identifies by its true place as a part of the past (me dijo "he said to me"). He shifts to present tense (le digo "I say to him") to bring the listener closer to the dialogue, which occurred in a past time to which he has returned. At this point, he and the listener (and by now, the reader) "are there," if only through the use of language. He continues with present tense of the verb decir ("to tell") to indicate the preacher's talk, as the interaction proceeds, but alternates between present and past tense when producing his talk. I propose that shifts in tense, from present (normative tense, once the stage is set) to past, mark “turning points" for him in which he acted and/or spoke in good faith. Notwithstanding the predicament he created, these turning points should have been culturally correct behavior where he acknowledged transgressions. Continuation of the present tense (again, the normative tense within the internal dialogue) marks a turning point of another kind, one to which he cannot return and one which he cannot make different. This is the turning point of his effort to negate the main agenda of the preacher's statements at two points, first, denial that he knows the girl, and, second, denial that he sinned and broke the trust placed in him. Rather than alter the preacher's interpretation Well, there were two or three times when I was killing myself (ya me anda matando). To feel death right up close (sentir la muerte cerquitas) and all, like it has never touched me. (.) And when I came to this program, (.) I sent a request to the Lord ((draws out sound)) (.) that He remove me from drinking (que me quitara de tomar), (.) ((firmly)) that he take me from drinking was the one thing I most wanted! (.) To never drink no more (no tomar na‘). Cause I couldn't stand it. Yes, that alcohol sure controlled me like a companion (sí me dominó así machin). But, not drugs. Alcohol, not drugs, it was that little adorable beer (esa cervezita sí). That entangles (esa sí amarrame), that really raked me over (sí me arrastró), raked me over but BAD! (me arrastró pero FEO) ((inhales)) Real bad for seventeen years. That's why I told Samuel, my nephew, he shouldn't drink so much. (.) He tells me, “No.” He says, “I don't drink." He says, "only when, (.) when I get off from work. That’s when we drink a six-pack (un seis) together. (.) It's me, and for sure, my dad and I, and there's Chico," he's my other nephew, “together we drink only one." He says, "Right now it's not as much." D.N. pulls together several points in this excerpt, which he begins by a statement of 155 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 “I once abused thinner, (.) one day. I was young. But you know that, that (.) I really gazed upon (.) death. (.) It seems that I, when my friend told me, “Take that bag for yourself and this bag for me." There were four of us; the room was like this. It didn't have furniture. (.) I started, to d-do th-the bag (bolsa). All of a sudden I’m looking at a coffin (de repente yo miro un cajón de muerto). (.) And the cadaver was I. I was in the box (el cajón), like this ((hands crossed on chest, body stiffens)). They were going to bury me in the cemetery, and they told me, “Are you aware that, uh, you died and if you continue this and another that we are going-you are going to die, ((rise in tone)) and I went, "NO!! I'm not dead!, I'm not." ((chuckles)) There I was shouting that I wasn’t dead. And I left (salí). I was about twelve. I experienced a lot of dizziness (duré mucho mareo), and symptoms like one gets (.) when playing football or basketball. The thought came to me, but I mean real strong, that right there I was going to die. (.) And I went running home.” awareness of how close to death he has been. The most recent time was prior to his entry in the treatment program, where I first met him. He implicitly credits an answered prayer for how he made the program work for him, and he likens drinking to a close companion (machin) at the same time that he recognizes the devastation that he has experienced. He is thankful that his experience with substance use never entailed use of hard drugs5 other than alcohol, that "little adorable beer." His use of a diminutive (-ita) is common in Spanish to indicate something that engages one's attention, as well as one’s affection. D.N. ignores efforts family and friends made in the past to have him stop drinking, and he proceeds to describe his efforts to dissuade his nephew to quit drinking. His comment on alcohol's domination of his life provides an implicit suggestion that advice to his nephew will not be effective, at least not immediately. It is telling that he replicates a generalization common among abusers that their particular use is insufficient to warrant its definition as "abuse." Like confrontation with the preacher, D.N. uses the past tense to initiate internal dialogue with his nephew and he marks the immediacy of that dialogue with present tense. Where this dialogue differs is its onesidedness; it shows only the nephew's responses, all of which are marked by present tense of “say” (decir). In this case, D.N. is alluding to the nephew's error in not heeding his advice, similar to D.N.’s growing awareness of his own errors. His own silence in the dialogue with his cousin fits with Greenspan's concept of "the traumatic silence of memory" (1992) for people who experience severe trauma (also Bar-On 1990). In the case of D.N., he is reflecting on a silence of 17 years in which he left unheeded the advice of many concerned people who sought to discourage him from alcohol consumption as well as other forms of substance use. One of the few times that D.N. used the term "abuse" (abusar) is in introducing his experience with an inhalant, in this case paint thinner, which produced a reaction that he equates with death. Death carries a significant valence in Mexican culture, and D.N. is communicating the force of this experience with the singular mention of placement of his person in a coffin, which "they" were going to bury in the cemetery. His again uses past tense to set the stage for the listener: his friend "told" (dijo) him before they prepared the bags to inhale. Following this, he continues past tense to describe talk and actions within each component of the narrative. What is at variance with this narrative segment is use of present tense to describe his "gazing upon" (miro) death, the only time present tense is used as he describes its (hers? his?) warning to desist in using inhalants. These variants occur according to the same principle of a turning point that I proposed. He is communicating to the listener that what he saw had meaning for The first time D.N. encountered “death” was an experience with an illicit substance. The experience occurred in Mexico at age twelve, when he and some friends experimented with paint thinner. 156 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 arrive in motor vehicles to request the pills and those who walk (the maids and poor women) to purchase legitimate vegetables and fruits. He is making a subtle commentary on possession and use, distinguishing between use for illicit ends (pills) and use for legitimate ends (food). Participating in each as seller and consumer, D.N.’s tactics in his narrative provide a creative space outside the standard dichotomy of “rich” and “poor.” him. D.N. is giving testimony to the aberrance of substance use; he suggests that its repetition led to inappropriate behavior that was counterproductive to everyday activities and destructive to social relationships. Having come full circle in a long process of substance abuse, from a first encounter with “death” to a final encounter in a coma before treatment, D.N. drew upon a cultural idiom to explain a cessation of one form of life (se murió "you died") and his inscription into a way of life that encased him in substance use. Told that he "died" (past), he also was told that, if he continued, "you are going to die" (se va a morir). The Past and the Future were both contained in the message that he received through this experience. The recent experience of hospitalization prior to initiation of his treatment [not described here] was the culmination of seventeen years of use and inculcation of a way of life where securing and using substances became a driving force, a moment that is an ever-present residual from an earlier event from another time (Rosenwald 1992, p. 285-286), further transformed by his experience as a migrant laborer within and outside agriculture across several states. Papa has an alternative meaning. It may mean "sufficient to meet one's needs." Tener la papa en la mano y no saber pelarla ("to have a potato in one's hand and not know what to do with it") refers to someone who seeks or wants more than they need, that is, to have sufficient for one's needs but not recognize it, and/or uses things given in the course of daily life in ways that were unintended. This expression was in vogue among men in recovery with whom I spent time. The idiom of potatoes was thus a part of the cultural capital available for incorporation into D.N.’s life story. His long journey through substance use is a case of access and misapplication of "potatoes," as much as a form of resistance to the ways of a society wherein he worked, resided, sought recreation and lived a storied life that he is currently re-scripting and transforming in text and behavior. Seeds for change were sown more than once in D.N.’s life: formally through an acquaintance with the preacher, and informally by family and friends. He is fortunate that these seeds took sprout within his inner person, albeit after many years, that led him into, and through, a successful process of substance use treatment and recovery. The lesson of this story for those who provide services and counseling to farm workers or other populations of minimal resources is a gentle but firm persistence in educating and training. One never knows where or how the essence of one’s teaching and instruction will take root and benefit the recipient. Discussion and Conclusion I return to the setting of street selling in D.N. 's hometown in Mexico. I set the stage by describing a customer who desires to purchase "potatoes," as he walks to the street vendors (plataformistas) and signals interest by asking them, "Hey, who has potatoes?" (Quién tiene papas?). Papas is the codeword for "pills" (also called pastas, shortened from pastillas "pills"), a contraband commodity in Mexico and this country. Despite the voracity and culture-encoded message when he first tried an inhalant, D.N. began use of pills as an older adolescent at the time that he was a vegetablefruit seller. He began to sell pills to lessen the cost of purchasing pills for personal use; his regular earnings as a vendor he shared with his parents for household expenses. Since vendors cannot afford pills on their meager earnings, they developed networks for obtaining pills in quantity. Some were consumed and some were sold. D.N. contrasts the world of those who 157 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 Notes 1. D.N. completed two interviews in treatment (one during his first week, a second on his 45th day of treatment), and two in the community where he worked (third at six months of recovery, fourth just short of seven months). 2. Uxorilocal refers to residence on a wife's family's land, virilocal refers to residence on a husband's family's land. For D.N., “uxorilocal” is used to demonstrate respect for his father who devoted himself to raising a family without transgressing in ways that D.N. self-perceived for himself. D.N. once recalled his father’s lack of funds to return to his home province when D.N.'s grandfather (father’s father) died. One remittance, after D.N. renewed contact with his family, went to his father with the instruction that his father should visit his home province (earlier remittances went to his mother, both for household use and to bank for him on his behalf). 3. This was the census-estimated count of the population in 1990 (fewer people in the 1980s and 1970s). 4. D.N. never was told when Guadalupe was hospitalized to give birth. By coincidence, he went to the same hospital when he took a change of clothes for a buddy who had been shot and was ready for release. Like a scene from a movie, Guadalupe and her family were exiting the elevator: Que venían saliendo con, (.) y yo no más me hice a un lado, (.) no me hablaron ni las hablé. "They were exiting with, (.) I just stepped to one side, (.) they didn't speak to me, I didn't speak to them." One unfinished phrase in this short excerpt is instructive: con [la niña]) "with [our newborn daughter]." 5. D.N. briefly used crack-cocaine outside the state of Texas. Owing to the nausea and vomiting he experienced when he smoked the crack that he was offered, he discontinued after one week of irregular use. References Bar-On, D. (1990). Children of perpetrators of the holocaust: Working through one's own moral self. Psychiatry, 53, 229-245. Chavez, L. R. (1992). Shadowed lives: Undocumented immigrants in American society. New York: Holt, Rinehart and Winston. Chavira-Prado, A. (1992). Work, health, and the family: Gender structure and women's status in an undocumented migrant population. Human Organization, 51, 53-64. Davis, M. P. (1990). Mexican voices, American dreams: An oral history of Mexican immigrants to the United States. New York: Henry Holt and Company. Denzin, N. K. (1989). Interpretive biography. Newbury Park: Sage Publications. Gonzalez, R. (Editor). (1994). Currents from the dancing river: Contemporary Latino fiction, nonfiction, and poetry. New York: Harcourt, Brace and Company. Greenspan, H. (1992). Lives as texts: Symptoms as modes of recounting in the life histories of holocaust survivors. In C. Rosenwald and R. L. Ochberg (Ed.), Storied lives: The cultural politics of self-understanding, pp. 145-164. New Haven: Yale University Press. Griffith, D., and E. Kissam. (1995). Working poor: Farmworkers in the United States. Philadelphia: Temple University Press. Gutmann, M. C. (1996). The meanings of macho: Being a man in Mexico City. Berkeley: University of California Press. Heppel, M. L., and S. L. Amendola. (1992). Immigration reform and perishable crop agriculture: Compliance or circumvention? New York: University Press of America. 158 K. V. Bletzer / Californian Journal of Health Promotion 2003, Volume 1, Issue 2, 148-159 Hondagneu-Sotelo, P. (1994). Gendered transitions: Mexican experiences of immigration. Berkeley: University of California Press. Limón, J. (1998). American encounters: Greater Mexico, the United States, and the erotics of culture. Boston: Beacon Press. Limón, J. (1994). Dancing with the devil: Society and cultural poetics in Mexican-American South Texas. Madison: University of Wisconsin Press. Marcus, G. E. (1998[1997]). The uses of complicity in the changing mise-en-scène of anthropological fieldwork. Ethnography through thick and thin, pp. 105-131. Princeton: Princeton University Press. Palerm, J. V. (1992). A season in the life of a migrant farm worker in California. Western Journal of Medicine, 157, 362-366. Riessman, C. K. (1993). Narrative analysis. Newbury Park: Sage Publications. Rosenwald, G. C. (1992). Conclusion: Reflections on narrative self-understanding. In George C. Rosenwald and Richard L Ochberg (Eds.), Storied lives: The cultural politics of selfunderstanding, pp. 265-289. New Haven: Yale University Press. Rothenberg, D. (1998). With these hands: The hidden world of migrant farmworkers today. New York: Harcourt Brace and Company. Valle, I. (1994). Fields of toil: A migrant family's journey. Pullman WA: Washington State University Press. Vélez-Ibáñez, C. G. (1997). Border visions: Mexican cultures of the southwest United States. Tucson: University of Arizona Press. Weatherby, N. L., McCoy, H. V., Bletzer, K. V., McCoy, C. B., Inciardi, J. A., McBride, D. C., and Forney, M. A. (1997). Immigration and HIV among migrant workers in rural southern Florida. Journal of Drug Issues, 27, 155-172. Wells, M. J. (1996). Strawberry fields: Politics, class, and work in California agriculture. Ithaca: Cornell University Press. Acknowledgments Fieldwork for this paper was supported by a small research grant from the Wenner-Gren Foundation for Anthropological Research (#6206), as well as a Faculty Grant-in-Aid (M003) from Arizona State University. The author acknowledges both the generosity and instructive interactions with men of "the rented house," where he stayed while conducting field research outside his main research site. Thanks also to Al Gonzalez, Rosemary Quagan, Lourdes Pérez, Andrea Cruz, and Santos Osarnio. Author Information Keith V. Bletzer, PhD, MPH Department of Anthropology P.O. Box 87-2402 Arizona State University Tempe, AZ 85287-2402 E-Mail: Keith.bletzer@asu.edu 159
https://openalex.org/W2891223802	https://europepmc.org/articles/pmc6213242?pdf=render	English	null	The Role of Matrix Gla Protein (MGP) Expression in Paclitaxel and Topotecan Resistant Ovarian Cancer Cell Lines	International journal of molecular sciences	2,018	cc-by	12,712	Received: 24 August 2018; Accepted: 21 September 2018; Published: 25 September 2018 Abstract: The major cause of ovarian cancer treatment failure in cancer patients is inherent or acquired during treatment drug resistance of cancer. Matrix Gla protein (MGP) is a secreted, non-collagenous extracellular matrix protein involved in inhibition of tissue calciﬁcation. Recently, MGP expression was related to cellular differentiation and tumor progression. A detailed MGP expression analysis in sensitive (A2780) and resistant to paclitaxel (PAC) (A2780PR) and topotecan (TOP) (A2780TR) ovarian cancer cell lines and their corresponding media was performed. MGP mRNA level (real time PCR analysis) and protein expression in cell lysates and cell culture medium (Western blot analysis) and protein expression in cancer cells (immunoﬂuorescence analysis) and cancer patient lesions (immunohistochemistry) were determined in this study. We observed increased expression of MGP in PAC and TOP resistant cell lines at both mRNA and protein level. MGP protein was also detected in the corresponding culture media. Finally, we detected expression of MGP protein in ovarian cancer lesions from different histological type of cancer. MGP is an important factor that might contribute to cancer resistance mechanism by augmenting the interaction of cells with ECM components leading to increased resistance of ovarian cancer cells to paclitaxel and topotecan. Expression found in ovarian cancer tissue suggests its possible role in ovarian cancer pathogenesis. Keywords: matrix Gla protein; ovarian cancer; paclitaxel resistance; topotecan resistance International Journal of Molecular Sciences International Journal of Molecular Sciences The Role of Matrix Gla Protein (MGP) Expression in Paclitaxel and Topotecan Resistant Ovarian Cancer Cell Lines Karolina Sterzy´nska 1,* , Andrzej Klejewski 2,3, Karolina Wojtowicz 1, Monika ´Swierczewska 1, Małgorzata Andrzejewska 1, Damian Rusek 4, Maciej Sobkowski 5, Witold K˛edzia 6, Jacek Br ˛azert 3, Michał Nowicki 1 and Radosław Januchowski 1 1 Department of Histology and Embryology, Poznan University of Medical Sciences, ´Swi˛ecickiego 6 St., 61-781 Pozna´n, Poland; kwojtowicz@ump.edu.pl (K.W.); mswierczewska@ump.edu.pl (M.S.); mandrzej@ump.edu.pl (M.A.); mnowicki@ump.edu.pl (M.N.); rjanuchowski@ump.edu.pl (R.J.) 2 Department of Nursing, Poznan University of Medical Sciences, Smoluchowskiego 11 St., 1 Department of Histology and Embryology, Poznan University of Medical Sciences, ´Swi˛ecickiego 6 St., 61-781 Pozna´n, Poland; kwojtowicz@ump.edu.pl (K.W.); mswierczewska@ump.edu.pl (M.S.); mandrzej@ump.edu.pl (M.A.); mnowicki@ump.edu.pl (M.N.); rjanuchowski@ump.edu.pl (R.J.) 2 Department of Nursing, Poznan University of Medical Sciences, Smoluchowskiego 11 St., j p p ( ) p p ( ) j p p ( ) 2 Department of Nursing, Poznan University of Medical Sciences, Smoluchowskiego 11 St., 60-179 Pozna´n, Poland; aklejewski@ump.edu.pl j p p 3 Department of Obstetrics and Women’s Diseases, Poznan University of Medical Sciences, Polna 33 St, 60-535 Pozna´n, Poland; jbrazert@ump.edu.pl 4 Department of Pathomorphology, Non-public Health Care Facility Alfamed, Jana Pawła II 10 St, 22-400 Zamo´s´c, Poland; d.rusek@vp.pl p p 5 Department of Mother and Child Health, Poznan University of Medical Sciences, Polna 33 St, 60-535 Pozna´n, Poland; msobkow@ump.edu.pl 6 Department of Gynecology, Poznan University of Medical Sciences, Polna 33 St, 60-535 Pozna´n, Poland witold.kedzia@ump.edu.pl * Correspondence: k.olejniczak@ump.edu.pl; Tel.: +48-61-8546455 1. Introduction The major cause of ovarian cancer treatment failure is tumoral heterogeneity and occurrence of drug resistance in ovarian cancer patients. There are two mechanisms of inherent or acquired during treatment chemoresistance that involve cellular and/or tissue speciﬁc response. The cellular Int. J. Mol. Sci. 2018, 19, 2901; doi:10.3390/ijms19102901 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms 2 of 17 Int. J. Mol. Sci. 2018, 19, 2901 mechanisms include reduced drug accumulation and changes in drug distribution in the cell, faster inactivation of the drug and more efﬁcient mechanisms of DNA and cellular membranes repair caused by the drug [1]. However, one of the principal mechanisms of cellular chemoresistance is multiple drug resistance (MDR). The cancer cell acquires the ability to actively pump cytotoxic drugs out of the cell via drug transporters. The best known are ABC family transporters, with glycoprotein P (P-gp) and BCRP (breast cancer resistant protein) as the most extensively studied ones [2,3] Those proteins facilitate the outﬂow of chemotherapeutic agents and thereby contribute signiﬁcantly to cancer cell resistance. On the other hand, the knowledge concerning a cancer tissue speciﬁc mechanism of chemoresistance is still deﬁcient. The tumor environment is now identiﬁed as a leading factor that promotes cancer progression and resistance to anticancer therapy [4]. It is composed of cellular (cancer cells, cancer associated ﬁbroblasts (CAFs), and tumor associated macrophages (TAMs)) and non-cellular constituents including extracellular matrix components (ECM). Increased expression of proteoglycans or collagens in tumor microenvironment create a barrier and inhibits the diffusion of anti-cancer agents [5]. It has been reported that changes in ECM can modulate drug resistance by preventing the penetration of anticancer drugs through tumor tissue [6,7]. Changes in the microenvironment induce tumor progression, malignancy and anticancer drug resistance through integrin signaling as the result of ECM tumor-associated remodeling [8]. Cancer cells show decreased sensitivity to apoptosis inﬂuenced by ECM components that leads to induction of their resistance [9]. The development of drug resistance caused by compounds of extracellular matrix has been described as cell adhesion-mediated drug resistance (CAM-DR) [10] and noted in small cell lung cancer in vivo and in ovarian cancer in vitro [11,12] However, different molecules of ECM have also been described as produced by cultured cells in vitro, e.g., drug resistant breast [13] and ovarian cancer cell lines [14,15]. 1. Introduction p y g g Matrix Gla protein (MGP) is a secreted, non-collagenous extracellular matrix protein containing post-translationally modiﬁed γ-carboxyglutamic acid residues resulting from vitamin K-dependent carboxylation [16]. The protein was initially isolated from bone and cartilage [17,18] but in human it is found also in soft tissues such as kidney, lung, heart and vascular smooth muscle cells [19]. The physiological role of this protein is to inhibit tissue calciﬁcation [20], but it is also implicated in pathological calciﬁcations [21] as well as abnormal angiogenesis that can promote tumor progression [22]. It has been additionally suggested that MGP expression is related to cellular differentiation and tumor progression [23,24]. However, MGP expression may be tumor type dependent. On the one hand, there is a negative correlation of MGP expression with tumor progression and metastasis in renal and prostate carcinoma [25]. On the other hand, up-regulation of MGP transcript in breast tumors and glioblastomas is associated with tumor progression and poor prognosis [24,26]. Little is known about the mechanisms linking upregulation of MGP with migration ability, metastases formation or drug resistance, and, more generally, the function of MGP in tumors. Recently, it was proved that MGP binds ﬁbronectin and augments cell adhesion and spreading of cancer cells [27]. Increased expression of MGP in glioblastoma cells is associated with increased tumor cells migratory properties in vitro and tumor spreading in vivo, whereas MGP-knock down leads to opposite effect [26]. Overexpression of MGP in osteosarcoma cells leads to statistically signiﬁcant number and size of lung metastasis in mouse model. Furthermore, MGP serum level is associated with lung metastasis in osteosarcoma patients [28]. To gain a better understanding of the role of MGP in drug resistance development, we used an ovarian cancer model, which is the most lethal gynecological malignancy [29]. Although ovarian cancer is sensitive to chemotherapy, especially at early stages, most patients will eventually develop resistance to anti-cancer drugs. The ﬁrst-line chemotherapy regimen for ovarian cancer treatment combine chemotherapy of platinum (cisplatin (CIS)) and taxanes (paclitaxel (PAC)) [29]. The second line chemotherapy includes doxorubicin (DOX) and topotecan (TOP), among others [30]. y g In this study, we used two different cell lines resistant to PAC and TOP. Paclitaxel belongs to the family of antimitotic anticancer agents that block mitosis through stabilizing microtubules, Int. J. Mol. Sci. 2018, 19, 2901 3 of 17 which consequently prevents cell division and leads to apoptotic cell death [31]. 1. Introduction On the other hand, topotecan is semi-synthetic derivative of camptothecin [32] and acts as an inhibitor of DNA topoisomerase I. Inhibition of DNA topoisomerase I results in inhibition of DNA replication and transcription that eventually leads to cancer cell death [33]. Unfortunately, cancer cells can develop resistance to both cytotoxic agents. The mechanism of resistance to PAC and TOP is related to altered expression of β-tubulin [34] or Topoizomerase I [35], respectively. However, it is considered that the most important mechanism responsible for the resistance to PAC and TOP is the overexpression of drug transporters from ABC family [3,36]. The results of our previous microarray tests indicated that MGP was overexpressed in ﬁve of eight drug-resistant ovarian cancer cell lines [37]. In this study, we performed detailed expression analysis of MGP at mRNA and protein levels in sensitive (A2780) and resistant to paclitaxel (A2780PR1) and topotecan (A2780TR1 and A2780TR2) ovarian cancer cell lines and their corresponding media. Additionally, analysis of parafﬁn sections conﬁrmed the presence of MGP in ovarian cancer tissue. 2. Results 2.1. Analyses of MGP Gene Expression in Drug-Resistant Ovarian Cancer and Breast Cancer Cell Lines 2.1. Analyses of MGP Gene Expression in Drug-Resistant Ovarian Cancer and Breast Cancer Cell Lin According the ENSEMBL database (www.ensembl.org), MGP is expressed in two main transcript variants (Table 1) corresponding to two protein isoforms with molecular weight of 15.32 kDa (128 aa) (MGP-201) and molecular weight of 12.35 kDa (103 aa) (MGP-203). To determine expression of both transcripts, we designed two pairs of primers that speciﬁcally recognize both variants (Table 1). To determine whether the development of drug resistance in A2780 drug-resistant sublines is associated with MGP overexpression, expression of the MGP mRNAs was assessed. We observed a statistically signiﬁcant increase of both MGP transcripts in A2780PR1 (p < 0.01 for MGP-201 and p < 0.05 for MGP-203), A2780TR1 (p < 0.01 for MGP-201 and p < 0.01 for MGP-203) and A2780TR2 cell lines (p < 0.001 for MGP-201 and p < 0.01 for MGP-203) (Figure 1A,B). We observed proportional increase of both transcripts level in investigated cell lines (R2 = 0.998), however the expression of MGP-201 was higher than MGP-203 in all resistant cell lines; 189- vs. 77-fold (about 2.5-fold) for A2780PR1, 155- vs. 43-fold (about 3.5-fold) for A2780TR1 and 1098- vs. 428-fold (about 2.5–fold) for A2780TR2 cell line. Table 1. Oligonucleotide sequences used for RQ-PCR analysis. Transcript Sequence (5′-3′ direction) ENST Number http://www.ensembl.org Product Size (bp) MGP CTGATCCTTCTTGCCATCCT CCATCTCTGCTGAGGGGATA 00000228938.5 00000539261.5 141 bp MGP-201 GTGCCCAGGAATCACATGAAAG ACAGGCTTAGAGCGTTCTCG 00000228938.5 142 bp MGP-203 AAGAGAGGATCCGAGAACGCC AGCGTTCGCAAAGTCTGTAG 00000539261.5 81 bp GADPH GAAGGTGAAGGTCGGAGTCA GACAAGCTTCCCGTTCTCAG 00000229239 199 bp β-actin TCTGGCACCACACCTTCTAC GATAGCACAGCCTGGATAGC 00000331789 169 bp HRPT1 CTGAGGATTTGGAAAGGGTG AATCCAGCAGGTCAGCAAAG 00000298556 156 bp β2M CGCTACTCTCTCTTTCTGGC ATGTCGGATGGATGAAACCC 00000558401 133 bp Table 1. Oligonucleotide sequences used for RQ-PCR analysis. Int. J. Mol. Sci. 2018, 19, 2901 β2M CG 4 of 17 p C 00000558401 133 bp 4 of 1 C 00000558401 133 bp Figure 1. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts in the A2780 and drug resistant sublines. The figure presents the relative gene expression in the resistant cell lines (grey bars) with respect to that in the sensitive cell line (white bars), which was assigned a value of 1. The values were considered significant at * p < 0.05, p < 0.01 and * p < 0.001. Figure 1. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts in the A2780 and drug resistant sublines. 2. Results The ﬁgure presents the relative gene expression in the resistant cell lines (grey bars) with respect to that in the sensitive cell line (white bars), which was assigned a value of 1. The values were considered signiﬁcant at * p < 0.05, p < 0.01 and * p < 0.001. nt. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 17 β2M CGCTACTCTCTCTTTCTGGC ATGTCGGATGGATGAAACCC 00000558401 133 bp Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW β2M CGCTACTCTCTCTTTCTGGC ATGTCGGATGGATGAAACCC Figure 1. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts in the A2780 and drug resistant sublines. The figure presents the relative gene expression in the resistant cell lines (grey bars) with respect to that in the sensitive cell line (white bars), which was assigned a value of 1. The values were considered significant at * p < 0.05, p < 0.01 and * p < 0.001. Figure 1. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts in the A2780 and drug resistant sublines. The ﬁgure presents the relative gene expression in the resistant cell lines (grey bars) with respect to that in the sensitive cell line (white bars), which was assigned a value of 1. The values were considered signiﬁcant at * p < 0.05, p < 0.01 and * p < 0.001. To check if both MGP transcripts are also present in another cancer cell line, we compared their expression between A2780 cell line and breast cancer cell T47D, which is known to express MGP at very high level [38]. We observed statistically signiﬁcant increase in expression of both transcripts in T47 cell line. Here, we also observed higher increase of MGP-201 transcript (about 2500-fold, p < 0.01) than MGP-203 (about 1100-fold, p < 0.01) (Figure 2A,B). However, in comparison to control A2780 cell line, increase in both transcripts level was higher in T47D cell line than in A2780T2 cell line (MGP-201, 2500-fold vs. 1098-fold; MGP-203, 1100-fold vs. 428-fold). Figure 1. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts in the A2780 and drug resistant sublines. The figure presents the relative gene expression in the resistant cell lines (grey bars) with respect to that in the sensitive cell line (white bars), which was assigned a value of 1. 2. Results The values were considered significant at * p < 0.05, p < 0.01 and * p < 0.001. Figure 2. Expression analysis (Q-PCR) of the MG presents the relative gene expression in the brea that in the control ovarian cancer cell line A2780 values were considered significant at p < 0.01. .2. Immunofluorescence Analysis of the MGP Protei To confirm the presence of the MGP pro luorescence analysis of its expression in A2780 Figure 2. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts. The figure presents the relative gene expression in the breast cancer cell line T47D (grey bars) with respect to that in the control ovarian cancer cell line A2780 (white bars), which was assigned a value of 1. The values were considered significant at p < 0.01. 2.2. Immunofluorescence Analysis of the MGP Protein Expression To confirm the presence of the MGP protein in the investigated cell lines, we perform fluorescence analysis of its expression in A2780, A2780PR1, A2780TR1 and A2780TR2 cell lines. low fluorescence signal was present in the A2780 cell line. In the A2780PR1, A2780TR1, an A2780TR2 cell lines, we observed increase in fluorescence intensity (Figure 3). Figure 2. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts. The figure presents the relative gene expression in the breast cancer cell line T47D (grey bars) with respect to that in the control ovarian cancer cell line A2780 (white bars), which was assigned a value of 1. The values were considered significant at p < 0.01. Figure 2. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts. The ﬁgure presents the relative gene expression in the breast cancer cell line T47D (grey bars) with respect to that in the control ovarian cancer cell line A2780 (white bars), which was assigned a value of 1. The values were considered signiﬁcant at p < 0.01. GP1-201 (A) and MGP-203 (B) transcripts. The figure ast cancer cell line T47D (grey bars) with respect to 0 (white bars), which was assigned a value of 1. The in Expression otein in the investigated cell lines, we perfor 0 A2780PR1 A2780TR1 d A2780TR2 ll li orescence analysis of its expression in A2780, A2780PR1, A2780TR1 and A2780TR2 cell line w fluorescence signal was present in the A2780 cell line. 2. Results In the A2780PR1, A2780TR1, 780TR2 cell lines, we observed increase in fluorescence intensity (Figure 3). Figure 2. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts. The figure presents the relative gene expression in the breast cancer cell line T47D (grey bars) with respect to that in the control ovarian cancer cell line A2780 (white bars), which was assigned a value of 1. The values were considered significant at p < 0.01. Figure 2. Expression analysis (Q-PCR) of the MGP1-201 (A) and MGP-203 (B) transcripts. The ﬁgure presents the relative gene expression in the breast cancer cell line T47D (grey bars) with respect to that in the control ovarian cancer cell line A2780 (white bars), which was assigned a value of 1. The values were considered signiﬁcant at p < 0.01. 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression The elevated expression of MGP at the protein level was confirmed by Western blot analysis. In cell lysates, we observed increase in MGP bands intensity in both PAC- (p < 0.05) and TOP-resistant A2780 cell lines (p < 0.05 for A2780TR1 and p < 0.01 for A2780TR2). However, we observed only one b d di l l f 15 32 kD (Fi 4A) The elevated expression of MGP at the protein level was conﬁrmed by Western blot analysis. In cell lysates, we observed increase in MGP bands intensity in both PAC- (p < 0.05) and TOP-resistant A2780 cell lines (p < 0.05 for A2780TR1 and p < 0.01 for A2780TR2). However, we observed only one band corresponding to molecular mass of 15.32 kDa (Figure 4A). band corresponding to molecular mass of 15.32 kDa (Figure 4A). In contrast in the corresponding medium we observed band with mass of 12.35 kDa. This band was clearly present in A2780PR1 and A2780TR2 cell lines and very weak signal was observed in A2780TR1 cell line and signal was not present in A2780 cell line (Figure 4B). Next, we analyzed expression of MGP protein in T47D cell line. Clear increase in band intensity with molecular weight of 15.32 kDa was observed in T47D cell line in comparison to A2780 cell line (p < 0.05). In T47D cell line, we also observed additional band corresponding to molecular weight of 12.35 kDa, although the intensity of this band was very low (Figure 4C). Additionally, in all lysates of investigated cell lines we observed additional band with molecular mass about 110 kDa. Bands intensity correlated ith MGP t a i t le el i all i e ti ated ell li e (Fi u e 4D) In contrast in the corresponding medium we observed band with mass of 12.35 kDa. This band was clearly present in A2780PR1 and A2780TR2 cell lines and very weak signal was observed in A2780TR1 cell line and signal was not present in A2780 cell line (Figure 4B). Next, we analyzed expression of MGP protein in T47D cell line. Clear increase in band intensity with molecular weight of 15.32 kDa was observed in T47D cell line in comparison to A2780 cell line (p < 0.05). 2 2 Immunofluorescence Analysis of the MGP Protein Expression 2.2. Immunoﬂuorescence Analysis of the MGP Protein Expression I u of uo e e e A a y i of e o ei E p e io To confirm the presence of the MGP protein in the investigated cell lines, we performed fluorescence analysis of its expression in A2780, A2780PR1, A2780TR1 and A2780TR2 cell lines. A low fluorescence signal was present in the A2780 cell line. In the A2780PR1, A2780TR1, and A2780TR2 cell lines we observed increase in fluorescence intensity (Figure 3) To conﬁrm the presence of the MGP protein in the investigated cell lines, we performed ﬂuorescence analysis of its expression in A2780, A2780PR1, A2780TR1 and A2780TR2 cell lines. A low ﬂuorescence signal was present in the A2780 cell line. In the A2780PR1, A2780TR1, and A2780TR2 cell lines, we observed increase in ﬂuorescence intensity (Figure 3). 5 of 17 5 f 17 Int. J. Mol. Sci. 2018, 19, 2901 I t J M l S i 2018 19 FOR Figure 3. Immunofluorescence visualization of MGP protein expression in the A2780, A2780PR1, A2780TR1, A2780TR2 cell lines. MGP was detected using the anti-MGP antibody and MFP488-conjugated secondary antibody (green). To visualize the cell nuclei, the cells were mounted with a DAPI-containing mounting medium (blue). Scale bar = 20 μm. Figure 3. Immunoﬂuorescence visualization of MGP protein expression in the A2780, A2780PR1, A2780TR1, A2780TR2 cell lines. MGP was detected using the anti-MGP antibody and MFP488-conjugated secondary antibody (green). To visualize the cell nuclei, the cells were mounted with a DAPI-containing mounting medium (blue). Scale bar = 20 µm. Figure 3. Immunofluorescence visualization of MGP protein expression in the A2780, A2780PR1, A2780TR1, A2780TR2 cell lines. MGP was detected using the anti-MGP antibody and MFP488-conjugated secondary antibody (green). To visualize the cell nuclei, the cells were mounted with a DAPI-containing mounting medium (blue). Scale bar = 20 μm. Figure 3. Immunoﬂuorescence visualization of MGP protein expression in the A2780, A2780PR1, A2780TR1, A2780TR2 cell lines. MGP was detected using the anti-MGP antibody and MFP488-conjugated secondary antibody (green). To visualize the cell nuclei, the cells were mounted with a DAPI-containing mounting medium (blue). Scale bar = 20 µm. g g 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression The cellular proteins were separated using 7% PAGE and transferred to a PVDF membrane, which was then immunoblotted with either primary Ab or HRP-conjugated secondary Ab. A primary anti-GADPH Ab was used as a loading control for the cell lysates. The graphs show the results of the densitometric quantification of the Western blot analysis optical density, which is presented as a MGP/GADPH ratio (with exception of (B), presenting MGP optical density). The values were considered significant at * p < 0.05, ** p < 0.01 and Figure 4. MGP protein expression analysis: in the A2780 and drug-resistant cell lines (A); in the corresponding media (B); in the A2780 and T47D cell lines (C); and analysis of band with high molecular mass in all investigated cell lines (D). The cellular proteins were separated using 7% PAGE and transferred to a PVDF membrane, which was then immunoblotted with either primary Ab or HRP-conjugated secondary Ab. A primary anti-GADPH Ab was used as a loading control for the cell lysates. The graphs show the results of the densitometric quantiﬁcation of the Western blot analysis optical density, which is presented as a MGP/GADPH ratio (with exception of (B), presenting MGP optical density). The values were considered signiﬁcant at * p < 0.05, p < 0.01 and * p < 0.001. *** p < 0.001. 2.4. Early Response to PAC and TOP Treatment in Ovarian Cancer Cell Lines 2.4. Early Response to PAC and TOP Treatment in Ovarian Cancer Cell Lines The second part of our study focused on the early response to PAC and TOP treatment. In these experiments, drug-sensitive cell line A2780 was treated with low concentrations of PAC (20 and 25 ng/mL) or low concentration of TOP (10 and 20 ng/mL) for 24, 48 and 72 h. Then, changes in gene expression were investigated. We used primers that recognize both MGP transcript variants. We observed dose and time dependent increase in MGP transcript level (p < 0.05 with exception of 20 ng/mL and 24 h) in response to PAC treatment with maximum increase in transcript level—about The second part of our study focused on the early response to PAC and TOP treatment. In these experiments, drug-sensitive cell line A2780 was treated with low concentrations of PAC (20 and 25 ng/mL) or low concentration of TOP (10 and 20 ng/mL) for 24, 48 and 72 h. 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression In T47D cell line, we also observed additional band corresponding to molecular weight of 12.35 kDa, although the intensity of this band was very low (Figure 4C). Additionally, in all lysates of investigated cell lines we observed additional band with molecular mass about 110 kDa. Bands intensity correlated with MGP transcripts level in all investigated cell lines (Figure 4D). 6 of 17 Int. J. Mol. Sci. 2018, 19, 2901 J Figure 4. MGP protein expression analysis: in the A2780 and drug-resistant cell lines (A); in the corresponding media (B); in the A2780 and T47D cell lines (C); and analysis of band with high molecular mass in all investigated cell lines (D). The cellular proteins were separated using 7% PAGE and transferred to a PVDF membrane, which was then immunoblotted with either primary Ab or HRP-conjugated secondary Ab. A primary anti-GADPH Ab was used as a loading control for the cell lysates. The graphs show the results of the densitometric quantification of the Western blot analysis optical density, which is presented as a MGP/GADPH ratio (with exception of (B), presenting MGP optical density). The values were considered significant at * p < 0.05, p < 0.01 and * p < 0.001. Figure 4. MGP protein expression analysis: in the A2780 and drug-resistant cell lines (A); in the corresponding media (B); in the A2780 and T47D cell lines (C); and analysis of band with high molecular mass in all investigated cell lines (D). The cellular proteins were separated using 7% PAGE and transferred to a PVDF membrane, which was then immunoblotted with either primary Ab or HRP-conjugated secondary Ab. A primary anti-GADPH Ab was used as a loading control for the cell lysates. The graphs show the results of the densitometric quantiﬁcation of the Western blot analysis optical density, which is presented as a MGP/GADPH ratio (with exception of (B), presenting MGP optical density). The values were considered signiﬁcant at * p < 0.05, p < 0.01 and * p < 0.001. 2.4. Early Response to PAC and TOP Treatment in Ovarian Cancer Cell Lines Figure 4. MGP protein expression analysis: in the A2780 and drug-resistant cell lines (A); in the corresponding media (B); in the A2780 and T47D cell lines (C); and analysis of band with high molecular mass in all investigated cell lines (D). 2 5 Immunohistochemistry 2.5. Immunohistochemistry 2.5. Immunohistochemistry y Immunohistochemical analysis of the MGP protein was performed in ovarian cancer patients. The purpose of this analysis was to verify whether the expression of the analyzed MGP gene and protein that was observed in cell lines could also be confirmed in real cancer patient tissues. Few cases of endometrioid, serous and mucinous ovarian cancer was analyzed to determine differences in MGP expression in various subtypes of cancer. All ovarian cancer subtypes cells expressed MGP, with lower expression in serous and mucinous carcinoma (mild IRS score) (Figure 6A,B) when compared to endometrioid adenocarcinoma (moderate IRS score) (Figure 6C). Additionally, in the endometrioid adenocarcinoma, we could observe two subpopulations of cancer cells with different levels of MGP protein expression (high and weak/moderate). There was no difference in MGP expression in stroma of analyzed specimens where all cancer subtypes showed weak expression Immunohistochemical analysis of the MGP protein was performed in ovarian cancer patients. The purpose of this analysis was to verify whether the expression of the analyzed MGP gene and protein that was observed in cell lines could also be conﬁrmed in real cancer patient tissues. Few cases of endometrioid, serous and mucinous ovarian cancer was analyzed to determine differences in MGP expression in various subtypes of cancer. All ovarian cancer subtypes cells expressed MGP, with lower expression in serous and mucinous carcinoma (mild IRS score) (Figure 6A,B) when compared to endometrioid adenocarcinoma (moderate IRS score) (Figure 6C). Additionally, in the endometrioid adenocarcinoma, we could observe two subpopulations of cancer cells with different levels of MGP protein expression (high and weak/moderate). There was no difference in MGP expression in stroma of analyzed specimens, where all cancer subtypes showed weak expression. Immunohistochemical analysis of the MGP protein was performed in ovarian cancer patients. The purpose of this analysis was to verify whether the expression of the analyzed MGP gene and protein that was observed in cell lines could also be confirmed in real cancer patient tissues. Few cases of endometrioid, serous and mucinous ovarian cancer was analyzed to determine differences in MGP expression in various subtypes of cancer. All ovarian cancer subtypes cells expressed MGP, with lower expression in serous and mucinous carcinoma (mild IRS score) (Figure 6A,B) when compared to endometrioid adenocarcinoma (moderate IRS score) (Figure 6C). Additionally, in the endometrioid adenocarcinoma, we could observe two subpopulations of cancer cells with different levels of MGP protein expression (high and weak/moderate). 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression Then, changes in gene expression were investigated. We used primers that recognize both MGP transcript variants. We observed dose and time dependent increase in MGP transcript level (p < 0.05 with exception of 20 ng/mL and 24 h) in response to PAC treatment with maximum increase in transcript level—about 3.5-fold—after 72 h of treatment (Figure 5A). Similar time dependent increase in MGP transcript 7 of 17 f 7 of 17 Int. J. Mol. Sci. 2018, 19, 2901 I t J M l S i 2018 19 FOR level was observed after TOP treatment (p < 0.05 or p < 0.01) with maximum increase in transcript level—about 8-fold—after 72 h of treatment (Figure 5B). level was observed after TOP treatment (p < 0.05 or p < 0.01) with maximum increase in transcript level—about 8-fold—after 72 h of treatment (Figure 5B). level was observed after TOP treatment (p < 0.05 or p < 0.01) with maximum increase in transcript level—about 8-fold—after 72 h of treatment (Figure 5B). Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). The figure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered significant at * p < 0.05, and ** p < 0.01. Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). The ﬁgure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered signiﬁcant at * p < 0.05, and ** p < 0.01. Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). The figure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered significant at * p < 0.05, and ** p < 0.01. Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). 2 5 Immunohistochemistry 2.5. Immunohistochemistry 2.5. Immunohistochemistry There was no difference in MGP expression in stroma of analyzed specimens, where all cancer subtypes showed weak expression. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 μm. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 μm. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 µm. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 μm. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 μm. Figure 6. Immunohistochemical expression of MGP in serous adenocarcinoma patient (weak) (A); mucinous ovarian cancer patient (weak) (B); and endometrioid adenocarcinoma patient (moderate/strong) (C). Arrows point the cytoplasmic MGP expression in cancer cells. Sections were counterstained with hematoxylin. Scale bar = 50 µm. 2 3 Western Blot Analysis of MGP Protein Expression 2.3. Western Blot Analysis of MGP Protein Expression The figure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered significant at * p < 0.05, and ** p < 0.01. Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). The ﬁgure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered signiﬁcant at * p < 0.05, and ** p < 0.01. Figure 5. Expression analysis of MGP gene in A2780 cell line after short time exposure to PAC (A) and TOP (B). The figure presents relative genes expression in PAC or TOP treated cells (grey and black bars) with respect to the untreated control (white bars) assigned as 1. The values were considered significant at * p < 0.05, and ** p < 0.01. 3 Discussion 3. Discussion 3. Discussion The most significant problem with low efficiency of chemotherapy in cancer patients results from development of drug resistance to cytotoxic agents. For many years, most research has focused on cellular mechanisms of chemoresistance. However, an increasing body of evidence indicates that expression of ECM proteins in the tumor environment can play even more important role in drug The most significant problem with low efficiency of chemotherapy in cancer patients results from development of drug resistance to cytotoxic agents. For many years, most research has focused on cellular mechanisms of chemoresistance. However, an increasing body of evidence indicates that expression of ECM proteins in the tumor environment can play even more important role in drug The most signiﬁcant problem with low efﬁciency of chemotherapy in cancer patients results from development of drug resistance to cytotoxic agents. For many years, most research has focused on cellular mechanisms of chemoresistance. However, an increasing body of evidence indicates that expression of ECM proteins in the tumor environment can play even more important role in drug Int. J. Mol. Sci. 2018, 19, 2901 8 of 17 resistance, especially from the clinical point of view [10,39]. It is worth mentioning that expression of ECM and related proteins is limited not only to tumor stroma and cancer-associated ﬁbroblasts (CAF) but was also observed in tumor cells in vivo [10,12,15,40,41] and in cancer cell lines [13,37,42]. Recently, with the use of the RNA microarray technique, we have identiﬁed many new genes with increased expression in drug resistant cell lines [37]. Among them, we identiﬁed matrix Gla protein (MGP) with elevated expression levels in ovarian cancer cell lines resistant to PAC and TOP. Because nothing is known about the mechanisms linking upregulation of MGP and resistance to chemotherapeutic drugs and, more generally, about the function of MGP in ovarian cancer, we hypothesized that MGP promotes ovarian cancer progression and resistance to chemotherapy. Therefore, we decided to investigate the expression proﬁle of MGP in PAC- and TOP-resistant ovarian cancer cell lines in more detail. Since MGP is expressed in two main transcript variants corresponding to two protein isoforms with molecular weight of 15.32 kDa (128 aa) (MGP-201) and 12.35 kDa (103 aa) (MGP-203), we compared the expression levels of both MGP transcripts in ovarian cancer drug sensitive and resistant cell lines. 3 Discussion 3. Discussion 3. Discussion We observed high increase in expression of MGP in the cell lines resistant to PAC (A2780PR1) and TOP (A2780TR1 and A2780TR2), with the highest level for A2780TR2 cell line. Both transcript variants were upregulated in drug resistant sublines although expression of MGP-201 was always higher (2.5–3.5-fold) than expression of MGP-203. To verify if expression of both transcript variants is speciﬁc only to drug resistant cell lines or can be expressed also in other cancer cell lines, we investigated expression in breast cancer cell line T47D that is known to present MGP expression at high level [38]. Similar to results in drug resistant cell lines, we also observed increased expression of both transcript variants, with higher level of MGP-201, in T47D cell line as well (about 2.5-fold). We have also compared expression of both transcripts between A2780TR2 and T47D cell lines and we discovered that expression any of them was about two times higher in T47D cell line. Thus, proportion between both transcript expression seems to be a general feature of cancer cells. Immunoﬂuorescence analysis conﬁrmed elevated expression of MGP protein in investigated ovarian cancer cell lines, which indicates that MGP is equally expressed in all cells. Since this method does not allow distinguishing protein isoforms, we evaluated Western blot analysis to compare the protein expression levels of MGP between investigated cell lines. The differences in expression of two MGP variants were also conﬁrmed at the protein level. In breast cancer cell line, both variants of MGP proteins was clearly visible although isoform with molecular weight of 15.32 kDa seems to be expressed at much higher level. These results correlate with expression on transcript level and additionally indicate that the antibody used for the experiment is able to recognize both protein isoforms. In ovarian cancer cells only one form with molecular weight of 15.32 kDa was noted. This form was overexpressed in cell lines resistant to PAC and TOP. Since we previously observed that COL3A1, LUM [14,15] and MYOT (under review) are secreted to cell culture media in drug resistant ovarian cancer cell lines, we were interested whether MGP can also be presentin the culture medium. Indeed, we have noticed that MGP protein was present in culture media from investigated drug resistant cell lines. As opposed to cell lysates, the MGP isoform with a mass of 12.35 kDa was observed in the corresponding media. 3 Discussion 3. Discussion 3. Discussion Other research describes the increased expression of ECM, especially collagens that limit drug diffusion in cancer tissue and in in vitro study [5,7,44,45]. On the other hand, all investigated cell lines expressed different ECM molecules including different collagens as we reported previously [37]. Even in 2D cell culture condition we observed that COL3A1 was present extracellularly forming structure similar to a spiderweb [14]. Since it was already proved that MGP may incorporate into ﬁbronectin multimers [27], e it is also possible that this small molecule may place between collagen ﬁbers and in this way limit drug delivery to the cancer cells. To prove this hypothesis, further analysis with the use of 3D cell culture condition should be applied. The third possibility of MGP in drug resistance involvement is the role of extracellular MGP in CAM-DR [10,41]. Interaction of cancer cells with their microenvironment can lead to inhibition of drug induced apoptosis. Cancer cells interact with ECM compounds mainly by integrin receptors. CAM-DR has been observed both in vivo and in vitro. The interaction of β1-integrin with ECM was proven to be responsible for resistance to DOX and melphalan in SCLC [11]. On the other hand, collagen type I is considered as involved in inducing chemoresistance by upregulating microtubule associated protein tau in paclitaxel resistant ovarian cancer cell lines [46]. In in vitro study with A2780 ovarian cancer cell line revealed that those cells cultured on COL6A3 coated dishes were more resistant to CIS [12]. CAM-DR can be induced not only by collagen molecules but was also observed for other ECM compounds like ﬁbronectin and laminin. In this case, pancreatic cancer cells became resistant to DOX, CIS and 5-ﬂuorouracil (5-FU) [47] when growing on ﬁbronectin and laminin and breast cancer cells were more resistant to PAC on surfaces coated with collagen type I [48]. This might indicate a coordinated response between tumor cells and its microenvironment to protect cancer cell against drugs and to facilitate malignancy progress. The role of secretory MGP in CAM-DR seems to be related rather to its interaction with other ECM proteins. It has already been proven that MGP binds to ﬁbronectin and vitronectin and augments cell adhesion and spreading of cancer cells. The protein itself has no adhesive activity and its role is to alter the ability of cells to bind ﬁbronectin [27]. 3 Discussion 3. Discussion 3. Discussion In all investigated cell lines, we could also observe additional bands with molecular mass about 110 kDa. We were not sure whether these bands are speciﬁc, but we paid attention to them since their intensity clearly correlated with MGP transcript and protein level in investigated cell lines. This result is difﬁcult to interpret because molecular mass of MGP is about 10-fold lower. On the other hand, clear correlation with MGP expression suggests two possibilities. There could be another protein with coordinate expression and recognized by the same antibody or a different formation of multimeric MGP composed of several subunits. Because a Western blot experiment is conducted under denaturating and reductive condition, these subunits should bind to each other by covalent bonding, however this is only a hypothesis. To our knowledge, this is the ﬁrst report about MGP expression in drug resistant cell lines. We undertook a comprehensive literature search and did not ﬁnd any research concerning MGP 9 of 17 Int. J. Mol. Sci. 2018, 19, 2901 expression in cancer cells resistant to cytotoxic drugs. However, different levels of MGP transcripts were observed in different human and rat glioblastoma cell lines [26,43] and MGP protein released to culture medium was observed in glioblastoma cell line as well [26]. These ﬁndings conﬁrm that MGP is expressed in different neoplastic cell lines and can be secreted to cell culture medium. However, in those studies, the primers speciﬁc to transcript variants was not considered and the protein level was assessed by the ELISA test. Therefore, we could not compare or refer our results to other research and our result need further validation and explanation. However, different localization of MGP-201 and MGP-203 suggest their different role in drug resistance. The general question is what is the role of MGP in drug resistance. There can be three different possibilities taken under consideration. The ﬁrst one presumes that MGP molecules, found both intracellularly and extracellularly, can bind PAC and TOP molecules limiting their availability for cancer cells. Direct binding of PAC, DOX and VIN by ECM compounds has been reported previously by others [5]. The potential role of MGP in PAC and TOP binding require further investigation using other methods. The second possibility is a limited drug diffusion. It is assumed that MGP does not play a role as a single protein in this action but rather in coordination with other ECM components. 3 Discussion 3. Discussion 3. Discussion On one side, ﬁbronectin interacts with cells via integrins which connect to the actin cytoskeleton and, on the other, with ECM constituents such as collagens and laminins. MGP presence augments this effect and this property is related to migration-promoting activity that was demonstrated for glioblastoma [26] or osteosarcoma cells [28]. Since the interaction of cells with ﬁbronectin lead to increased resistance to different cytotoxic drugs (CAM-DR) and MGP augments this interaction, we have formulated a hypothesis that MGP–ﬁbronectin interaction can intensify CAM-DR in cancer cells (Figure 7). Int. J. Mol. Sci. 2018, 19, 2901 10 of 17 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 17 Figure 7. Schematic drawing of interaction between cell and ECM mediated by fibronectin and MGP. MGP augmented binding of integrins to ECM compounds activates intracellular signaling that results in: (1) cytoskeleton reorganization leading to increased cell proliferation and migration; and (2) upregulation of anti-apoptotic molecules and downregulation of pro-apoptotic molecules that inhibits drug-induced apoptosis. Figure 7. Schematic drawing of interaction between cell and ECM mediated by ﬁbronectin and MGP. MGP augmented binding of integrins to ECM compounds activates intracellular signaling that results in: (1) cytoskeleton reorganization leading to increased cell proliferation and migration; and (2) upregulation of anti-apoptotic molecules and downregulation of pro-apoptotic molecules that inhibits drug-induced apoptosis. Figure 7. Schematic drawing of interaction between cell and ECM mediated by fibronectin and MGP. MGP augmented binding of integrins to ECM compounds activates intracellular signaling that results in: (1) cytoskeleton reorganization leading to increased cell proliferation and migration; and (2) upregulation of anti-apoptotic molecules and downregulation of pro-apoptotic molecules that inhibits drug-induced apoptosis. Figure 7. Schematic drawing of interaction between cell and ECM mediated by ﬁbronectin and MGP. MGP augmented binding of integrins to ECM compounds activates intracellular signaling that results in: (1) cytoskeleton reorganization leading to increased cell proliferation and migration; and (2) upregulation of anti-apoptotic molecules and downregulation of pro-apoptotic molecules that inhibits drug-induced apoptosis. To further prove the significance of MGP expression in PAC and TOP resistance, we performed experiments with short time exposure of cancer cells to investigated drugs. Most research concerning drug resistance is based on “established” mechanism of resistance to cytotoxic agents and comparison of sensitive and resistant pairs of cell lines. It is difficult to find papers that demonstrate the response of cancer cells to cytotoxic drugs during first hours of treatment. 3 Discussion 3. Discussion 3. Discussion Recently, we have published a few papers describing the expression of genes in response to PAC [49], TOP [50,51] and CIS [51] treatment in drug sensitive ovarian cancer cell lines. In the present study, we could observe a dose- and time-dependent increase of MGP mRNA in response to PAC and TOP treatment. The increased expression during the first days after contact with cytotoxic drugs confirms that MGP can indeed be involved in PAC and TOP resistance. Along with our previous results [49–51], this finding indicates that the genes expressed at the beginning of the treatment, a short time after drug exposure, are further expressed in established drug resistant cell lines (at much higher level). Thus, “first response” can be an indicator of “established” response after long time to To further prove the signiﬁcance of MGP expression in PAC and TOP resistance, we performed experiments with short time exposure of cancer cells to investigated drugs. Most research concerning drug resistance is based on “established” mechanism of resistance to cytotoxic agents and comparison of sensitive and resistant pairs of cell lines. It is difﬁcult to ﬁnd papers that demonstrate the response of cancer cells to cytotoxic drugs during ﬁrst hours of treatment. Recently, we have published a few papers describing the expression of genes in response to PAC [49], TOP [50,51] and CIS [51] treatment in drug sensitive ovarian cancer cell lines. In the present study, we could observe a dose- and time-dependent increase of MGP mRNA in response to PAC and TOP treatment. The increased expression during the ﬁrst days after contact with cytotoxic drugs conﬁrms that MGP can indeed be involved in PAC and TOP resistance. Along with our previous results [49–51], this ﬁnding indicates that the genes expressed at the beginning of the treatment, a short time after drug exposure, are further expressed in established drug resistant cell lines (at much higher level). Thus, “ﬁrst response” can be an indicator of “established” response after long time to cytotoxic drug exposure. cytotoxic drug exposure. Previously, upregulation of MGP expression in ovarian cancer tissue was noted by Hough et al. but only at transcript level [52]. To verify MGP expression on protein levels in real cancer patients, we performed immunohistochemistry using paraffin sections. All of the ovarian cancer subtypes cells were positive for MGP. 3 Discussion 3. Discussion 3. Discussion However, the intensity of immunohistochemical staining in our study was different and dependent on the type of ovarian cancer (endometrioid, serous and mucinous ovarian cancer). The weaker expression was present in serous and mucinous carcinoma (mild IRS score) when compared to endometrioid adenocarcinoma where moderate to strong signal was detected. Additionally, in the endometrioid adenocarcinoma we could observe subpopulations of Previously, upregulation of MGP expression in ovarian cancer tissue was noted by Hough et al. but only at transcript level [52]. To verify MGP expression on protein levels in real cancer patients, we performed immunohistochemistry using parafﬁn sections. All of the ovarian cancer subtypes cells were positive for MGP. However, the intensity of immunohistochemical staining in our study was different and dependent on the type of ovarian cancer (endometrioid, serous and mucinous ovarian cancer). The weaker expression was present in serous and mucinous carcinoma (mild IRS score) when compared to endometrioid adenocarcinoma where moderate to strong signal was detected. Additionally, in the endometrioid adenocarcinoma we could observe subpopulations of cancer cells with different levels of MGP protein expression (high and weak/moderate). cancer cells with different levels of MGP protein expression (high and weak/moderate). Similar observations were made by other researchers. In samples with cervical squamous cell Similar observations were made by other researchers. In samples with cervical squamous cell carcinoma (SCC), clear cytoplasmic MGP staining was seen in the tumor cells but in cells seen at the 11 of 17 Int. J. Mol. Sci. 2018, 19, 2901 borders of the tumor ﬁelds the staining was more intense. Moreover, they could also observe a clear strong staining for individual tumor cells of SCC. In the same study, they could also observe elevated MGP expression only in the lower layers of epithelium of high grade cervical intraepithelial neoplasia (CIN) lesions [53]. The analysis and interpretation of immunohistochemical stainings must be done with a great caution since the MGP expression level of individual cancer cells can vary even within a single cancer cell nest. The expression of MGP was noted in some other cancers. In line with our ﬁndings concerning ovarian cancer, increased levels of MGP were found in glioblastomas, breast, cervical and skin cancer where positive correlation with tumor progression and survival was observed [26,54–56]. Yoshimura K et al. proved the correlation between mRNA levels of MGP and a poor prognosis of breast cancer patients [24]. 4.1. Reagents and Antibodies PAC and TOP were obtained from Sigma (St. Louis, MO, USA). RPMI-1640 and MEM medium, fetal bovine serum, antibiotic–antimycotic solution, and L-glutamine were also purchased from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-MGP Ab was obtained from Proteintech (Manchester, UK). Donkey anti-goat horseradish peroxidase- (HRP) conjugated Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ﬂuorescent secondary antibody, Alexa Fluor®488 Donkey Anti-Rabbit IgG, was obtained from Jackson ImmunoResearch Laboratories (Cambridgeshire, UK). The mounting medium with DAPI was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 4.2. Cell Lines and Cell Culture A2780 human epithelial ovarian cancer cell line was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and drug-resistant derivatives of A2780 cells were developed by exposing them to cyclic drug treatment. Paclitaxel resistant subline (A2780PR1) and topotecan resistant sublines (A2780TR1 and A2780TR2) were developed by subjecting A2780 cells to incremental doses of paclitaxel or topotecan. The ﬁnal concentrations used for selecting the resistant cells were 300 ng/mL of PAC and 24 ng/mL of TOP. The increase in resistance according to parental drug sensitive cell lines were as follow: 146-fold for A2780PR1 vs. A2780; 59.6-fold for A2780TR1 vs. A2780; and 48.5-fold for A2780TR2 vs. A2780 as described previously [3]. All of the cell lines were maintained as monolayers in the MEM medium supplemented with 10% fetal bovine serum, 2 pM L-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL) and amphotericin B (25 µg/mL) at 37 ◦C in a 5% CO2 atmosphere. 3 Discussion 3. Discussion 3. Discussion The increased expression of MGP correlated with increased migration of tumor cells in vivo and increased spreading and metastases formation in mouse model of osteosarcoma [28]. Migratory activity of cells with elevated levels of MGP was also observed in vitro. Among the glioma cell lines used in the experiment, those with knocked down MGP gene presented decreased cell migration ability when compared to cells overexpressing MGP [26]. Additionally, the role of circulating levels of MGP was described for osteosarcoma patients who revealed a signiﬁcant increase of this protein at the time of diagnosis and then developed lung metastases [28]. The role of MGP produced by cancer cells remains still not clear and its main function seems to be limited mainly to tumor environment. Therefore, the role of MGP in tumor progression should be considered from the stromal point of view where MGP–ECM interactions could trigger changes and rearrangements of tumor microenvironment. 4.5. SDS-PAGE and Western Blot Analysis of MGP Protein samples (20 µg each) were resuspended in 4× loading buffer (Bio-Rad Laboratories, Hemel Hempstead, UK) and incubated at RT for 20 min. Next, they were loaded onto a four 20% mini-PROTEAN® TGX™precast gels using the SDS-PAGE technique and ﬁnally transferred onto nitrocellulose membrane. This step was followed by blocking in 5% milk in TBS/Tween (0.1 M Tris-HCl, 0.15 M NaCl, 0.1% Tween 20) and immunodetection using rabbit anti-MGP Ab at 1:1000 dilution, and the appropriate HRP-conjugated secondary Ab. Membrane was washed in TBST and develop during a chemiluminescence detection system (ECL, Femto Super Signal Reagent) and Hyperﬁlm ECL (GE Healthcare, Buckinghamshire, UK). To normalize protein loading in the lanes, the membranes were stripped and reblotted with rabbit anti-GADPH Ab, from Santa Cruz Biotechnology, at a 1:1000 dilution and goat anti-rabbit HRP-conjugated Ab. The relative density of MGP to that of GADPH was analyzed with ImageJ Java-based image processing program developed at the National Institutes of Health (University of Wisconsin, Madison, WI, USA). 4.4. Protein Isolation from Cell Culture and Media Whole cell lysates from drug-sensitive and drug-resistant ovarian cancer cells (1 × 106 cells/20 µL lysis buffer) were lysed using containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min on ice. The lysates were centrifuged at 8000× g for 10 min at 4 ◦C, and protein concentration was quantiﬁed using the Bradford protein assay system (Bio-Rad Laboratories, Hemel Hempstead, UK). The isolation of proteins from media was preceded by culturing cells in serum-free media for 72 h. After that, the media was centrifuged at 15,000 rpm for 30 min at RT. Then, the supernatants were transferred to Amicon Ultra-15 3K centrifuge ﬁlter devices and centrifuged according the manufacturer’s protocols (40 min, 4000× g, RT, swinging-bucket rotor). 4.3. Examination of Gene Expression Using QPCR Total RNA was extracted from A2780 and drug-resistant cell lines using the GeneMATRIX Universal RNA puriﬁcation kit (EURx Ltd. Gdansk, Poland) as described by the manufacturer’s 12 of 17 Int. J. Mol. Sci. 2018, 19, 2901 protocol. Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen by Thermo Fisher Scientiﬁc, Waltham, MA, USA) and a thermal cycler (Veriti 96-well Thermal Cycler) according the manufacturer’s instructions. Two micrograms of RNA were used for cDNA synthesis. Real-time PCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientiﬁc, Waltham, MA, USA) and the sequence-speciﬁc primers that are indicated in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GADPH), β-actin, hypoxanthine-guanine phosphoribosyltransferase 1 (HRPT1) and beta-2-microglobulin (β2M) served as internal control housekeeping genes to normalize the PCRs (geometric mean) for the gene expressions being analyzed. Reactions were performed in a total volume of 24 µL, including 12.5 µL of Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientiﬁc, Waltham, MA, USA), 1 µL of each primer (Oligo, Warsaw, Poland) (Table 1), 9.5 µL of water and 1 µL of the reverse-transcribed cDNA template. One RNA sample from each preparation was processed without the RT-reaction to provide a negative control in the subsequent PCR reaction. After ampliﬁcation, melting curves were used to determine the speciﬁcity of the gene products, which was conﬁrmed by running the PCR products on 3% agarose gel. Gene expressions were analyzed using the relative quantiﬁcation (RQ) method. The RQ method estimates the differences in gene expression against a calibrator (drug-sensitive line) (RQ of the calibrator = 1). The drug-sensitive A2780 cell line was used as the calibrator. The analysis was conducted using the following standard formula: RQ = 2−∆∆Ct (where ∆∆Ct = ∆Ct of the sample (drug-resistant line) −∆Ct of the calibrator (drug sensitive line)). The graphs were plotted using Sigma Plot. 4.9. Statistical Analysis Statistical analysis was performed using Microsoft Excel software. The statistical signiﬁcance of the differences was determined using the Student’s t-test, and p-values of 0.05 or less were considered statistically signiﬁcant. 4.7. Incubation of Cells with PAC and TOP in Time-Course experiment The drug-sensitive A2780 cells were seeded into 6-well plates at 0.5 × 106 in 1 mL of medium per well. Cells were treated with low concentrations of PAC (20 and 25 ng/mL) or TOP (10 and 20 ng/mL) for 24, 48 and 72 h. After each period of incubation, cells were harvested and the RNA isolation was conducted. 4.8. Immunohistochemistry Immunohistochemical staining was conducted for formalin-ﬁxed, parafﬁn embedded human ovarian carcinomas lesions. The analysis of MGP expression was performed using the polymer-based immunohistochemical (IHC) technique [57] using an EnVisionTM HRP-polymer anti-mouse/rabbit IHC Kit (Dako REAL EnVisionTM Detection System peroxidase/DAB+, Rabbit/Mouse, Dako, Glostrup, Denmark) according to the manufacturer’s guidelines. Brieﬂy, prior to immunhistochemical staining, the slides were dewaxed and hydrated. The blocking of the activity of endogenous peroxidise with 1% H2O2 for 30 min was followed by incubation with the primary antibody at 4 ◦C/overnight (rabbit polyclonal anti-MGP antibody, 1:100 Proteintech, Manchester, UK). Then, the incubation with EnVision Detection System for 30 min at room temperature was followed by hematoxylin counterstaining. Finally, the sections were dehydrated, mounted and examined under the optical Olympus BH-2 microscope coupled to a digital camera. Color microscope images were recorded using LUCIA Image 5.0 computer software (Nikon, Tokyo, Japan). The expression of MGP was established by mean proportion of immunopositive cancer cells as well as surrounding stroma, for 10 microscope ﬁelds at magniﬁcation of 400×. For each specimen, the immunoreactivity score (IRS) was derived by multiplying intensity score by distribution score. Intensity for cancer cell staining was scored as negative (0), mild (1+), moderate (2+), or strong (3+). The distribution of staining of cancer cells was scored as 0 (no positive cells), 1+ (<10% of cells staining), 2+ (10–50% of cells staining), 3+ (51–80% of cells staining) or 4+ (>80% of positive cells). IRS score was interpreted as negative (IRS 0-1), mild (IRS 2-3), moderate (IRS 4-8) or strongly positive (IRS 9-12). Staining intensity was also determined in stromal tissues adjacent to cancer cells. Intensity of stroma staining was scored as negative (0), mild (1+), moderate (2+), or strong (3+). 4.6. Immunoﬂuorescence Analysis The cells were cultured on microscopic glass slides and grown to a near-conﬂuent state. Immunoﬂuorescence analysis was conducted following the previously established protocol [15]. Brieﬂy, the cells were ﬁxed with 4% PFA, permeabilized in ice-cold acetone/methanol (1:1) and then washed Int. J. Mol. Sci. 2018, 19, 2901 13 of 17 with PBS. After blocking with 3% BSA the cells were incubated with the anti-MGP primary antibody (rabbit polyclonal anti-MGP antibody, 1:100 Proteintech, Manchester, UK) for 2 h at room temperature. Next, the cells were washed with PBS and incubated with the secondary antibody for 1 h at room temperature (Alexa Fluor®488 Donkey Anti-Rabbit IgG, Jackson ImmunoResearch Laboratories, Cambridgeshire, UK). Afterwards, the cells were washed with PBS and sealed with DAPI-containing mounting medium. The cells were imaged for MGP expression analysis using ﬂuorescence microscope (Zeiss Axio-Imager.Z1, Oberkochen, Germany). The pseudo-color representation of ﬂuorescence intensity was assessed for DAPI at 365 nm excitation and 420 nm emission wavelengths (blue) and for Alexa Fluor®488 at 470 nm excitation and 525 nm emission wavelengths (green). Abbreviations Abbreviations TOP Topotecan PAC Paclitaxel MGP Matrix Gla Protein ECM Extracellular Matrix CAM Cell Adhesion Molecule TR Topotecan Resistance PR Paclitaxel Resistance References 1. Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Snyder, N.; Sarkar, S. Drug resistance in cancer: an overview. Cancers (Basel) 2014, 6, 1769–1792. [CrossRef] [PubMed] l 1. Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Snyder, N.; Sarkar, S. Drug resistance in cancer: an overview. Cancers (Basel) 2014, 6, 1769–1792. [CrossRef] [PubMed] 2. Leonard, G.D.; Fojo, T.; Bates, S.E. The role of ABC transporters in clinical practice. Oncologist 2003, 8, 411–424. [C R f] [P bM d] 1. Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Snyder, N.; Sarkar, S. Drug resistance in cancer: an overview. Cancers (Basel) 2014, 6, 1769–1792. [CrossRef] [PubMed] 2. Leonard, G.D.; Fojo, T.; Bates, S.E. The role of ABC transporters in clinical practice. Oncologist 2003, 8, 411–424. [CrossRef] [PubMed] 3. Januchowski, R.; Sterzy´nska, K.; Zaorska, K.; Sosi´nska, P.; Klejewski, A.; Br ˛azert, M.; Nowicki, M.; Zabel, M. Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines. [CrossRef] [PubMed] 3. Januchowski, R.; Sterzy´nska, K.; Zaorska, K.; Sosi´nska, P.; Klejewski, A.; Br ˛azert, M.; Nowicki, M.; Zabel, M. Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines. J. Ovarian Res. 2016, 9, 65. [CrossRef] [PubMed] 3. Januchowski, R.; Sterzy´nska, K.; Zaorska, K.; Sosi´nska, P.; Klejewski, A.; Br ˛azert, M.; Nowicki, M.; Zabel, M. Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines. J. Ovarian Res. 2016, 9, 65. [CrossRef] [PubMed] 3. Januchowski, R.; Sterzy´nska, K.; Zaorska, K.; Sosi´nska, P.; Klejewski, A.; Br ˛azert, M.; Nowicki, M.; Zabel, M. Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines. J. Ovarian Res. 2016, 9, 65. [CrossRef] [PubMed] 4. Sung, S.Y.; Hsieh, C.L.; Wu, D.; Chung, L.W.; Johnstone, P.A. Tumor microenvironment promotes cancer progression, metastasis, and therapeutic resistance. Curr. Probl. Cancer 2007, 31, 36–100. [CrossRef] [PubMed] 4. Sung, S.Y.; Hsieh, C.L.; Wu, D.; Chung, L.W.; Johnstone, P.A. Tumor microenvironment promotes cancer progression, metastasis, and therapeutic resistance. Curr. Probl. Cancer 2007, 31, 36–100. [CrossRef] [PubMed] 5. Di Paolo, A.; Bocci, G. Drug distribution in tumors: mechanisms, role in drug resistance, and methods for 4. Sung, S.Y.; Hsieh, C.L.; Wu, D.; Chung, L.W.; Johnstone, P.A. Tumor microenvironment promotes progression, metastasis, and therapeutic resistance. Curr. Probl. Cancer 2007, 31, 36–100. [CrossRef] [P progression, metastasis, and therapeutic resistance. Curr. Probl. 5. Conclusions In summary, to our knowledge, this is the ﬁrst report about MGP expression in drug resistant cell lines and in ovarian cancer tissue at protein level. The research found elevated levels of MGP expression in PAC and TOP resistant ovarian cancer cell lines and corresponding media as well as in response to short time PAC and TOP treatment. For the ﬁrst time, we have described the coordinate expression of two MGP isoforms in ovarian and breast cancer cell lines. Additionally, as secreted Int. J. Mol. Sci. 2018, 19, 2901 14 of 17 outside the cell, the MGP protein could be implicated in cell adhesion-mediated drug resistance (CAM-DR). Therefore, we have demonstrated that MGP is an important factor that might contribute to cancer resistance mechanism by augmenting the interaction of cells with ECM components leading to increased resistance of ovarian cancer cells to paclitaxel and topotecan. The results of present research may improve our understanding of the role of MGP in ovarian cancer biology and help identify new therapeutic targets. Author Contributions: Conceptualization, methodology, investigation, and writing—original draft preparation, K.S.; methodology, investigation, and validation, A.K.; methodology and investigation, K.W., M.´S., M.A. and D.R.; resources and data curation, M.S., W.K. and J.B.; validation and supervision, M.N.; conceptualization, supervision, and writing—original draft preparation, review and editing, R.J. Funding: This research was funded by the National Science Centre, Kraków, Poland, grant number 2016/22/E/NZ5/00381. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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Mathematische Geographie zusammenhängend ent wickelt und mit geordneten Denkübungen versehen Nr. 92. von Kurt Geissler. 1 / Y.' J: f У чМ V• : ■ii h,!>3 V , '-'J. Г Sammlung Göschen Ebene und sphärische Trigonometrie von Dr. Gerhard Hessenberg in Charlottenburg-Berlin Mit 69 ein- und zweifarbigen Figuren ЖрК-взЬ^ 'Ь KSIĘGARNIA \ Leipzig G. J. Göschen’sche Verlagshandlung 04 1899 ЪоЛ ЬЪЬ KD ’bKUoZi) Alle Rechte, insbesondere das Uebersetzungsrecht, von der Verlagshandlung Vorbehalten. BIBLIOTEKA PCLITECIIIIIBZU / a. i '' î KäAKÖW i Y- / * \ ? 2 g В s k> Ш : ■ Äke. Mr. Druck und Einband von Carl Inhaltsverzeichnis. Einleitung, §§ 1—3 Seite 9 Erster Teil. Ebene Trigonometrie. оо 00 § to § ^ Ю ÎÛ b § м MM м os f-» < юел Erstes Kapitel: Das rechtwinklige Dreieck. Definition der trigonometrischen Funktionen Auflösung der rechtwinkligen Dreiecke . « Verlauf und Verallgemeinerung des Sinus und Cosinus Verlauf der Tangente . Algebraische Gleichungen zwischen den Funktionen des selben Winkels .... t £S£2S Zweites Kapitel: Das schiefwinklige Dreieck. § 9. Cosinusformeln und Cosinussatz . 10. Anwendung des Cosinussatzes .... 11. Sinussatz ...... 12. Rechnerischer Beweis des Sinussatzes . § 13. Anwendung des Sinussatzes .... § 14. Tangenten der halben Winkel .... Drittes Kapitel: Die Additionstheoreme der trigono metrischen Funktionen. § 15. § 16. § 17. § 18. § 19. § 20. Erste Form der Additionstheoreme Beweis der Additionstheoreme . Zweite Form der Additionstheoreme Bedeutung der Additionstheoreme Additionstheorem der Tangente Doppelte und halbe Winkel 48 50 56 59 60 61 Inhaltsverzeichnis. 6 Seite Viertes Kapitel : Anwendung der Additionstheoreme auf das schiefwinklige Dreieck. 63 65 § 21. Tangentensatz § 22. Weiteres Formelmaterial Fünftes Kapitel : Berechnung der Vierecke. § 23. § 24. § 25. § 26. § 27. § 28. Allgemeines ..... Berechnung durch Teildreiecke Die vollständigen Beziehungen zwischen den Winkeln Berechnung der Winkel aus vier gegebenen Das Sehnenviêreck .... Das Trapez .... Zweiter Teil. Sphärische Trigonometrie. Sechstes Kapitel : Einleitendes. Aufgabe der sphärischen Trigonometrie Nebendreiecke und Scheiteldreieck Inhalte von Zwei- und Dreiecken Das Polardreieck M to oft (M 00 00 00 05 § 29. § 30. § 31. § 32. Siebentes Kapitel: Das schiefwinklige sphärische Dreieck. § 33. § 34. § 35. § 36. Erster Cosinussatz . . . Funktionen der halben Winkel. Sinussatz Eeciproke Formeln ..... Eechnerische Herleitung des zweiten Cosinussatzes. Cotangentensatz .... § 37. Uebergang in die ebene Trigonometrie . § 38. Auflösung der sphärischen Dreiecke . 95 98 100 100 101 103 Achtes Kapitel : Das rechtwinklige sphärische Dreieck. § 39. Auflösung der rechtwinkligen sphärischen Dreiecke § 40. Die Napier’sche Eegel ..... § 41. Geometrischer Beweis der Fundamentalformeln des recht winkligen Dreiecks ..... § 42. Das Quadrantendreieck . . § 43. Das schiefwinklige Dreieck im Zusammenhang mit dem rechtwinkligen . . . . 107 111 114 116 116 Inhaltsverzeichnis. 7 Seite Neuntes Kapitel : Weiteres Formelmaterial für das sphärische Dreieck. § 44. Die Gauss’schen Gleichungen und die Napier’schen Ana logien . . . . . § 45. Formeln für s, sa, o', ffa, q, (>a, r, ra . . § 46. Die I/Huilier’schen Formeln § 47. Logarithmische Rechnung zum Auflösen der Dreiecke . 118 120 122 125 Dritter T eil. Berechnung und algebraische Anwendung der trigonometrischen Funktionen. Zehntes Kapitel : Elementare Berechnung der trigono metrischen Funktionen. §^48. Die regulären Polygone § 49. Funktionen sehr kleiner Winkel 126 128 Elftes Kapitel : Der Moivre’sche Satz. . Begriff des Vektors Addition von Vektoren . Multiplikation von Vektoren Zerlegung der komplexen Zahl in reellen und imaginären Teil ....... § 54. Summation der Sinus und Cosinus einer arithmetischen Reihe von Winkeln . . . . ' . § 55. Der Moivre’sche Satz. Funktionen der vielfachen eines Winkels ...... § 60. § 51. § 52. § 58. 181 133 135 137 138 140 Zwölftes Kapitel: Unendliche Reihen und Produkte zur Darstellung der trigonometrischen Funktionen. 143 § 56 . Dreizehntes Kapitel: Die Methode der Hilfswinkel. § 57. § 58. §59. § 60. Beispiele . . .... Trigonometrische Auflösung der Gleichung zweiten Grades Trigonometrische Auflösung der Gleichung dritten Grades Beispiele zu §§ 58 und 59. 145 149 150 152 8 Inhaltsverzeichnis. Seite Anhang: Uebungsbeispiele. Tafel I: Rechtwinklige ebene Dreiecke я II: Schiefwinklige ebene Dreiecke „ III : Rechtwinklige sphärische Dreiecke . я IV : Schiefwinklige sphärische Dreiecke . я V : Die Sinus und Cosinus der vielfachen von 3° etc. 20 Textaufgaben ..... 155 157 158 159 160 16 L Anmerkung des Verfassers : Der zweite Beweis der Additionstheoreme (§ 16, pag. 53 ff.) ist zwar vom Verfasser selbständig gefunden, aber nicht neu. Er ist ein anscheinend in Vergessenheit geratener Beweis der Baur’schen Schule. Einleitung. § i. Eine der wichtigsten Aufgaben der elementaren Geometrie ist die Konstruktion einer Figur aus einer hinreichenden Anzahl gegebener Stücke, speciell die des Dreiecks aus drei Stücken. An diese Aufgabe schliesst sich naturgemäss die Frage nach der Grösse der übrigen Stücke an. Dieselbe kann ohne weiteres beantwortet werden, wenn die Aufgabe der Konstruktion der Figur gelöst ist : Man zeichnet die Figur und misst mit Mass stab und Transporteur die gesuchten Stücke nach. Dieses Verfahren nennt man das „mechanische“, „konstruktive“ oder „graphische“. Früher vielfach unterschätzt, findet es in neuerer Zeit ausgedehnte An wendung in der Technik (Graphostatik). Es ist nicht auf ebene Figuren beschränkt : in der darstellenden Geometrie wird die Aufgabe behandelt, die einzelnen Stücke räumlicher Figuren durch ebene Konstruktionen zu ermitteln. Dagegen ist der Anwendbarkeit des graphischen Verfahrens durch folgende zwei Haupt punkte eine Grenze gesetzt: Erstens setzt es die konstruktive Lösung der Aufgabe voraus. Diese aber ist mit Zirkel und Lineal durchaus nicht immer möglich und, wenn Einleitung. 12 Endlich bezeichne г den Radius, M das Centrum des Umkreises, q den Radius, 0 das Centrum des Inkreises, J den Inhalt des Dreiecks. ,Л А О с С К м \ В V h Оа X Са Иа —'V Figur 2. Figur 1. Die Formeln für das ebene Dreieck sind dann: (I) c2 = a2 -f- ba + 2aba (Projektionssatz), (II) J = )/s.Sa.Sb.Sc, (III) J = -k all» = f blib = ^- eh«, (IV) J =pS = e& Sa = 9b Sb = Qc Sc, abc (V) .1 Tr-’ (VI) J = ]/ p . pa . pb . Qc , (VII) Q Qa + pb + (>c Ist beispielsweise a = 13 ; b = 14 ; c = 15, so wird s = 21 ; sa = 8 ; Sb = 7 ; sc = 6, also nach (II) J = 84 und nach (III) Einleitung. lo Sr 168 ha = — = 12,9280... ; hb = 12 ; hc = 13 = 11,2. Ferner ist nach (IV) я P = 4; pa = — 10,5 ; eb = 12; ec = 14 und nach (V) 65 8 -8,125. 003 Wie gross sind ba, Ca, ab u. s. w. ? (Ans (I) zu be rechnen.) Der Leser überzeuge sich, dass auch (VI) untf (VII) erfüllt sind, und konstruiere das Dreieck, um die Stücke nachzumessen. Die Winkel finden sich in Кар. II, § 14, berechnet. 3. Die Aufgabe der Trigonometrie kann man nun mehr folgendermassen definieren: Zunächst für das ebene Dreieck, dann aber auch für beliebige ebene und räumliche Figuren den Zu sammenhang zwischen den Strecken, Flächeninhalten und Winkeln zu ermitteln. Das wesentlich neue Moment liegt dabei in der Betrachtung der Winkel, da diejenigen Beziehungen, in denen keine Winkel Vorkommen, durch die elementare Geometrie gefunden werden können. Es werden jedoch auch solche Beziehungen mit trigonometrischen Hilfs mitteln oft einfacher und kürzer herzuleiten sein. Die Gleichungen zwischen den Strecken und Flächen inhalten sind algebraisch, d. h. sie können auf eine Form gebracht werden, in der nur Additionen, Subtraktionen und Multiplikationen, und zwar in endlicher Anzahl, Vor kommen. So kann für (II) geschrieben werden J . J = s . sa . Sb . sc oder 16.J.J = (a + b + c) (—a + b + c) (a — b + c) (a + b —c). 14 Einleitung. Man sagt daher, dass der Inhalt und die geradlinigen Stücke des Dreiecks algebraische Funktionen der Seiten sind. Das Entgegengesetzte gilt von den Winkeln. Sie sind zwar auch durch die drei Seiten bestimmt, d. h. Funktionen der drei Seiten; aber der Zusammenhang zwischen Seiten und Winkeln lässt sich nicht durch eine endliche Anzahl von Additionen, Subtraktionen und Multiplikationen defi nieren; die Winkel sind also „nicht-algebraische“, oder, wie man dafür sagt, „transcendent e“ Funktionen der Seiten. Erster Teil. Ebene Trigonometrie. Erstes Kapitel. Das rechtwinklige Dreieck. § 4. Definition der trigonometrischen Funktionen. Wenn in einem bei C rechtwinkligen Dreieck ABC ein spitzer Winkel, etwa a, bekannt ist, so kennt man auch den zweiten Winkel, ß = 90° — «. Man kennt В В' а __ г V A&- ь Figur 3. also das Dreieck seiner Gestalt nach. Ist in irgend einem anderen rechtwinkligen Dreieck A'B'C' der Winkel а* gleich a, so sind die Dreiecke ABC und A'B'C' ähn lich und folglich auch a : b : c = a' : b' : c'. Die Уerhältnisse der Seiten eines recht winkligen Dreiecks sind also durch einen Ebene Trigonometrie. 16 der spitz en Winkel völlig bestimmt, sie sind, wie der Mathematiker dafür sagt, „Funktionen“ desselben. D O Ist z. В. a = 30°, so ist, a = c, also nach dem Pythaи уз goras b = — c, mithin hat man I i-H C M а c 0,5; = 0,8660 ; = y<T= 1,7321. Ist « = 45°, so ist c —• а ]/ 2, also а =0,7071 ;-L = ^ =0,707!; ■; = 1. с Man nennt den Quotienten den Sinus des л P Winkels a: Sinus = Gegenkathete durch Hypotenuse sin а — — (spr. Sinus alpha). b ist die Gegenkathete von ß, d. h. des Comple ments von a ; ^ ist daher der Sinus des Complements von a; kürzer nennt man diesen Quotienten den Co sinus von a (entstanden durch die Abkürzung со. sin. für complementi sinns). Cosinus = Gegenkathete des Complements durch Hypotenuse. b cos a = c (spr. Cosinus alpha). Nach dieser Definition ist cos a — sin (90° — a), sin a = COS (90° — a). er p Кар. I. Das rechtwinklige Dreieck. Den Quotienten - - а: с b:c 17 sin a nennt man die cos« Tangente des Winkels a. Tangente = Sinus durch Cosinus — Gegenkathete durch andere Kathete. sin а а *а-ц COS а (spr. Tangens alpha.) ist hiernach die Tangente von ß und wird als solche die Cotangente von a genannt. Die Bezeichnung entstand aus co. tang. = complementi tangens. er Cotangente = Gegenkathete des Complements durch andere Kathete. cotg a =----- = tg (90° - a) COS a » sin a (spr. Cotangens alpha). Nach dieser Definition sind Tangente und Cotangente reciproke Werte : a 1 b cotg a = weil cotg a Aga = tg«’ a '-b-=h ! Im ganzen giebt es 6 Seitenverhältnisse: -jp » er Aus diesem Grunde wird cotg a nur selten gebraucht, da es sich so einfach durch tga ersetzen lässt. у b . —Л • Bie Namen der 4 ersten sind soeben angec geben worden. Von den beiden letzten nennt man ? TT die Secante und dementsprechend — a die Cosecante von a und scnreibt dafür sec a und cosec a. Offenbar ist cosec a Hessenberg, Ebene und sphärische Trigonometrie. 2 Ebene Trigonometrie. 18 1 -------. Daher werden die Funktionen sec sin a sec a = cos a und cosec im allgemeinen nicht benutzt. Dagegen präge sich der Leser die Definition der Funk tionen Sinus, Cosinus, Tangens und Cotangens aufs sorg fältigste ein. Hierbei sind folgende Merkmale dienlich : Im Zähler steht stets die Gegenkathete, und zwar die des Winkels selbst bei den Funktionen ohne Co, dagegen die des Complements bei den Funktionen mit Co. Im Nenner steht die Hypotenuse bei Sinus und Cosinus. Sinus, Cosinus, Tangens und Cotangens heissen die „trigonometrischen Funktionen“. Sie. sind absolute unbenannte Zahlen; ihr Wert ist unabhängig davon, ob die Seiten des Dreiecks nach Centimetern, Zoll oder sonst irgend einem Masse gemessen sind. Man kann ihre Werte für jeden Winkel mit beliebiger Genauigkeit berechnen, und zwar mit elementaren Hilfs mitteln, aber auf Grund von Eigenschaften, die erst später zu entwickeln sein werden. Wesentlich einfachere Methoden liefert jedoch die höhere Mathematik. Näheres hierüber findet sich im dritten Teil dieses Buches. Man hat, wie für die Logarithmen der Basis 10, auch für die trigonometrischen Funktionen Tabellen auf gestellt. Diese finden sich aber, da sie wenig gebraucht werden, gar nicht oder nur in beschränktem Umfange den logarithmischen Tabellen werken beigegeben. Da gegen enthalten diese Werke ausführliche Tabellen der Logarithmen der trigonometrischen Funktionen. Die sichere Handhabung dieser Tabellen ist für das Weitere unumgänglich notwendig, und zwar übe sich der Leser zunächst an der für die meisten Zwecke ausreichenden fünfstelligen Tabelle. Кар. I. Das rechtwinklige Dreieck. 19 § 5. Auflösung der rechtwinkligen Dreiecke. Wenn man bedenkt, dass die zu einem Winkel а gehörigen Zahlen sin et, cos et, tgct, cotga nichts anderes • sind, als die Seitenverhältnisse eines beliebigen recht winkligen Dreiecks, dessen einer spitzer Winkel gleich et ist, so erkennt man, dass mit den trigonometrischen Tabellen vollständig das Material zur Auflösung der rechtwinkligen Dreiecke gegeben ist. In einem solchen kann gegeben sein: 1) ct und а 3) a und c 2) ce und b 4) a und b 5) a und c. R œt Ó qL S a о" & J IZ Il ^ C fQ ZI. ce II sin ct log c = log a — log sin cc -BP а = sin ct, also с й -О о O P Im ersten Falle hat man: Im zweiten Falle ist: b cos cf ce LQ b — = COS Cf J c = tg а, а = b tg et log a = log b + log tg a. Im dritten Falle ist: a . . — = sin a, a = c . sm a c log a = log c + log sin a - - = cos «, Ъ = с cos а О log c = log b — log cos a log Ъ — log с + log cos а. C T " P Im vierten Falle ist: = tgct log a — log b == log tg a oder auch c a sin ct log c = log a—: log sin et c— 20 Ebene Trigonometrie. Im fünften Falle ist: a b — a cotg cc — = sin c log a — log c — log sin a log b = log a -f- log cotg a oder b '== У c2 — a. Die Berechnung des zweiten Winkels ist hierbei nicht berücksichtigt. Es ist ja ß = 90°— «. Die An wendung der Formeln c = )/a2+ b2, b = ]/ ca — а2 ist nur zu empfehlen, wenn a, b resp. c, a ganzzahlig sind, oder wenn man eine ausführliche Quadratzahlentabelle zur Verfügung hat. Erstes Beispiel, а — 3, c 1 5. b = ]/2ö — 9 = 4. log а = 0,47712 log c = 0,69897 log sin а = 0,77815 — 1 = 9,77815 — 10. а = 36° 52' 11" ß = 90° — а = 53° 7' 49". Der Leser rechne für dieses Dreieck auch die andern 4 Fälle durch. Die Bestimmung der Sekunden ist nicht ganz genau. Man findet denselben log sin für alle Winkel von 36° 52' 9" bis 36° 52' 12". Mit 7stelligen Logarithmen findet man. dass der letzte Wert der richtige ist. Zweites Beispiel, a = 12, а = 67° 22' 49" log с = log a — log sin a log b = log a -f- log cot a log a = 1,07918 log a = 1,07918 — log sin a — — 9,96524 -f 10 log cota = 9,61971 — 10 log c = 1,11394 c = 13,000 Probe: 122 4- 58= 139. log b = 0,69897 b = 5,0000 Кар. I. Das rechtwinklige Dreieck. 21 Drittes Beispiel. c = 21,746; a = 38° 2Г 47" log а = log c -f- log sin а log b === log c -f- log cos а log c = 1,33738 log sin a = 9,79285 -f-10 loge =1,33738 log cos a = 9,89437 — 10 log b = 1,23175 log a = 1,13023 a = 13,497 b= 17,051 Probe mit Hilfe der Quadratzahlentabelle : a2 = 182,17, b2 = 290,73, c2 = 472,89. Das zweite und dritte Beispiel sind der Tafel I am Schlüsse des Buches entnommen. § 6. Verlauf und Verallgemeinerung von Sinus und Cosinus. 1. Um von dem Verlauf des Sinus und Cosinus ein anschauliches, geometrisches Bild zu erhalten, denke man sich die Hypotenuse des bei C rechtwinkligen Drei ecks ABC als Längeneinheit gewählt. Dann ist sin a = a, cos a = b, weil c = 1 wird. Denkt man sich nun Q B2j By BA A C2 c c; p Figur 4. den Winkel a veränderlich, den Schenkel АС aber fest gehalten, so beschreibt der Punkt В einen Viertelskreis PBQ. Das Lot von В auf den Radius AP, der durch Ebene Trigonometrie. 22 C geht, ist der Sinus, die Entfernung seines Fusspunktes von A der Cosinus, vorausgesetzt, dass der Radius des Kreises die Längeneinheit ist. Man sieht aus der Figui unmittelbar, wie der Sinus von 0 bis 1 wächst, wäh rend der Cosinus von 1 bis 0 abnimmt. 2. Es hindert uns aber nichts, den Schenkel AB über die Lage AQ hinauszudrehen, wenn wir nur AP über A hinaus rückwärts verlängern. Dann wandert C auf dem zu AP entgegengesetzten Radius A R weiter, das Lot В C nimmt von 1 bis 0 wieder ab, und AC wächst wieder von 0 bis 1, ist aber jetzt entgegengesetzt gerichtet wie vorher. B' B\ X \ R А p Figur 5. Man bezeichnet nun auch weiter BC, ge messen durch AB, als den Sinus von PAB und hat damit die Definition des Sinus auf stumpfe Winkel erweitert. Ebenso nennt man auch weiter AC, gemessen durch AB, den Cosinus von a, erteilt ihm aber, da AC entgegengesetzt ge richtet istу wie die Cosinus des ersten Qua dranten, das negative Vorzeichen. Кар. I. Das rechtwinklige Dreieck. 23 Die so definierten Sinus und Cosinus stumpfer IVinkel lassen sich leicht durch die ihrer spitzen Neben winkel ausdrücken. Ist nämlich < B'AP = <§C BAB, = 180° — a, so ist В' C' gleich und gleichgerichtet, wie В C, d. h. es ist sin а = sin (180° — a). (I) Dagegen ist AC gleich und entgegengesetzt gerichtet, wie А' C, d. h. es ist COS а = — cos (180 ° — a) (П) Nebenwinkel haben gleiche Sinus und entgegen gesetzt gleiche Cosinus, 3. Man kann AB noch weiter drehen, durch den dritten und vierten Quadranten, bis nach AP. Dabei wandert C von R nach P zurück und wenn man А C, gemessen durch AB, wieder mit dem negativen Vorzeichen, wenn C auf AR, mit dem positiven, wenn C auf AP liegt, auch weiterhin als Cosinus von « bezeichnet, so durchläuft der Cosinus die Werte von —1 bis -}-l wieder zurück. Ebenso bezeichnet man BC, gemessen durch AB, auch weiterhin als den Sinus, erteilt ihm aber das negative Vor zeichen, da В C entgegengesetzt gerichtet ist, wie im ersten und zweiten Quadranten. B’ B' С А В 1В Figur 6. Figur 7. 24 Ebene Trigonometrie., Die Sinns und Cosinus der Winkel des dritten und vierten Quadranten lassen sich wieder in einfacher Weise durch die des ersten und zweiten ausdrücken. Verlängert man nämlich B A über A hinaus bis zum zweiten Schnitt punkt B' mit dem Kreis, so ist <£: P А В' = а — 180°, В' С' ist gleich und entgegengesetzt gerichtet, wie В C : sin а = — sin (a — 180°). (ui) C'A ist gleich und entgegengesetzt gerichtet, wie CA: cos а =- — cos (a — 180°). (IV) Winkel, die sich um 180° unterscheiden, lmben ent gegengesetzt gleiche Sinus und Cosinus. 4. Hierdurch erhält man folgendes Bild von dem Ver lauf des Sinus und Cosinus in den 4 Quadranten : а sin а cos а =0 =0 = 1 geht von 0 bis R geht von 0 bis 1 geht von 1 bis 0 == R =1 =0 geht von R bis 2 R geht von 1 bis 0 geht von 0 bis —1 =2R =0 — —1 geht von 2 R bis 3 R geht von 0 bis —1 geht von —1 bis 0 =3R =0 geht von3Rbis4R geht von —1 bis 0 4R 0 geht von 0 bis 1 1 Lässt man den Punkt В von irgend einer Lage aus einen vollen Umlauf auf dem Kreise machen, so vermehrt sich zwar der Winkel a um 360°, aber В und mit ihm BC und AC kehren in ihre vorige Lage zurück. Man sagt da her, Sinus und Cosinus seien „um 360° periodisch46: Кар. I. Das rechtwinklige Dreieck. sin (a -J- 360°) = sin а 25 (V) (VI) COS (a -f- 360°) = COS а Q 90 R (iso sin + sin + cos - cos + о 0 )P sin - sut - cos- cos + 2 70 s Figur 8. эо° °\ 180 \o° 270 ° Figur 8 a. 5. Bisher war angenommen, dass sich В auf dem Kreise im entgegengesetzten Sinne wie der Uhrzeiger drehe. Dreht man А В nach der anderen Seite, bis es mit А P zusammen 26 Ebene Trigonometrie. fällt, so wird a null und der bei weiterer Drehung ent stehende Winkel ist konsequenterweise als ein negativer Winkel zu bezeichnen. Die Funktionen negativer Winkel definiert man durch die zuletzt aufgestellte Gleichung, in dem man zu dem Winkel so oft 360° hinzufügt, bis man X Ж УВ Figur 9. einen positiven Winkel erhält. Auch überzeugt man sich an der Figur leicht, dass sin (— a) = — Sin a, COS (— a) = COS a ist. (VII) § 7. Verlauf der Tangente. Die Tangente definiert man für beliebige Winkel am besten immer als Quotienten von Sinus und Cosinus: sin« tgo = cos а Durch Division der Formeln sin а = sin (180° — a), cos а = — cos (180° — а) erhält man tga = —tg (180° — «) (VIII) Nebenwinkel haben entgegengesetzt gleiche Tangenten. Durch Division der Gleichungen sin a — — sin (а — 180°), cos a = — cos (a — 180°) erhält man tg а = tg (а — 180°). (IX) Кар. I. Das rechtwinklige Dreieck. 27 Die Tangenten der Winkel im dritten und vierten Qua dranten stimmen wieder mit denen der Winkel im ersten und zweiten überein, die Tangente ist also bereits um 180° periodisch. Für negative Winkel erhält man durch Division von sin (— a) = — sin a, cos (— a) = cos a (X) tg (— a)= — tg a. Um von dem Verlauf der Tangente ein geometrisches Bild zu erhalten, errichte man in Fig. 10 in P das Lot auf AP und verlängere AC bis zum Schnitt T mit A r, T By А Ci С Figur 10. demselben. Dann ist TP: AP = tga, und da AP die Längeneinheit ist, ist TP das Mass der Tangente. Uebrigens stammt die Bezeichnung „Tangente“ davon her, dass TP in P den Kreis berührt. Die Tangente wächst von 0 bis 90° stetig und kann jeden Wert annehmen. Für 90° ist sie nicht definierbar, da sich dann AC und das Lot in P nicht schneiden. Auch wird cos 90° == 0, so dass der Quotient Ebene Trigonometrie. 28 sin 90° keinen Sinn hat. Man schreibt aber cos 90° tg 90° = oo (gesprochen : unendlich), um damit anzudeuten, dass tga bei der Annäherung von a an 90° jeden beliebig grossen Wert annehmen kann. § 8. Algebraische Gleichungen zwischen den Funktionen desselben Winkels. Nach dem Satz des Pythagoras ist für jede Lage des Punktes B: (AC)2 + (B C)2 — (AB)2 oder, da AB = 1, AC = + cosa, BC = + sina ist: (XI) sin2 a -)- COS2a = 1. В с А а л Ь Figur 11. Dies ist eine der wichtigsten Fundamentalformeln der Trigonometrie. Mit der andern Gleichung sin cg tgcg = cos« zusammen gestattet sie, jede der drei Funktionen sin, tg, cos durch eine andere auszudrücken. Eliminiert man irgend eine, etwa sin, aus beiden Gleichungen, so erhält man eine Beziehung zwischen den beiden andern, etwa cos und tg. Der Leser übe dies zunächst an einigen I __ Zahlenbeispielen, z. B. tg«= —, уз, 3 u. a. m., und А leite sodann folgende 6 Formeln ab: Кар. I. Das rechtwinklige Dreieck. sina = sin a У1 —cos2 a cos a = У1 — sin2 a cos a tg a = sin a У1 — sin2 a У1 — cos2 a cos a 29 tg« V1 ~ł~ tg2 a 1 ]/l + tg“a tget Zweites Kapitel. Das schiefwinklige Dreieck. § 9. Cosinusformeln und Cosinussatz. In jedem schiefwinkligen Dreieck gelten die drei Cosinusformeln : a = b cos у + c cos ß b = c cos а + a cos у с = a cos ß + h cos a (I) Ъ a к ß А& kВ Figur 12. Beweis. 1. a und ß seien spitz. Dann ist in dem rechtwinkligen Dreieck ACHc AEC = AC cos a = b cos а und in dem rechtwinkligen Dreieck BCHc BHC = BC cos/? = a cos/?. Ebene Trigonometrie. 30 Durch Addition folgt AHc + BHc = AB=:C = acos/?+b cos a, w. z. b. w. 2. Einer der Winkel a und /?, etwa a, sei stumpf. Dann ist wie vorher В Hc = В C cos ß == a cos ß. Dagegen A Hc == А C cos a4 ^ b cos a', wo a4 —180° — a der Aussenwinkel an der Ecke A ist. Ar Дс а V ct\cl А Ä 'В- Figur 13. Durch Subtraktion folgt B ïï0 — AHC = AB = c = a cos ß — b cos a4 und nach Formel (II) des ersten Kapitels ist — cos a' = cos a, also c = a cos ß -f- b cos ce, w. z. b. w. Man kann die drei Cosinusformeln als drei Glei chungen ersten Grades mit den drei Unbekannten cos ce, cos/?, cos/ auffassen und nach diesen Unbekannten auflösen. Dann erhält man : — a2 + b2 + c2 cos a = 2bc + a2 —b2 + c2 cos/? = 2ac a2 + b2 — c2 cos / = (П) 2ab Кар. IL Das schiefwinklige Dreieck. 31 Man schreibt nach WegschaffuDg der Brüche diese Formeln meist in der Gestalt: a2 — b2 + c2 — 2 b c cos a, b2 = c2-j-a2 — 2ca cos/?, c2 — a2 + b2 — 2 a b cos/. (III) Diese Formeln enthalten den sogenannten Cosinus satz. Weiss man eine der Formeln (III) auswendig, so leitet man die anderen durch Vertauschung der Seiten und Winkel aus ihr ab. Will man den Cosinussatz unmittelbar aus den Cosinusformeln erhalten, so multi pliziere man die erste mit a, die zweite mit b, die dritte mit c, addiere zwei von ihnen und subtrahiere die dritte davon. Umgekehrt erhält man durch Addition zweier der Gleichungen (III) die Cosinusformeln. Addiert man z. B. die beiden letzten, so hebt sich b2 -{- c2 auf beiden Seiten und es bleibt n0 = 02 а 2 — о2 ca cos ßл — 02 abu cosy. Dividiert man durch 2 a und bringt die negativen Glieder auf die linke Seite, so erhält man die erste Cosinusformel. Die Gleichungen (III) erhält man auch unmittelbar aus dem Projektionssatz oder verallgemeinerten Pythagoras [For- , mel (I) der Einleitung]. Ist nämlich у spitz, so ist c2 = a2 + b2 — 2aba und ba = b cos y. Ist aber у stumpf, so ist c2 “ a2-j- b2-f-2aba und ba = bcos(180°—y) = — bcos/. § 10. Anwendung des Cosinussatzes. Der Cosinussatz gestattet die Berech nung der fehlenden Stücke des Dreiecks, wenn drei Seiten oder zwei Seiten und ein Winkel gegeben sind. 32 Ebene Trigonometrie. Erster Fall: Gegeben die 3 Seiten a, b, c. Man erhält die Winkel aus (II). Erstes Beispiel. a = 13, b = 4, c = 15. 1. Berechnung von a. — a8 + b8 + c* = - 169 + 16 + 225 = 72, 3 72 0,6, cos a = 2.4.15 5 log cos a = 9,77815 — 10, a = 53° 7' 49". 2. Berechnung von ß. а8 — b8 + c2 = 169 — 16 + 225 = 378, 63 378 cos/? 2.13.15 65 * log cos ß = log 63 — log 65, log 63 = 1,79934 log 65 = 1,81291 log cos ß = 9,98643 — 10, ß = 14° 15' 0". 3. Berechnung von y. a2 + b8 —c8= —40, 5 40 cosy 13' 2.13.4 у ist also ein stumpfer Winkel. Da stumpfe Winkel nicht in der Tabelle stehen und negative Zahlen keine (reellen) Logarithmen haben, bestimme man statt у den Winkel y'=180°— y, dessen Cosinus nach Formel (II) des 5 Кар. I gleich -f- -jg- ist. Man hat log 5 = 0,69897 log 13 = 1,11394 log cos y' — 0,58503 — 10. y' = 67° 22' 48" у = 180° — у' = 112° 37' 12". Кар. IL Das schiefwinklige Dreieck. 33 Probe : « + /? + 7 = 180° 0' 1". Der Fehler von 1" ist sehr gering. Wenn a, b, c nicht ganzzahlig gegeben sind, wird die Rechnung wohl umständlicher, bleibt aber im Prinzip die gleiche, wie bei dem ersten Beispiel. Zweites Beispiel. a = 13,509, b = 16,470, c = 21,746. Logarithmisch oder mit einer Quadratzahlentabelle be rechne man zunächst a2, ba, c2. Die Quadratzahlentabelle ergiebt a2 = 182,49, ba = 271,26, c2 = 472,89. 1. Berechnung von a. — a2 + b2 + c2 = 561,66 log cos a — log 561,66 — log 2 — log b — log c ausgerechnet = 9,89437 — 10 a = 38° 21' 48". 2. Berechnung von ß. a2 — b2+c2 = 384,12 log cos ß = log 384,12 — log 2 — log а — log c ausgerechnet = 9,81543 — 10 ß = 49° 10' 24". 3. Berechnung von 7. a2 + b2 —c2 = — 19,14 7 ist stumpf. 7' = 180° — 7. cos 7' = -f 19,14 2 ab log cos 7' = log 19,14 — log 2 — log а — log b ausgerechnet = 8,63360 — 10 7' = 87° 32' 5" 7 = 180° — 7' = 92° 27' 55". Probe : « + ß + 7 = 180° 0' 7", hinreichend genau. Zweiter Fall: Gegeben zwei Seiten und der ein geschlossene Winkel: a, b, 7. Hesseuberg, Ebene und sphärische Trigonometrie. 3 34 Ebene Trigonometrie. Man findet c aus der dritten Formel (III) dieses Kapitels, sodann a und ß wie im ersten Fall oder nach dem Sinussatz, § 13. Drittes Beispiel. y spitz, a = 13, b = 14, y = 67° 22' 48". log 2 ab cos y = log 2 + log a + log b -f- log cos y ausgerechnet = 2,14613 2 ab cos y — 140,00 c8 = a2 -f- b2 -- 2 ab cos y = 169 + 196 — 140 = 225 c = 15. Für a und ß erhält man nach den Formeln (II) : a = 53° 7' 49", ß = 59° 29' 23". Probe : « + ß + y = 180° 0' 0". Viertes Beispiel. y stumpf, а = 13,509, b = 16,470, у = 92° 27' 55". у* = 180° — y = 87° 32' 5", cos y1 = — cos y c2 — аа bа —2 ab cos y1 log 2 ab cos y' = log 2 + log a -f- log b + log cos y' ausgerechnet = 1,28194 2 ab cos yl = 19,14 [a2, b2 und c sind aus a, b und a2 = 182,49 ca mit der Quadratzahlentabelle b2 = 271,26 ermittelt.] c2= 472,89 c = 21,746. Dritter Fall: Gregeben zwei Seiten und ein nicht eingeschlossener Winkel: a, b, a. Dieser Fall ist einfacher nach dem Sinussatz (siehe § 13) zu lösen, lässt sich aber auch nach dem Cosinus satz behandeln. Man hat für c die quadratische Gleichung a2 == b2 -f- c2 — 2bccosa oder c2 — 2 b c cos a = a 2 — h . Кар. И. Das schiefwinklige Dreieck. 35 Ihre Wurzeln sind c = b cos а + \|/ а — b2 + b2 cos2 a = b cos a + У a2 — b* sin2a (IY) 1st a > b, so ist nur das obere Vorzeichen brauch bar; das untere würde einen negativen Wert ergeben. Ist a < b, so sind beide Vorzeichen brauchbar, es muss aber b2 sin2 a kleiner als a2 sein, weil sonst der Aus druck unter dem Wurzelzeichen negativ wird. Fünftes Beispiel. a = 13, b=4, a = 53° 7'49". log b cos a = log b -f- log cos a log b sin a — log b + log sin a log b = 0,60206 log cos a = 9,77815 —10 log sin a = 9,90309 — 10 log b cos a = 0,38021, . . . b cos a — 2,4000 log b sin a = 0,50515 2 log b sin a = 1,01030 ... b2 sin2 a = 10,240 a2_b2 sin2 a = 158,70 j/a2 — b* sin2 a = 12,600 c = 2,4+12,6 = 15 oder — 10,2. Die negative Wurzel ist nicht brauchbar. Aus a = 13, b = 4, c = 15 berechnet man die fehlenden Winkel nach Fall 1. Sechstes Beispiel. c = 21,746, a = 13,509, « = 38° 21' 48". Man hat für b die quadratische Gleichung a3=b2-\|-c2 — 2bc cos«, deren Wurzeln b = c cos a + j/a2— c2sin2a sind. Es wird wie im fünften Beispiel: c cos a = 17,051, c2 sin2 a = 182,16 a2 — c2 sin2 a = 182,49 — 182,16 = 0,33 У а2 — с2 sin2 а = 0,575 Ъ = 17,051 + 0,575 = 17,627 oder 16,476. 36 Ebene Trigonometrie. Beide Werte sind brauchbar; aber die letzte Stelle ist um 6 Einheiten falsch, wie genauere Berechnung zeigt. Der Fehler entsteht dadurch, dass a2 — c2sin2a nur auf zwei Stellen genau gefunden wird. Er kann noch bedeutend grösser werden. Weitere üebungsbeispiele enthält Tafel II am Schluss des Buches. § 11. Sinussatz. Die Seiten eines Dreiecks verhalten sich, wie die Sinus der gegenüberliegenden Winkel: a : b : c = sin a : sin ß : sin y. 00 Beweis. In dem rechtwinkligen Dreieck ACHc liegt bei A der Winkel a, wenn er spitz ist, dagegen der Winkel (180° —a) = a', wenn a stumpf ist. Nach Formel (I) des ersten Kapitels haben a' und a denselben Sinus CHc hc sin a = AC b b a K ß A&- ‘B Figur 14. Es ist also hc = b sin a und ebenso im Dreieck В С Hc hc — a sin/? folglich : a sin ß = b sin « a : b = sin а : sin/? w. z. b. w. (VI) Кар. II. Das schiefwinklige Dreieck. 37 C hc % a V d\ći L А A 'В Figur 15. Erweiterter Sinussatz : а Ъ с = 21(VU) sin a sin ß sin 7 (VIII) oder a = 2 r sin a ; b = 2 r sin ; c = 2r sin y. Beweis. Man ziehe durch В den Durchmesser des Umkreises. A' sei sein zweiter Endpunkt. Das А A' / ■ ri' м в а Figur 16 а. Dreieck А' В С hat nach dem Satz des Thaies bei C einen rechten Winkel. Also ist: BC а sin°/=BÄ‘=27 Ebene Trigonometrie. 38 Ist aber a spitz, so steht es mit a' über demselben Bogen, ist also nach dem Satz vom Peripheriewinkel gleich a'. Ist dagegen a stumpf, so ergänzen sich die A A С В а / âL А' Figur 16 b. Bogen, über denen a und а' stehen, zu 360°, die Winkel a und ce' also zu 180°. Es ist daher nach Formel (I) des ersten Kapitels sin a' gleich sin «, mithin unter allen а Umständen sin а = 2г Zweite Form des erweiterten Sinussatzes: sin а sin ß sin у 2J (IX) a c abc b oder J = -g- bc sin a = -g- ca sin ß = ab sin y (X) (Inhaltsformel). Erster Beweis. Nach Formel (VII) dieses Kapitels sina 1 ist----- = Drückt man r nach Formel (V) der а 2r ’ sina Einleitung durch J und die Seiten aus , so wird------а 2J abc* Кар. II. Das schiefwinklige Dreieck. 39 Zweiter Beweis. Nach Formel (VI) dieses Kapitels ist hc = b sin a, also J = 2 c hc = — b c sin а, ж. z. b. w. § 12. Rechnerischer Beweis des Sinussatzes. Bei den soeben angegebenen geometrischen Beweisen des Sinussatzes mussten die Fälle stumpfer und spitzer Winkel unterschieden werden. Dies ist nicht notwendig, wenn man mit Hilfe der Formel (XI) des ersten Kapitels : sin12 a + cos2 a = 1 sich den Sinus aus dem in § 9, Formel (II), angegebenen Ausdruck des Cosinus ausrechnet. Man hat dabei drei mal die Formel p2 — q2 = (p — q) (p + q) aus der Algebra anzuwenden. Zunächst ist sin2 a = 1 — cos2 a = (1 — cos a) (I + cos a). Die beiden Faktoren der rechten Seite berechnen wir einzeln. Es ist — a2+b2 + c2 1 — cos a = 1 2bc a2 — (b2 — 2bc-\|-c2) 2b c a2 — (b — c)2 2bc ,_[a—(b — c)] [a+(b — c)] 2bc 2 \ se 1 — COS a — (XI) bc * Ebenso wird — a2 + b2 + c2 1 —J- cos a — 14 2bc Ebene Trigonometrie. 40 b2 + 2bc + c2 — a2 2bc (Ъ + с2) —a2 2b c [b -j- c — a] [b -f- c -f- a] 2bc 1 + cos а = 2 8.8* (XII) bc Nunmehr ist sin2 а = (1 — cos а) (1 cos а) 2 ]/ s . Sa . Sb . SC bc 2J sin а 1 J=2bcsince 4 S . Sa . Sb . sc (bc)2 bc w. z. b. w. Dieser Beweis deckt sich im wesentlichen mit der Ab leitung der Formel J = j/ s. Sa . sb . sc in der ebenen Geometrie. § 13. Anwendung des Sinnssatzes. Der Sinussatz und seine Erweiterung dient zur Berechnung eines Dreiecks aus einer Seite und z wei Winkeln, oder aus zwei Seiten und einem nicht eingeschlossenen Winkel, oder aus drei Seiten. Erster Fall. Gegeben eine Seite und zwei Winkel. Man rechne zuerst nach dem Satz von der Winkel summe den dritten Winkel aus. Aus a, а, /?, у findet man dann b und c aus (V) a a b sin y sin/? sin a sin a logb = loga — log sin а 4“ log sin/? log c = log a — log sin a -f- log sin y. Кар. I Г. Das schiefwinklige Dreieck. 41 Erstes Beispiel, а — 13, а = 53° 7' 48", ß = 14° 15' 9", Zuerst ist а -f" ß — 67° 22' 48" / = 180 — (а + ß) = 112° 37' 12". 1. Berechnung von b. log b — log a — log sin « -)- log sin ß ausgerechnet = 0,60206 b = 4,000. 2. Berechnung von c. sin/ ist gleich dem Sinus des Nebenwinkels /' = 67° 22' 48" log c = log a — log sin a + log sin /' ausgerechnet = 1,17609 c = 15,000. Zweiter Fall. Zwei Seiten und ein gegenüber liegender Winkel: a, b, a. Nach dem Sinussatz ist sin a . b sin ß_b sin ß a sin a a Aus ß und a findet man у nach der Winkelsumme, c wieder nach dem Sinussatz: a sin/ с= sm а Zweites Beispiel, a = 13, b = 4, а = 53° 7' 49". log sin ß = log sin a — log a -f- log b, ausgerechnet = 9,39121 — 10, ß = 14° 15' 0" ß* — 180° — 14° 15' 0". oder Der zweite Fall ist aber ausgeschlossen, denn da ß‘ stumpf ist, könnte es nur der grössten Seite des Dreiecks gegenüberliegen, während b kleiner als a ist. Für / erhält man aus der Winkelsumme 112° 37' 12" und daraus für c 15,000. 42 Ebene Trigonometrie. Drittes Beispiel. a = 13,509, b = 17,632, a = 38° 21' 47". log sin ß = log sin a — log а -f- log b, ausgerechnet = 9,90852 — 10, ßi = 54° 6' 7" ß* = 180° — 54° 6' 7" = 125° 53' 53'" Da b grösser als a ist, liegt kein Grund gegen die Brauchbarkeit des zweiten Wertes vor. Es wird y1 =87° 32' 6", 72 = 15° 44' 20". a sin y2 a sin y1 c2 — sin а ci sin а ’ Die Berechnung ergiebt Cl =21,746, ca =5,9041. Viertes Beispiel, а = 13,509, c = 21,746, а = 38° 21' 47". Hier ist natürlich erst у zu berechnen. log sin 7 = log sin а — log a + log c, ausgerechnet = 9,99960 —10, 7j = 87° 32' 72 = 180° -7! = 92° 28'. Die Sekunden sind nicht zu bestimmen. Beide Winkel können um eine Minute falsch sein. Es wird A = 54° 6' 13", ß2 =: 49° 10' 13". Auch diese Winkel sind naturgemäss ungenau. Man findet mit ihnen \ = 17,632, b2 = 16,469. Man hätte ebensogut yl =87° 33' nehmen können und hätte dafür bt= 17,629, b2 = 16,473 erhalten. Noch stärker weichen die nach dem Cosinussatz (sechstes Beispiel in § 10) berechneten Werte ab. Wie verhält sich die graphische Lösung? Der Leser versuche sie mit den abgerundeten Werten a = 13,5 mm, c ~ 21,7 mm, a — 38°,5. Кар. II. Das schiefwinklige Dreieck. 43 Dritter Fall. Gegeben die 3 Seiten a, b, c. Hier ist der erweiterte Sinussatz, am besten Formel (IX) oder (X) anzuwenden. Man berechne dabei zuerst die den kleineren Seiten gegenüberliegenden Winkel, da diese sicher spitz sind, also der der Tabelle entnommene Wert der richtige ist. Fünftes Beispiel. a = 13, b = 4, с = 15. Es wird s = у (13 + 4 + 15) =16, sa = Sb = Sc = s -— a s—b s—c = 3, = 12, = 1, J2 =re . Sa . Sb . SC = 16.36, J = 4.6 = 24. Nun ist 2.24 - = 0,8, 4.15 log sin a — 9,90309 — 10, a = 53° T 48" ; 2.24 _16 1 J = c a sin ß, also sin ß 13.15 65’ log sin ß = 9,39121 — 10, ß = 14° 15' 0" ; 2.24_ 12 J = a b sin y, also sin y 4.13 13? log sin y = 9,96524 — 10. Aus der Tafel findet man für y zunächst 67° 22' 48". Da aber c die grösste Seite ist, kann y auch der Nebenwinkel von 67° 22' 48" sein. Der Satz von der Winkelsumme ergiebt in der That, dass У = 180° — 67° 22' 48" = 112° 37' 12" sein muss. toi Г С 1-^ Ü1 1 J = — b c sin a, also sin a = Ebene Trigonometrie. 44 Sechstes Beispiel. a = 13,509, b = 17,632, c = 21,746. s = 26,4435, sa = 12,9345, sb = 8,8115, sc = 4,6975 ; }=y[logs + logsa+logsb+logs0} 0gJ=ybg{ s • sa • sb • sc ansgerechnet = 2,07550 ; log sin a = log 2 -f- log J — log b — log c, ansgerechnet = 9,79285 — 10, « = 38° 21' 49". log sin ß = log 2 + log J — log c — log а, ansgerechnet = 9,90852 — 10, ß = 54° 6' 7". log sin у = log 2 -f- log J — log a — log b, ausgerechnet — 9,99960 — 10. у ist spitz, da а ß grösser als 90° wird. Die Tabelle giebt у nicht genau genug: 87°, 32'— 33'. Man wende da her die Winkelsumme an : у -87° 32' 4". § 14. Tangenten der halben Winkel. Zur Berechnung der Winkel aus den Seiten giebt es noch ein drittes Verfahren mit Hilfe eines ange schriebenen oder des eingeschriebenen Kreises. Fällt man von О aus auf die Seite b das Lot OV = so ist nach Sätzen der Planimetrie AV = sa, < OAV = \|. In dem rechtwinkligen Dreieck OAV ist aber а OV ,___ P AV Sa 2 (XIII,J Fällt man von Oa das Lot OaV' auf b, so ist, wie in der Planimetrie gezeigt wird : AV' = s, folglich CV' = s — b — sb« Кар. II. Das schiefwinklige Dreieck. 45 In dem rechtwinkligen Dreieck OaV'A ist а OaV' Jpa (XIII,,) tg2 AV' s ,Л % r c \ Oa V' Figur 17. In dem rechtwinkligen Dreieck OaV'C ist der Winkel bei C die Hälfte des Aussen winkeis von 7, also gleich 1 7 2"(180°—7) = 90°----g", folglich der Winkel bei Oa gleich X 2 Man hat also . y tg2 CV' Sb OaV' pa (XIII, 3) tg-\|-= ¥^ ^ Aus den drei Formeln (XIII> 1 > 2> 3 ) kann man durch Vertauschen der Seiten und Winkel folgende Tabelle hersteilen: а = =_Sb = _Sç t%j= Qc Çb __ _£L _ S Ç (XIII) tsi = Ç __ Qc,s __ Sa __ Sb Çb Ça Sc Drückt man die Radien durch die Seiten aus (Formel IV der Einleitung), so reduziert sich die An zahl dieser Formeln auf die Hälfte. Es wird z. B. Ebene Trigonometrie. 46 tg2 = J Sb. Sc J Setzt man hierin J = Vs . sa . Sb . sc, so werden auch diese beiden Ausdrücke miteinander identisch. Man /Sh • sc erhält : S.Sa «•ï-V ł-y s. sa : Są, • S,, S.Sb’ ‘4-У S.SC (XIV) Diese Formeln sind zur В erechnung der Winkel aus den Seiten sehr viel geeigneter als der Sinus- und Cosinussatz. Erstens ge statten sie durchgehende logarithmische Rechnung, was beim Cosinussatz nicht möglich ist. Zweitens sind die aufzuschlagenden Winkel sicher spitz. Drittens ist die Tafeldifferenz von log tg nie kleiner als 25 Einheiten der letzten Stelle, also kann man mindestens auf zwei Sekunden genau interpolieren. Erstes Beispiel. а = 13, b = 14, c = 15. s == 21 ; Sa = 8 ; Sb = 7 ; Sc = 6. а 776 1 -f-y 21.8 = T' ,og tg У = 9>69897 — 10- 4 tg- = l/ 8.6 ß ё 2 У 21.7 = у ; log tg j = 9,75696 - 10. , У т/8.7 2 tg¥=fäГб= Г logtg = 9,82391 — 10. ~ = 26° 33' 54" ; а = 53° 7' 48". Кар. II. Das schiefwinklige Dreieck. ß = 29° 44' 41" ; ß = 59°29'22". — jL = 33» 41' 25" ; 7 — 67°22'50". Probe : a + ß + y = 180° 0' 0". Zweites Beispiel. a = 13,509, b = 17,632, c = 21,746. s = 26,4435 ; sa = 12,9345 ; sb = 8,8115 ; sc = 4,6975. bg tg y = у {log Sb + log Sc — ausgerechnet = ^1,08285 — 2^ = 9,54143 — 10, Y = 19° 10' 54". log tg ■§■ = Y {log Sa + log se — log s — log sb J , = y { 1,41625 — 2 ] = 9,70813 — 10, ß Y = 27° 3' 6". log tg y = y {log Sa + log Sb — log s — log s0 ] , = Y { 1,96261 — 2 } = 9,98131 - 10, Zr = 43° 46' 3". 2 Somit ergiebt sich: « = 38° 21' 48" ß = 54° 6' 12" y = 87° 32' 6" Probe : a + ß + y = 180° 0' 6". 47 48 Ebene Trigonometrie. Drittes Kapitel. Die Additionstheoreme der trigonometrischen Funktionen. § 15. Erste Form der Additionstheoreme. (I) sin (f + tj) = sin I cos 17 + cos £ sin y sin (I — fj) = sin £ cos 1? — cos \| sin 17 (И) (III) cos (I + rj) = cos I cos y — sin I sin y (IV) cos (I — rç) = cos I cos 17 + sin £ sin 17. a) Die vorstehenden Formeln, die soge nannten Additionstheoreme der trigonometrischen Funktionen, müssen auswendiggelernt werden, am besten, ehe man ihre Beweise durch arbeitet. Auch empfiehlt es sich, ihre Richtigkeit an speciellen Bei spielen zu erproben (z. B. £ = 0; 90°, 180°, 270°, 360° oder für (I) und (III) £ = 90° — 7, 180° — y etc., für (II) und (IV) £ = 7, 90° + y etc.) Dabei ist zu be achten, dass (für Winkel des ersten Quadranten) der Cosinus des grösseren Winkels £ + у kleiner sein muss als der des kleineren Winkels £ — y\ hierdurch erklärt sich das Verhalten der Vorzeichen in (III) und (IV). b) Wenn man eine derFormeln(I)bis (IV) kennt, so kann man die anderen daraus her leiten. Setzt man z. B. in (I) у — — y\ so wird nach Кар. I, § 6, 5 »sin у = — sin y\ cos у — cos 7', wodurch Formel (I) in (II) und (III) in (IV) übergeht. Ferner ist cos (£ + y) = sin (90° — I + 17) = sin (90° — £) cos у cos (90° — £) sin у = cos £ cos у sin £ sin y. Кар. III. Die Additionstheoreme der trig. Funkt. 49 Der Leser versuche ebenso die Herleitung der übrigen Formeln aus (II), (III) und (IV). c) Man setze in (I) und (III) f = J' — tj und löse die so entstehenden Gleichungen nach sin (f'— 77) und cos (£' — 77) auf. Man wird (II) und (IY) erbalten. Umgekehrt setze man in (II) und (IV) \| = + ^ und löse nach sin (\|' -f- 77), cos (f ' 77) auf. d) Man setze in (I) \| = £' — 77, drücke sin (\|' — 77) nach (II) aus und berechne cos (\|' — 77). In ähnlicher Weise leite man (III) aus (I) und (II) und umgekehrt (I), (П) aus (III) und (IV) ab. Hieraus wird ersichtlich, dass es genügt, zwei oder auch bloss eine der Formeln (I) bis (IV) zu beweisen. Der Beweis wird noch vereinfacht durch folgende Sätze: e) Hat man eine der Formeln (I) bis (IV) für die Winkel zweier aufeinanderfolgen der Quadranten bewiesen, so gilt sie für beliebige Winkel. Man setze beispielsweise in (I) \| = -f-180°. Es wird sin\|'= — sinf; cosf' =— cosf; sin(\|'-f-?) =— sin(J-f-77), Die Formeln für sin (f ' -f- v) UQd sin (IH“ 77) sind dem nach gleichbedeutend und gehen ineinander über. Ist also (I) bewiesen für Winkel §' von 0 bis 180°, so ist es auch bewiesen für die um 180° grösseren Winkel \| von 180° bis 360° u. s. f. Der Leser untersuche auch (II), (III) und (IV) daraufhin. f) Hat man eines der Formelpaare (I), (ITT) und (II), (IV) bewiesen für die Winkel eines Quadranten, so gilt es für beliebige Winkel. Hessenberg, Ebene und sphärische Trigonometrie. 4 Ebene Trigonometrie. 50 Man setze beispielsweise in (I) und (III) \| = \|' + 90°. Es wird, da 90° — §' der Nebenwinkel von \| ist: COS S = — sin S' = COS S' sin S ebenso sin (I + v) = cos (§' + v)i cos (S + v) = — sin (S' +17), womit die Formeln für sin (S + v), cos (\| + rj) beziehlicb in die für cos (S' + ?), sin (£' + v) übergehen. Hat man also (I) und (III) für die Winkel §' des ersten Quadranten bewiesen, so gelten sie auch für die um 90° grösseren S des zweiten u. s. f. Der Leser untersuche auch (II), (IV) und den Winkel 17 daraufhin. § 16. Beweis der Additionstheoreme. Erster Beweis. a) Aus der Planimetrie ist der Satz des Ptolem ä u s bekannt1 : In einem Sehnenviereck, dessen Ecken in der Reihenfolge, wie sie auf dem Kreise liegen, mit PQRS bezeichnet seien, ist das Produkt der Diagonalen gleich der Summe der Produkte der Gegen seiten : PR.QS = QR.PS + PQ.RS. (A) Man kann diese Gleichung nach Division durch PR2 in der Form schreiben: Qß _ QR PS_ PQ B^S PB ~~ PB ‘ PR + PR PR (B) Wählt man PB als Durchmesser, so lassen sich diese sämtlichen Verhältnisse als Sinus und Cosinus auf 1 Ein trigonometrischer Beweis des Satzes unter Anwendung der Additionstheoreme iindet sich unten, § 27. Кар. III. Die Additionstheoreme der trig.;Funkt. 51 fassen. Die vier Quotienten auf der rechten Seite sind nämlich die Seitenverhältnisse der nach dem Satz des Tales bei Q und S rechtwinkligen Dreiecke P Q R und PSR. Zieht man noch durch den Mittelpunkt M des Kreises den Durchmesser QP', so ist in dem bei S rechtwinkligen Dreieck QP' S QS: P'Q = QS:PR. 9 P AI R / Figur 18. b) Man mache jetzt QPR = \|, <SPR = 7. Der Winkel QP'S steht mit QPS über gleichem oder entgegengesetztem Bogen, ist also ihm oder seinem Supplement gleich, so dass auf jeden Pall sin QP'S = sin QPS = sin (f + n). Mithin ist Q R : P R = sin \| R S : P R = sin tj Q P : P R = cos I P S : P R = cos tj QS : PR äsin (£ + ). In (B) eingesetzt ergiebt dies (I). c) Man mache Q P R = £, <^SRP==^# Dann ist P Q M = <£ Q P M — £ als Basiswinkel im gleich- Ebene Trigonometrie. 52 schenkligen Dreieck PQM und <£PQS = <£PRS — ц als Peripheriewinkel über dem gleichen Bogen PS; mit hin ist <SQP' = <PQM — <£PQS = \| — n RS:PR = cos 17 QR : PB = sin g Q P : P В = cos I P S : P R = sin ri Q S : P В = cos (£ — rj). In (B) eingesetzt ergiebt dies (IV). d) Schreibt man (A) in der Form QR __ PR QS _ PQ BS (C) PS “ PS * PS PS" PS und macht PS zum Durchmesser, zieht durch M den Durchmesser QP', so sind die fünf in (C) auftretenden Quotienten Verhältnisse der Seiten der rechtwinkligen Dreiecke PQS, PBS, QRP'. Q /7 / i s p Figur 19. Man mache <QPS = £, < BPS = tj. Es wird <£ QP'R = < QPB = £ — ij, als Peripheriewinkel über dem Bogen QB, mithin Кар. III. Die Additionstheoreme der trig. Funkt. P Q : PS = cos £ PB : P S — cos rj QP 53 Q S : P S = sin \| K S : P S — sin QP = sin (f — ri). PS QP' In (C) eingesetzt ergiebt dies (II). e) Man mache <£ Q S P = \|, < PPS = rç. Da die Bögen QP und PS zusammen kleiner als 180° sind, muss vorausgesetzt werden, dass I + V < 90°, also cos (I + rj) positiv ist. Es wird <£BQS = <£PPS = Ti (Peripheriewinkel über PS), ^MQS = MSQ = \| (Basiswinkel im gleichschenkligen Dreieck MQS), d. h. <PQP' = \| + rj, mithin P Q : P S = sin I Q S : P S = cos f PS : PS- sin V PP:PS = cos r\ QP:PS = QP: Q P'= cos (£+ ). Ist ! + ? >90°, so mache man <£ QPS = f, < PSP = rj. Es wird < PQP = 180° — PSP = 180° — n (Peripheriewinkel über den entgegengesetzten Bögen PP), <PQM = < QPM = £ (Basiswinkel im gleich schenkligen Dreieck QPM), <£ PQP' = 180° — £ — v. P Q : P S = cos £ Q S : P S = sin ! P P : P S = sin 7j P S : P S = cos rj 1 QR:PS = QP:QP' = cos(180° — ?) = —-cos (£+?). In (C) eingesetzt ergiebt dies in beiden Fällen (III). Hiermit sind die Formeln (I) bis (IV) für "Winkel des ersten Quadranten, somit nach § 15, f allgemein bewiesen. Zweiter Beweis, f) Setzt man in die Cosinusformel c — a cos ß -f- b cos a Ebene Trigonometrie. 54 aus dem erweiterten Sinussatz a = 2r sin a etc. ein, so hebt sich 2r fort und es bleibt sin y — sin a cos ß -f- cos a sin ß. Ф) Man trage jetzt in zwei Punkten А, В einer Ge raden an diese, auf derselben Seite, einander zugekehrt die Winkel f und rj an, die wir beide kleiner als 180° annehmen. Ist dann l + ^<180°, so schneiden sich die freien Schenkel in einem Punkte C, und in dem Dreieck ABC ist C г i А В Figur 20* « = Si ß У = 180° — (S + Setzt man diese Werte in (D) ein, so wird sin у = sin (I + y) = sin i cos rj -f- cos § sin rj. Ist I + V > 180°, so schneiden sich die freien Schenkel rückwärts verlängert in einem Punkt C, und im Dreieck ABC ist а = 180° — f, ß = 180° — n, y = i + v —180°, sin а = sin \|, sin ß = sin sin у = — sin (£+ 77) cos а = — cos I, cos /? = — cos 17. In (D) eingesetzt ergiebt dies wiederum (I). Кар. III. Die Additionstheoreme der trig. Funkt. 55 Ч ci /з X Figur 21. Hiermit ist (I) für alle Winkel von 0° bis 180°, mithin nach § 15, e allgemein bewiesen. Nach § 15, b erhält man daher durch einfache Umformungen auch (II) bis (IV) daraus. g) (II) kann in einfacher Weise ähnlich bewiesen werden. Man trage in A und В § und r\ auf derselben C d 4 Ä В Figur 22. Seite der Geraden an, aber nicht mehr einander zu Ebene Trigonometrie. 56 gekehrt, sondern beide mit der Oeffnung nach derselben Seite, den grösseren vor dem kleineren. Die freien Schenkel mögen sich in C schneiden. Es wird a = 180° — {, sin a = sin cos cc = — cos \|, womit (D) in (II) übergeht. — ß—V: y = i — V- Hiermit ist (II) für Winkel zwischen 0' und 180°, also nach § 15, e allgemein bewiesen. Die Formeln (III) und (IV) kann man jetzt etwa nach § 15, d herleiten. Ein weiterer Beweis findet sich im nächsten Para graphen, und ein ganz allgemeiner für Winkel beliebiger Quadranten im Кар. XI, der aber für Anfänger schwerer verständlich sein dürfte. § 17. Zweite Form der Additionstheoreme. Addiert man die Gleichungen (I) und (П) dieses Kapitels, so folgt: 2 sin \| cos rj = sin (\| + y) + sin (f — rj). Durch Subtraktion ergiebt sich 2 sin 7 cos \| = sin (f + rj) — sin (f — rj). Ebenso erhält man aus (III) und (IV) 2 cos I cos ^ = cos (! + ?) + cos (I — ri) (V) (VI) (VII) und « =ê+v ■r<r — 2 sin I sin ri = cos (\| + v) — cos (\| — rj). (VIII) Bezeichnet man die Winkel f -f- rj und \| — rj mit a und ß, so wird ß = S—v 4 = -j(<—ß)- ~~2 Кар. III. Die Additionstheoreme der trig. Funkt. 57 Durch Einsetzen in die soeben abgeleiteten Formeln erhält man folgende vier wichtige Beziehungen: cos a -\|- cos ß = a—ß cos —2 ß 2 sin a ^ ^ cos ct -f2ß-a—ß 2 cos a - cos —g cos a — cos /? = . a—ß — 2 sin a2 ^ Sïn—g—. sin a -f- sin ß — sin а — sin ß = 2 sin (IX) Sie werden öfter gebraucht, als die Formeln (I) bis (IV), sind aber mit diesen gleichbedeutend. Man kann sie auch sehr einfach geometrisch ableiten. Doch soll dieses Verfahren nur für den Fall angegeben werden, dass a und ß Winkel des ersten Quadranten sind. Es sei PB A ein Kreis mit dem Radius 1 und dem Centrum О. Winkel PO A sei gleich a, Winkel POB gleich ß. Von A und В seien die Lote AA' und BB' auf OP gefällt. Dann ist (Fig. 23 a. f. S.) AA' = sina, O A' = cos a, BB' = sin ß, OB' — cos/?. Sodann sei C der Halbierungspunkt der Sehne AB. Es ist OC senkrecht auf AB und <AOC = F < AOB-“ — ß Ji 2 ’ а +ß < COP = /? + ^=~/î 2 Fällt man noch das Lot С C' auf О P, so ist СС' = -\|'{АА< + ВВ'} = 7 {sina + sin,?} als Mittellinie des Trapezes AA'BB'. Ebene Trigonometrie. 58 Andererseits ist in dem rechtwinkligen Dreieck COC' C C' = O C. sin <£ C O P = O C sin Li und in dem rechtwinkligen Dreieck AOC О C = O A cos AOC = 1 . cos —- Somit wird cc — ß . a +ß g—sin 2—‘ Dies ist die erste der Formeln (IX). С C' = {sin а -[- sin ß j — cos А >X JA О A’ \ Yp Figur 23. Gleichzeitig ist OG' ~ ~2 {OA/-b О В7} — ~2 {cos а cos und andererseits OC' = CC' cos< COP = cos cs —ß 2 cos 2 ' Durch Vergleich beider Werte für OC' entsteht die dritte der Formeln (IX). Zieht man jetzt noch CX senkrecht zu AA' bis zum Schnitt mit AA' in X, so ist AX gleich der halben Differenz von AA' und BB', Кар. III. Die Additionstheoreme der trig. Funkt. 59 AX = ~ {sin a — sin /?}, <XAC = <COP, und weil XAiOP, AC_LOC. In dem rechtwinkligen Dreieck XAC ist aber: X A = AC cos < X А С = AC cos und in dem rechtwinkligen Dreieck AOC AC = OA.sin<AOC = l.ein Damit wird X A = ~ {sin а — sin ßj = sin — —ß 2 Dies ist die zweite der Formeln (IX). Gleichzeitig ist XC = A'C' =Iä'b'= cos +ß 2 ' ~ {cos/? — cos und andererseits XC =ACsinXAC = sin . « + /? sm 2 Durch Vergleich beider Werte für XC entsteht die letzte der Formeln (IX). § 18. Bedeutung der Additionstheoreme. Die Bedeutung der eben abgeleiteten Formeln ist eine vierfache. Erstens dienen sie vom rein rechnerischen Standpunkt vornehmlich in der Gestalt (V) bis (IX) zur Um formung der für logarithmische Rechnung ungeeigneten Summen von Sinus in Cosinus in bequeme Produkte. Anmerkung. Vor Erfindung der Logarithmen wur den die Formeln im umgekehrten Sinne zur Verwandlung von Produkten in Summen benutzt. Man nannte das Ver fahren, das bald durch die Logarithmen verdrängt wurde, „P г о s t h a p h ä r e s i s“. 60 Ebene Trigonometrie. Zweitens erweitern die Additionstheoreme das Ge biet der Anwendungen der trigonometrischen Funktionen bis ins Bereich der Algebra und Analysis. Man verwendet sie beispielsweise zur Auflösung algebraischer Gleichungen. Drittens bieten die Additionstheoreme das einzige Hilfsmittel zur elementaren Berechnung des Sinus und Co sinus eines Winkels, und viertens sind sie für die höhere Mathematik die fundamentale Eigenschaft der trigonometri schen Funktionen, aus der ihre Reihenentwicklungen abge leitet werden. § 19. Additionstheorem der Tangente. Durch Division der Formeln (I) und (III) erhält man sin £ cos ri + cos £ sin rj tg (!+?) = cos £ cos r\ — sin £ sin r[ Dividiert man Zähler und Nenner durch cos £ cos so wird daraus is(ê + v)-i-ëttitsv' (X) Ebenso erhält man durch Division aus (II) und (IV) : tg I — tg rj (XI) tg (!-) = 1 + tg £ tg rf Die Formeln (X) und (XI) enthalten das Additions theorem der Tangente. (XI) geht aus (X) hervor, wenn man —y für t\ setzt und beachtet, dass tg(—r\)~—tgrj ist. Dividiert man (III) durch (I) und dann auf der rechten Seite Zähler und Nenner durch sin £ sin so erhält man cot (! + ,) = —/ cot n ~1 cot £ -f cot ri und ebenso aus (IV) und (II) cot (g^«) = 1±cot^cot^. cot rj — cot I (XII) Кар. III. Die Additionstheoreme der trig. Funkt. 61 Diese beiden Formeln werden, wie die Cotangente selbst, selten gebraucht. Durch Division beider Seiten mit cos £ cos tj erhält man aus (I) bis (IY) folgende vier nützliche Beziehungen : sin (g + i?) COS £ COS 1J cosfg + g) COS £ COS 7J — g £ + tg V = 1 + tg I tg П (XIII) I Durch Division der beiden ersten folgt weiter sin(l+g) = tgf + tgq (XIV) 8in(\|— n) tgS — tgv’ ebenso aus der ersten und vierten sin(\|+^)^ tgg + tgi? cos(\|—i?) “ 1 + tgg tg 1?’ und aus den beiden letzten (XV) СОЗ (g+v)_ 1 — tgl tgi? 008(1—1?) ~ 1 + tgl tgl?', Aus den Formeln (IX) erhält man durch Division der ersten und zweiten sin a + sin ß sin a — sin ß der vierten und dritten: cos« — cos/9 cös^'+cosj? = tg (XVI) a—ß * ff 2 •tg „ 2 r > /VTlr 4 (XVI”) der ersten und dritten oder der zweiten und dritten: sin a + sin ß a+ß (XVII) cos a -f- cos ß = tg ~2~§ 20. Doppelte und halbe Winkel. Aus (I) und (II) erhält man, wenn man £ = rj setzt: Ebene Trigonometrie. 62 sin 2f = 2 sin I cos f, cos 2\| = cos21 — sin2 Ç. Aus (X) ebenso 2tg\| tg2\| l-tg2! Aus den Gleichungen (XVIII) (XIX) (XX) cos 2\| = cos21 — sin2 i 1 = cos2! + sin2! und erhält man durch Addition und Subtraktion: 1 + cos 2 ! = 2 cos2 !, 1 — cos 2\| = 2 sin2\|, also sin\| = j/1---- °°S2^, cos\| = j/ l+cos2! 2 •(XXI) Aus (XV) wird für ! = 17, da cosO=l ist: 1-tg2! 2tg! (XXII) sin 2 ! -, cos 2! = 1 + tg2! 1 + tg* I* Setzt man 2 ! = a, so wird aus (XXI) und (XXII) : ; cos 1 + COS a 2 • (XXI') 2tg\| sin a cos a — . (XXII') i+tg2\| Setzt man in (XVII) ß — 0, so wird noch sin a tgr\| (XXIII) 1 + cos a * Кар. IV. Anwendung der Additionstheoreme. 63 Viertes Kapitel. Anwendung der Additionstheoreme auf das schiefwinklige Dreieck. § 21. Tangentensatz. a = 2rsince, b = 2rsin/? Aus erhält man a + b 2 r (sin а + sin ß) sin а + sin ß а — b 2 r (sin а — sin /?) sin а — sin ß und nach den beiden ersten der Formeln (IX) oder nach Formel (XVI) des vorigen Kapitels а+b a—b . a—ß g Ist a kleiner als b, so schreibt man besser b+а b—а © (I) Чнг” Diese Formel enthält den Tangentensatz ; man nennt sie auch die „ Napier’sehe Gleichung“. Der Tangentensatz dient zur Auflösung eines Dreiecks, wenn a, b und у gegeben sind, und eignet sich dazu besser, als der Cosinussatz, weil er durchgehende logarithmische Rechnung gestattet. 1. Beispiel: а = 13,509, b = 17,632, у = 87° 32' 6". b + а = 31,141 b — a= 4,123 a + ß = 180° — у = 92° 27' 54" a ß 46° 13' 57". 2 Ebene Trigonometrie. 64 / , 2. tg log tg ß — a 2 . /? 4- ce b — a — tg 2 ' b + a - = log tg f log (b — a) — log (b + a logtg^T“ = 0,01869 log (b — a) = 0,61521 log (b + a) = 1,49333 log tg ^“ “ = 9,14057 — 10 = 7° 52' 10". 3. ß~Ya+ß~^ “ = ß = 54° 6' 7" /?-: « 2 ß—a = a = 38° 21' 47". 4. c nach dem Sinussatz, zur Probe etwa auf zwei Arten : a sin/ _ b sin / c C sin ß sin« Für die vier Kongruenz fälle empfehlen sich also im allgemeinen folgende Berechnungsmethoden, die durch gehende Logarithmierung gestatten: Erster Kongruenzfall: gegeben a, b,/. ce---ß Man berechnet —— aus dem Tangentensatz, sodann c aus dem Sinussatz. Aufzuschlagen sind die Logarithmen von a-j-b, a — b, tg—, a, since, sin/, die Numeri von log tga -g ^ und log c. Die Tabelle wird also mindestens 8mal benutzt. Zweiter Kongruenzfall: gegeben a und die Winkel. Кар. IV. Anwendung der Additionstheoreme. 65 Man erhält Ъ und с aus dem Sinussatz. Aufzu schlagen sind die Logarithmen von a, sin«, sin/?, sin/, und die Numeri von log b und c. Die Tabelle wird mindestens 6mal benutzt. Dritter Kongruenzfall: gegeben a, b, c. Man berechne nach § 14 die Tangenten der halben Winkel. Aufzuschlagen sind die Logarithmen von s, cc sa, Sb und sc, die Numeri von log tg und log tg , (log tg 2 nur zur Probe). Die Tabelle wird mindestens 6mal benutzt. Vierter Kongruenzfall: gegeben a, b, a. Man berechne ß aus dem Sinussatz, / durch die Winkelsumme und c wieder nach dem Sinussatz. Auf zuschlagen sind die Logarithmen von a, b, sin«, sin/; die Numeri von log sin ß und log c. Die Tabelle wird mindestens 6mal benutzt. К § 22. Weiteres Formelmaterial. In Кар. II, § 12, waren die Gleichungen 1 0SbSc , S Sa 1 — cosa=2^----, 1-4- cosa = 2-,— bc bc abgeleitet worden. Nach Formel (XXI') in Кар, III '§ 20) folgt daraus ohne weiteres: inf-)/Sb^c bc : a = 1 /--/ s SÄcos — 2 F be то Durch Division entsteht die Gleichung IHVrr (Kap- n’ § 14)r S Sa Hessenberg, Ebene und sphärische Trigonometrie. 5 Ebene Trigonometrie. 66 Aus den Beziehungen a==2rsina, b = 2rsin/? erhält man durch Addition und Subtraktion : a —ß cos 2 ’ a + b = 4 r sin ß a — b = 4 r sin a 2 ^ cos a-\2 ' Aus a-\- ß-\- y — 180' folgt aber = 90° — also y a-\-ß a~\~ß • 7 sin 2— = cos 2 ’ cos —^— = sin 2 ’ . und damit * a a + b = 4 r cos e 2~ " cos 27.> a — b = 4 r sin a ~2^ sin g-. Ferner ist c = 2 r sin 7 = 4 r sin 72 cos 7 (IV) Dividiert man (III) hierdurch, so entstehen die sogenannten Mollweide’schen Gleichungen a—ß . ci —ß cos sin § a+b а —Ъ (V) ; c c sin I cos 79 Ferner ergiebt sich aus (III) und (IV) durch Addition : 2s=a-f-b + c = 4r cos = 4r cos T -H "M (a-ß 7 — 8rcos~ cos l 2 jcos^4p+sin g} (__a - ß 2 a-\-ß} 2 a+ß\ l/ce+0 a—ßs _______ nr\a I _ _ _ !_ _ 1- - - - - - - - - - - - - - - - - - 2 2 2 2 Anwendung der Additionstheoreme. ß s = 4 r cos 2 cos 2 cos oder 67 toN Кар. IV. Ebenso durch Subtraktion: [ а — ß 2 8C = a + b — c = 4 r cos I \|cos 2 = 8 r cos oder cos a+ ß . ce —ß \ . 1 (a+ß sin g ^ce-[-ß 2 2 ) ЗШ2П Sc = 4 r sin 2 ! a—ß ) 2 y 2cos 2. 2 sin i Hierdurch entstehen die vier Formeln: s — 4 r cos g cos g cos g sa = 4 r cos g sin g sin g (VI) sb = 4 r sin g cos g sin g sc = 4 r sin I sin g cos L 2‘ Mit Hilfe der Gleichungen (XIII) des § 14 erhält man hierzu folgende Ergänzungen: Q = 4 r sin -g- sin ~ sin ~ а ß У (>a = 4rsin-g cos — COS — 2 2 Qb = 4 r cos g- sin g- cos L. 2 Qa = 4r cos ~ cos Ą g sin t 2 (VII) 68 Ebene Trigonometrie. Fünftes Kapitel. Berechnung der Vierecke. § 23. Allgemeines. Man unterscheidet wohl neben der Trigonometrie noch die „Polygon ometrie“ und speciell die „Tetragonometrie“, — die Lehre von den Vielecken, speciell den Vierecken. Diese sind aber keine selbständigen Wissenschaften, sondern der Trigonometrie untergeordnet. Durch Zerlegung einer geradlinigen Figur in Dreiecke und Aufstellung der Gleichungen für diese erhält man stets die notwendige und hinreichende Anzahl von Be- Щ Ziehungen zur Berechnung der unbekannten Stücke aus den gegebenen. Mit welchen Hilfsmitteln diese Glei chungen nach den Unbekannten aufzulösen sind, ist ein Problem der Algebra, nicht der Geometrie. Wir erörtern die wichtigeren Aufgaben über das Viereck, unter Ausschluss des überschlagenen oder ein springenden, für das übrigens ein prinzipieller Unter schied in der Berechnung nicht existiert. Die vier Ecken des Vierecks seien in der Beibenfolge, wie sie auf dem Umfang liegen, mit A, B, C, D, die zugehörigen Winkel mit «, /?, y, ô bezeichnet. Die Seiten AB, BC, CD, DA sollen bezw. d b, a, c, heissen. Ausser diesen 8 eigentlichen Stücken des Vierecks betrachten wir noch die Diagonalen Кар. V. Berechnung der Vierecke. 69 AC = e, BD = f und die Winkel, die sie mit den Seiten bilden: CAD = al9 CAB = «2 ACB = yj9 ACD = y2 KC r i) DBA = ^, DBC = Ą, BDC = BDA = ^. а s b а J f re dL4 d-2 ßz Al А Figur 24. Diese 18 Stücke, a, b, c, d ; e, f ; «, a,, «2 ; ß, ßt, ß2 ; y, Ун Y2 5 ^2 geboren den vier Teildreiecken ABC, BCD, CDA, DAC an, die durch Weglassen je einer Ecke entstehen. Kennt man von diesen Drei ecken zwei, so kennt man auch die beiden anderen. Entweder haben nämlich zwei dieser Drei ecke eine Seite, etwa a, oder aber eine Diagonale, etwa e, gemeinsam. Im ersten Fall kennt man aus Л ABC : a, b, e; A ABD: a, d, f; avß%y„ ßl9<}öt. folglich auch und dBCD aus b, f, ß2=ß—0j, A ACD aus d, e, al = a — a2. Ebene Trigonometrie. 70 Im zweiten Fall kennt man aus A ABC: a, b, e; A ADC: c, d, e; «2, ß,ryt, «,,<5, y2, folglich auch 4 BCD aus b, с, у = ух + у2 A BAD aus a, d, а = а1 -\- «2. und § 24. Berechnung durch Teildreiecke. Bestimmt ist ein Viereck durch 5 unab hängige Stücke. Am einfachsten ist offenbar der Fall, wo aus diesen 5 Stücken ohne weiteres zwei Teildreiecke berechnet werden können. Da für jedes Dreieck 3 Stücke notwendig sind, muss das gemeinsame Stück gegeben sein. Es sind also bei passender Bezeichnung zwei Hauptfälle möglich: 1. Gregehen a mit zwei von den Stücken b, e; a2, ß, yi d, f ; ßlJ a, <52. und „ 2. Gregeben e mit zwei von den Stücken a, b; ce2, /?, yx und „ c, d; «n^72. Am nächsteinfachsten ist der Fall, dass nur ein Te il dr eieck bestimmt ist, dass aber dessen 3 weitere Stücke die Berechnung eines zweiten gestatten. Es sind wieder dieselben beiden Fälle zu unterscheiden, wie oben: 3. Gegeben drei von den Stücken b, e; a2,/?, y^) und zwei „ „ d, f ; ß19 a, ^2. 4. Gegeben drei von den Stücken a, b; a2,/?, yi1) und zwei „ c>d; avö,y2. ») Natürlich nicht die 3 letzten. Кар. V. Berechnung der Vierecke. 71 Unter 3 und 4 gleichzeitig fällt die Bestimmung des Vierecks aus b, d; av av 0; (y1 = 180° — ß — a2). Man kann fragen, ob nach Berechnung eines Teil dreiecks auch stets die eines zweiten möglich ist. Dies ist nicht der Fall. Ist etwa ABC durch 3 seiner Stücke a, b, e; «2, ft yx bestimmt, und ist ausserdem gegeben eines der beiden Paare : 1. c, ft ; d, ft, 2. f, 6 ; 3 » ft ; 72> ^2 i 4. ß1 (und ß2 = ß — ft), Ô ; 5. ft, ft, so ist kein zweites Teildreieck bestimmt. Die unter 1 bezw. 3 aufgeführten Paare sind nicht verschieden, weil sie durch Vertauschung der Bezeichnungen A und C ineinander übergehen. Prinzipiell nicht verschieden sind ferner die Fälle 1, 2, bezw. 3, 4. Der letzte Fall ist als Pothenot’sche Aufgabe bekannt und findet ebenso wie 3 und 4 in § 26 seine Erledigung. 1 und 2 sind nicht von Interesse, eignen sich aber zu Konstruktionsaufgaben. Hiermit sind die Fälle vollständig aufgeführt, in denen sich ein Teildreieck oder mehrere berechnen lassen. § 25. Die vollständigen Beziehungen zwischen den Winkeln. Durch 4 unabhängige Winkel ist ein Viereck der Gestalt nach bestimmt, wie eine einfache Ueberlegung zeigt. Sind also 4 Winkel und eine Seite oder Dia gonale bekannt, so wird man versuchen, aus den 4 Ebene Trigonometrie. 72 Winkeln so viele der übrigen Winkel zu finden, dass man die der Seite oder Diagonale anliegenden Teil dreiecke berechnen kann. Wir hatten 12 Winkel unterschieden, zwischen denen also 8 Gleichungen bestehen müssen. Es ist zu nächst «= У = Уг + Г2 } (i) ß=ßi + /»„ 5 = <1 + <2 Diese 4 Gleichungen sind unabhängig und gestatten, uns auf die 8 Winkel cq, cq, ßl9 ß2f y1} y2, 02 zu be schränken. Für diese erhält man aus den Winkel summen der Teildreiecke: “2 + ßt + ßt + Уг = 180°, + +^s + “i = 180°, ß, + rt + Г, ЧЧ = 180° ) (Щ + “, +“s ~hß, — 180°) Yon diesen 4 Gleichungen ist aber jede eine Kon sequenz der 3 anderen. Auch die Gleichung cc + ß + y + ö = 360° folgt aus (I) und (II). Die fehlende 8. Gleichung lässt sich nur mit tri gonometrischen Hilfsmitteln angeben, und zwar in den ver schiedensten Formen. 1. In der Ecke b liegen drei Stücke: a, b, f. Man hat im Dreieck BAD : а : f = sin <52 : sin «, BDC : f : b = sin у : sin öy und BCA: b : а = sina2 : sin y1% Multipliziert man diese drei Gleichungen, so wird die linke Seite zu 1. Schafft man den Nenner der rechten Seite weg, so erhält man Кар. V. Berechnung der Vierecke. 73 sin а . sin yl . sin öt = sin a2 . sin 7 . sin <52 und analog aus den 3 anderen Ecken : (III) sin ß . sin ä1 . sin «, = sin ß2 . sin <5. sin a2 sin 7 . sin öj . sin ßx = sin y2 . sin a . sin ß2 sin ô . sin ßt. sin 7j = sin ä2 . sin ß . sin 72 2. Geht man von A über В, C, D nach A zurück, so durchläuft man die 4 Stücke a, b, c, d. Es ist aber a : b =■ sin yx : sin «2 im Dreieck ABC b : c = sin dj : sin ß2 „ BCD c : d = sin at : sin y2 „ „ CDA d : a = sin ßl : sin S2. „ „ DAB Mithin, wie oben, sin at sin ß1 sin уг sin ô1 = sin a2 sin ß2 sin y2 sin ô2. (IV) Durchläuft man ebenso die Stücke e, b, f, d, so er hält man: sin a sin ß sin y2 sin öt = sin a2 sin ßt sin 7 sin ô, (V) und aus den Stücken e, c, f, a: sin « sin ß2 sin yt sin ô = sin ax sin ß sin 7 sin ô2. (VI) Die Gleichungen (III) bis (IV) lassen sich mittels (I) und (II) aus einer einzigen unter ihnen herleiten. Geschickte Rechner mögen dies versuchen. Sieben beliebige der Gleichungen (I) und (II) und eine beliebige der Gleichungen (IV) bis (VI) bilden das vollständige System von Beziehungen zwischen den zwölf Winkeln. § 26. Berechnung der Winkel aus 4 gegebenen. Wenn 4 Winkel gegeben sind, so wird man bei passender Bezeichnung der Ecken stets einen der fol genden 7 Fälle erkennen : Ebene Trigonometrie. 74 Erste Hauptlage: Gegeben «„ «2; ßv ß2 oder «i» «3; 7v r2- Zweite Hauptlage: Gegeben a2, уг ; <5ly <52. Hauptlage: Gegeben a2, ; y2, <52 oder <2, 7, ;A,(5. Vierte Hauptlage: Gegeben al9 ßl9 y19 St oder «, ß, y2)t Dritte Auflösung: Erste Hauptlage: al9 «2 ; ßx, ß2» Aus (II) ergeben sich Yl = 180° — a2 — ßi—ßi S2 = 180° — «j — «2 — ßl9 so dass die 6 Winkel a1? «2; ßt, ß2\ yl9 à2 bekannt sind. Für (5j und y2 erhält man aus (II) noch ôi+72= «2 + ^i C D [B A Figur 25. Mehr Beziehungen können aus (II) nicht hergeleitet werden. Aus (IV) ergiebt sich aber sin _ sin a2 sin ß2 sin 6^ sin/2 ~~ sin«! sin/Jj sin/^ Setzt man die rechte Seite, die bekannt ist, gleich t und <i + /2 == «2 + ßi = 2 ° = 2? -У2 Кар. V. Berechnung der Vierecke. 75 so ist a bekannt, ç unbekannt und nach Кар. III, Formel (XVI): sin — sin y2 _ tg q sin àx -f- sin y2 tg а ’ woraus q zu berechnen ist. Sind , a2 • yt, y2 gegeben, so wird nach (II) und (I) 1 —t 1 +1 C D A В Figur 26. ß = 180° — «2 — У17 à = 180° — y2 — ßt —à, =y2—a2 = 2 ç, wo () bekannt, und nach (V) sin _ sin a sin /? sin y2 sin sin a2 sin y sin ^ Setzt man ß1-{- ö1=2a) so wird nach (II) und (I) tg<r _ 1 + t tgö ~l—t’ woraus sich <r berechnen lässt. Vergl. hierzu Кар. XIII. Zweite Hauptlage: C Gegeben a2, y^ <52. In der ersten Gleichung (III) kom^ men diese 4 Winkel vor, ausj serdem a und y\ sin« siny sina2 sin<52 sin/j sinój i t. A Figur 27. в Ebene Trigonometrie. 76 Aus (II) und (I) folgt aber a + V = 180° + «2 + У, — <, — <5, = 2<г, und a — у = 2 q ergiebt sich wieder aus tgQ __ 1 —t tg o' 1 -j-1 Wenn a2 + /i = 6, + 62 wird, ist, a = 90°, tg n = oo, unsere Methode versagt also. In der That ist dann а -J- у —180°, also das Viereck ein Sehnenviereck, mithin auch <5t = a2, 62=y1. Ist dies nicht so gegeben, so ist die Auf gabe unmöglich ; andernfalls ist sie unbestimmt ; jeder Punkt des Umkreises von ABC genügt den gestellten Anforderungen. Dritte Hauptlage: «2, yx ; y2, <?2. Die dritte der Gleichungen (III) ergiebt sin «2 sin (/, + уг) sin St ± sin a sin <5X --- --------------- ------------------- ---- t. sin/j л C D i А 4i Figur 28. Aus (II) folgt: dj + a, = 180° — y2 — ö2, also ^ + а = 180° — y2 ■— ö2 a2 = wo Л bekannt. Setzt man noch <5 — а = ,иу so wird sin a sin <5, =. rl cos^tt ---cos = t, also cos p = cos Л -f- 21, woraus sich /г ergiebt. Кар. V. Berechnung der Vierecke. 77 Ist gegeben: a2, y1 ; ßl,6l} so ist nach (II) ß2 = 180° — «2 — ß1 — yv C D s' Figur 29. also nach der zweiten Gleichung (III): oder . sin /?2 sin ô sin a2 sm at sm ö = —;—= t, 1 1 sm (ß1 + ß2) cos ((5j — cq) — cos (dj -f- cq) = 2 t. Nach (II) ist <5j — cq = <5 -j- <2 + ft — 180 = ^ also findet man ^ ~h eq = Я aus cos Л = cos — 2 t. Vergleiche hierzu Кар. XIII. In der vierten Hauptlage ist eine Auflösung mit quadratischen Gleichungen und eine Konstruktion mit Zirkel und Lineal nachweislich unmöglich. c C D D [B А Figur 30. А Ув Figur 31. Ebene Trigonometrie. 78 § 27. Das Selmenyiereck. Im Sehnenviereck wird auch die achte Beziehung zwischen den Winkeln von einfacher Gestalt. Es seien Л, [л, v, q die den Seiten a, b, c, d gegenüberliegenden Peripheriewinkel; dann ist Л -\~ i “f" v q — 180°, <5 D = £ 7i=ö2 = ßi = 72 = •4 at=ß2=v- C M » \ чП А Ць ßt 2 \ / \ D' Figur 32. Figur 33. Zieht man durch В den Durchmesser BD' des Kreises, so ist das Dreieck BD'A bei A rechtwinklig und der Winkel bei D' entweder Л oder 180° — also, wenn r der Badius des Kreises ist: a = 2 r sin Л und ebenso b = 2rsin^; c~ 2rsinv; d = 2rsin^. Ferner ist das Dreieck В DD' bei D rechtwinklig und hat bei D' entweder den Winkel oder £-f- Л-, beide ergänzen sich zu 180°, so dass ihre Sinus gleich sind. Es ist also f = 2 r sin -f- v) = 2 r sin (Л + q) und ebenso e = 2 r sin (Л = 2 r sin (^ + (>). Kap. Y. Berechnung der Vierecke. 79 Man beweist mit Hilfe der Formeln des dritten Kapitels leicht, dass unter der Voraussetzung Л —J— [Л, —J- V + Q === 180° sin Л sin V + sin p sin q = sin (Л + sin (Л + q) ist. Multipliziert man beide Seiten mit 4 ra, so wird daraus: ac + bd = ef, der bekannte Ptolemäische Lehrsatz. Im Dreieck ABC ist e2 = a2 + b2 — 2 a b cos ß und im Dreieck ADC e2 = c2 + d2 — 2 c d cos ö. Da 6 + ß == 180°, ist cos <5 = — cos ß. Vergleicht man beide Ausdrücke für e2, so wird daraus: (aa + ba)-(c2 + da) (VH) со^2(ab + cd) • Also 1 + 003/9= 2 cos2 -f = (a+4b-+Cod7d) — Setzt man a + b + c + dj = sa, у ^a — b -f- c + dj — Sb, у \|a+b —c + dj = sc, -у ^a + с + c — dj = so folgt daraus: ß c°s2T = und analog aus 1 — cos/?: ß sm2- Sd, Sc Sd ab + cd’ sa Sb ab + cd ‘ Mithin ist: Sb Sc sTSa -T-VSd Sft • “-И »bTSc S* Sb (VIII) Ebene Trigonometrie. 80 2 cos ß 2 sin 10 to Andererseits wird - = sin ß = 2 j/s» Sb Sc s,\|~ ab + cd (IX) Es ist aber sin ß — sin ô und ^ ab sin ß der Inhalt von ABC, cd sin(5 der Inhalt von ADC, somit der Inhalt J des ganzen Vierecks: J= Sb Sc S<\| • (X) (Wie erhält man hieraus die Inhaltsformel des Dreiecks?) Wenn man noch den Wert von cosß in den Aus druck für e2 einsetzc, erhält man nach einigen leichten Umformungen ê e2 = (ac + b d) (ad + b c) ab + cd (XI) Ebenso ist ” (ac + bd) (ab + cd) ad + bc Durch Ausmultiplizieren erhält man wieder den Ptolemäischen Lehrsatz. Im Dreieck ABC folgt aus dem Sinussatz: a sin ß sin Л = e und aus (IX) und (XI) 2a J sin Л = . (XII) У (a b + c d) (ac + bd) (ad + bc) Durch Vergleich mit der Formel = 2 r sin^ ergiebt sich daraus noch _ У (a b + с d) (ac + bd) (ad + b c) . 4J (XIII) Kap. Y. Berechnung der Vierecke. 81 § 28. Das Trapez. Ein Trapez ist durch 4 unabhängige Stücke be stimmt. Durch 3 Winkel ist es der Gestalt nach ge geben. Es seien die parallelen Seiten mit AB und CD bezeichnet. Dann ist ct2 = y2, ßl — (h, 05 ~f“ ^ = 180°, ß -f- y —180°. (XIV) D 6, J, HX\ Щ ! У Figur 34. Diese Relationen ergeben sich aus einer von ihnen mit Hilfe der in § 24 aufgestellten Gleichungen (I) und (II). Die Sinusgleichungen lassen sich nicht wesentlich ver einfachen. (IY) und (Y) werden zu sin ax sin2/?, sin yx — sina«2 sin ß2 sin ö2 ) (XV) sin2 a sin ß2 sin yx = sin a, sin® ß sin <52 Wenn 3 Winkel des Trapezes gegeben sind, so kann man aus (XIY) stets einen vierten dazu finden, dass sich eine der in § 25 angegebenen Hauptlagen ergieht. Ausgenommen ist nur folgender Fall: Gegeben: <S,2, ß2; y, = 180° — «, — ß2 — <?2. Dann hat man aus der ersten der Gleichungen (XY): sin q2 i /sin a, siny, = t p sin/?2 sin<52 sin ß2 TJnd nach den Beziehungen zwischen den Winkeln selbst: a2+ßi = 180° — (eh + d2) = 2a. Hessenberg, EbeDe und sphärische Trigonometrie. 6 Ebene Trigonometrie. 82 Setzt man ß2 — a2 = 2 <S, so wird, wie in § 25 : —t tg<5=t^T+-t' Zur Lösung anderer Trapezaufgaben sind ausser den vier Dreiecken ABC, BCD, CDA und DAB noch folgende zwei zu verwenden: Man ziehe durch C die Parallelen zu DA und DB. Sie mögen AB und A' und B' schneiden. Im Dreieck A'BC ist dann D Ф B' Ä Figur 35. der Winkel bei A' gleich «, bei В gleich /9, bei C gleich y+S—180° = 180°— («+/?), die Seite BC =b, CA' = d, A'B= а — c. Im Dreieck A CB' aber ist der Winkel bei A gleich «2, bei B' gleich ßlf bei C gleich 180° — (a2 + ^i) = 180°- (/,+ <,), die Seite B'C =f, CA=e, AB'=a + c. Mit Hilfe dieser Dreiecke werden in der elemen taren Geometrie verschiedene Konstruktionsaufgaben gelöst. Zweiter Teil. Sphärische Trigonometrie. Sechstes Kapitel. Einleitendes. 41^ Sb Y § 29. Aufgabe der sphärischen Trigonometrie. Nachdem die Berechnung ebener geradliniger Figuren auf ein algebraisches Problem zurückgeführt ist, stehen wir vor der Aufgabe, ebenso für räumliche Figuren alle zu ihrer Berechnung notwendigen Glei chungen herzuleiten. Die dem Dreieck analoge ein fachste Figur ist im Baum die dreiseitige körper liche Ecke. Ihre drei Kanten bilden zu je zweien \ Figur 36. insgesamt drei Winkel, ebenso die drei Seiten (Ebenen). Die Kantenwinkel bezeichnen wir mit a, b, c, die gegen überliegenden Neigungswinkel der Seiten entsprechend mit a, ß, y. Durch drei dieser sechs Stücke sind die Sphärische Trigonometrie. 84 übrigen mitbestimmt. Denkt man sich z. B. Figur 37 ausgeschnitten, längs der Kanten OQ und OR gebrochen und so zusammengebogen, dass OP' auf OP fällt, so О b AV p\ л Ш p I R Figur 37. entsteht eine körperliche Ecke mit der Spitze 0. Die Winkel a, b, c waren gegeben; die Winkel, die die Ebenen I, II, III einschliessen, sind fest, unveränderlich ? von der Länge der Kanten OP, OQ, OR unabhängig, also lediglich Funktionen von a, b, c. Ein anschaulicheres Bild von der Lage der sechs Stücke erhält man, wenn man um die Spitze 0 der A \ r\C '/Û & Figur 38. Ecke eine Kugel legt. Dieselbe wird von den drei Seiten der Ecken in drei Bögen grösster Kreise, BC, Кар. VI. Einleitendes. 85 CA und AB geschnitten, deren Längen, in Graden, Minuten und Sekunden gemessen, a, b und c sind. Die Winkel aber, die diese Bögen einschliessen, sind a, ß und y. So erhalten wir für jede Ecke ein sphärisches Dreieck; aber umgekehrt entspricht auch jedem Drei eck auf der Kugelfläche eine Ecke mit der Spitze im Centrum der Kugel. Die Aufgabe der Berech nung der dreiseitigen Ecke ist also identisch mit der der Berechnung des sphärischen Dreiecks, dessen Seiten grösste Kreise sind. Die letztere ist eines der ältesten Probleme der Geodäsie und Astronomie. Die ebene Trigonometrie ist zu Erdmessungen nur auf kleine Entfernungen tauglich, nämlich nur solange man die Krümmung der Erdober fläche vernachlässigen kann. Grössere Dreiecke auf der Erdoberfläche müssen dagegen als sphärische behandelt werden. Die Formeln des sphärischen Dreiecks müssen, wie man zugleich erkennt, eine Verallgemeinerung derer des ebenen Dreiecks sein. Wenn man den Radius der Kugel grösser und grösser werden lässt, müssen sie in diese übergehen. Die Lehre von den Beziehungen am sphärischen Dreieck bezeichnet man als die sphärische Trigonometrie. Ihre Aufgab e ist, aus drei Stücken desselben die drei anderen durch Rechnung zu finden. Die Hilfsmittel der sphärischen Trigonometrie sind dieselben, wie die der ebenen. Die trigonometrischen Funktionen reichen auch hier zur Darstellung aller Be ziehungen aus. Während aber in der ebenen Trigono metrie nur die Sinus, Cosinus, Tangenten und Cotangenten Sphärische Trigonometrie. 86 der Winkel vorkamen, treten in der sphärischen auch die der Seiten auf. Dies ist nicht erstaunlich, ja sogar von vornherein zu vermuten, wenn man wieder an die dreiseitige Ecke denkt, in der ja alle sechs Stücke des sphärischen Dreiecks als Winkelgrössen er scheinen. Aus der Stereometrie ist bekannt, dass die Summe der drei Kantenwinkel einer konvexen körperlichen Ecke kleiner als 360° ist. Wir beschränken die Betrachtung auf solche Ecken und damit auf sphärische Dreiecke, die ganz auf einer Hälfte der Kugel liegen. Offenbar ist in solchen Dreiecken keine Seite und kein Winkel grösser als 180°. § 30. Nebendreiecke und Scheiteldreieck. Ä Ć В B'] c_ Ä Figur 39. Кар. VI. Einleitendes. 87 Denkt man sich die drei grössten Kreise, denen die Seiten des Dreiecks ABC angehören, vollständig ausgezogen, so zerlegen się die Kugeloberfläche in acht Dreiecke. Die durch A gehenden Kreise schneiden sich in einem zweiten Punkt A', die durch В und C gehenden in B', bezw. C'. Die Dreiecke A'BC, AB'C, ABC', welche mit ABC je eine Seite gemeinsam haben, heissen die „Nebendreiecke11 von ABC. Sie stim men mit ABC in je zwei Stücken überein: in der ge meinsamen Seite und deren Gegenwinkel. Die anderen vier Stücke ergänzen die gleichbenannten Stücke von ABC zu 180° (sie sind ihre Supplemente), z. B. ist im Dreieck A'BC: <BA'C = a, < A'BC = 180° — ß, <£ A'CB = 180° —, BC = a, A'B = 180° — c. A'C = 180°— b, Das Dreieck A'B'C', welches mit ABC keine Ecke und keine Seite gemeinsam hat, heisst das „Scheitel dreieck11 von ABC. Es stimmt mit ABC in allen Stücken überein. Die anderen drei Dreiecke, die mit ABC nur eine Ecke gemeinsam haben, sind die Scheitel dreiecke der Nebendreiecke und zugleich die Nebendreiecke des Scheiteldreiecks von ABC. Sie führen keine besondere Bezeichnung. Das Scheiteldreieck A'B'C' kann mit dem ursprüng lichen nicht zur Deckung gebracht werden. Schiebt man es z. B. an dem grössten Kreis A'C'AC um die Kugel herum, so fällt A' auf А, C' auf C, aber B' nicht auf B, vielmehr auf die andere Seite von AC, auf B". Wenn in der Ebene zwei Dreiecke so gelegen sind, kann man sie durch Umklappen um die ge meinsame Seite zur Deckung bringen. Aber auf der Sphärische Trigonometrie. 88 Kugel geht dies wegen ihrer Krümmung nicht. Man darf daher Scheiteldreiecke nicht kongruent nennen und A i ; В" 0 B'\ -----\c / / / Figur 40. muss neben dem Begriff der Kongruenz auch den der Symmetrie anwenden. Scheiteldreiecke sind symmetrische Figuren. § 31. Inhalte von Zwei- und Dreiecken. А Ä Figur 41. Кар. VI. Einleitendes. 89 1. Zwei grösste Kreise durch A und seinen Gegen punkt A' teilen die Kugel in vier Streifen, die man Kugelzweiecke nennt. Ein Kugelzweieck ist vollständig bestimmt, wenn man den Winkel an einer seiner Ecken kennt. Die Fläche eines Kugelzweiecks verhält sich zu der Fläche der ganzen Kugel, wie der Winkel an der Spitze des Zweiecks zu 360°. Es ist also die Fläche F des Zweiecks mit dem Winkel a, wenn R der Radius der Kugel ist: R2 7Г a° F = 360»45lR2 2. Scheiteldreiecke haben gleichen In halt. Dieser Satz ist unmittelbar durch die Anschauung gegeben. Der Beweis gestaltet sich aber sehr viel umständlicher, da man die Dreiecke nicht au feinander legen kann. A' A C СЛ в' В Figur 42. Ist zunächst А В = А C, so ist auch im Scheiteldreieck A'B' = A'C'. Wenn man AB mit A'B' zur Deckung bringt und dann das eine Dreieck um den Winkel a dreht, so kommt A mit C', C mit B' zur Deckung ; es ist also ABC kongruent A'C'B' : Ein gleichschenkligesDreieck istmit seinem Scheiteldreieck inhaltsgleich. Sphärische Trigonometrie. 90 In einem beliebigen sphärischen Dreieck konstruiere man sich nunmehr den sphärischen Mittelpunkt M des Um kreises. Sein Gegenpunkt M' ist der Mittelpunkt des Um kreises des Scheiteldreiecks. Die Dreiecke ABM, В CM ÄA Ä 'Ж r JT VB' в c Figur 43. é CAM sind gleichschenklig, also nach dem zuletzt Bewiesenen ihren Scheiteldreiecken A' B' M#, B' C' M', С' A' M' inhaltsgleich. Da aber ABC aus den ersteren ebenso (durch Addition oder Subtraktion) zusammengesetzt ist, wie A'B'C' aus den letzteren, so ist auch ABC mit A'B'C' Inhalts gleich. Die Flächengleichheit der Scheiteldreiecke ist not wendig zum Beweis einer wichtigen Formel über den Inhalt eines beliebigen sphärischen Dreiecks. Konstruiert man zu ÀBC die Nebendreiecke und bezeichnet die Inhalte von ABC, ABC, AB'C und ABC' mit F, X, Y und Z, so ist der Inhalt des Zweiecks ABAC: В,2 я; F+X ebenso F+Y F+Z В2уг 90° A R2 л 90° Г' Andererseits erfüllen die Dreiecke ABC, A'BC, AB'C und B'A'C die Halbkugel, die durch den grössten Кар. VI. 9t Einleitendes. Kreis А В А'В' begrenzt ist, und da die Fläche von A'B'C gleich der von ABC', seinem Scheiteldreieck, F + X + Y + Z = 2B2tt. ist, hat man Addiert man die drei ersten Gleichungen und sub trahiert diese davon, so bleibt: 90° {• •C ö B2 71 2 F == —180° . I Л Л В 0 В' c_ Ä Figur 44. Man bezeichnet den Ueberschuss « + /? + /—180° der Winkelsumme über 180° als den „sphärischen Excess des Dreiecks“, e. Der Inhalt eines sphärischen Dreiecks ist dem sphärischen Excess proportional: R2tv F = 180 €' Die Winkel sum me im sphärischen Drei eck wächst also mit dem Inhalt des Dreiecks und ist immer grösser als 180°. Sphärische Trigonometrie. 92 § 32. Das Polardreieck. Wenn die Ebene eines grössten Kreises und der zu einem Punkt P gehörige Durchmesser aufeinander senkrecht stehen, so nennt man den grössten Kreis die Polare des Punktes P und diesen einen Pol des grössten Kreises. Jeder grösste Kreis zerlegt die Kugel in zwei Halbkugeln. Der Mittelpunkt jeder dieser Halb kugeln ist ein Pol des grössten Kreises. Der Aequator ist die Polare des Nordpols wie des Südpols, Nord- und Südpol sind die Pole des Aequators. Wir empfehlen dieses Beispiel dem Leser zur Veran schaulichung der nachfolgenden Sätze: 1. Jeder grösste Kreis durch P steht senkrecht auf der Polare von P. i 9/ Ф dY Tm A ту JS) ж B Figur 45. 2. (Umkehrung.) Jeder auf einem grössten Kreis senkrechte Kreis geht durch den Pol desselben. Кар. VI. Einleitendes. 93 3. Jeder Punkt der Polare hat vom Pol den Ab stand 90°. 4. (Umkehrung.) Jeder Punkt, der von P um einen rechten Winkel entfernt ist, liegt auf der Polare von P. 5. Liegen A und В auf der Polare von P, so ist AB = < APB. 6. P sei der Pol von А В und von A sei nach В hin AS = 90°, von В nach A hin BT = 90° auf AB aufgetragen. Dann ist P S die Polare von A, P T die Polare von В und <£ S P T soll als Winkel der Polaren von A und В bezeichnet werden. Es ist aber nach 5 <SPT = ST. S und T liegen ausserhalb oder innerhalb von AB, je nachdem AB < oder > 90° ist. Im ersten Fall ist ST=SA+AT=SA+BT—AB = 90° +90°—AB, nn zweiten ST = S A - AT = SA —AB + ВТ = 90°—AB+ 90°. Also auf alle Fälle: <SPT + AB= 180°, d. h.: Der sphärische Abstand zweier Punkte und derWinkel ihrer Polaren ergänzen sich zu 180°. 7. Zugleich erkennt man: Die Polaren SP und TP von A und В schneiden sich im Pol P von AB. Man konstruiere jetzt die Polaren der drei Ecken des sphärischen Dreiecks. Zu diesem Zweck trage man von A aus auf b nach C hin ABa = 90°, auf c nach В bin A Ca = 90° ab. Der durch BaCa gehende grösste Kreis ist die Polare von A. Ebenso konstruiere man sich die Polare von В und C. Ba Ca schneidet sich mit Ab Cb in einem Punkt C', CaBa mit ACBC in B', ^ Sphärische Trigonometrie. 94 ß4, y' die Winkel und Bc Ac mit Cb Ab in A'. Sind Ar GtK’' В I c Bc \ А гт<к \' Ba i # Ą Figur 46. a', b', c' die Seiten des Dreiecks A'B'C', so ist nach Satz 6 dieses Paragraphen: <£ Ca C' Cb + AB = 180°, d. h. у4 = 180° — c. Da А', В', C' umgekehrt (nach 7) die Pole von a, b, c sind, ist auch А'Аь = A'AC = 90° u. s. f., mithin wieder nach 6 : <AcCBc + A'B'=180°, d. h. c/ = 180° — y\ Hiermit erhalten wir den fundamentalen Satz: Giebt es ein sphärisches Dreieck mit den Stücken a, b, c; a, ß,y, so giebt es ein zweites mit den Stücken a' = 180° — a, b' = 180° — ß, c' = 180° — ya‘ = 180° — a, ß* = 180° — b, / = 180° —c. Das Dreieck А' В' C' heisst das Polar- oder Supplementendreieck des gegebenen, auch das reciproke Dreieck. Кар. VII. Das schiefwinklige sphärische Dreieck. 95 Die Nebendreiecke des Polardreiecks sind die Polar dreiecke der Nebendreiecke des ursprünglichen. Es giebt also auch ein Dreieck mit den Winkeln a, b, 180° — c und den Seiten a, ß, 180° — y. Der sphärische Excess des Polardreiecks, hat den Wert: e' = 180° — a + 180° — b + 180° — c —180° q — 360° — (a —f— b —}— c). Man nennt diese Grösse den sphärischen De fekt des ursprünglichen Dreiecks. Siebentes Kapitel. Das schiefwinklige sphärische Dreieck. § 33. Erster Cosinussatz. О < cos a = cos b cos c + sin b sin c cos a cos b = cos c cos a + sin c sin a cos ß (I) cos c = cos a cos b + sin a sin b cos y. cos a — cos b cos c COS a sin b sin c cos b — cos c cos a cos ß sin c sin a cos c — cos a cos b (П) cos y sin a sin b Beweis: 1. Die Seiten b und c seien kleiner als 90°. Die Tangenten in A an b und c treffen dann die verlängerten Radien und O C in zwei Punkten P und Q. Im Dreieck ist der Winkel bei А gleich a, und wenn P Q mit x bezeichnet wird, folgt nach dem ebenen Cosinussatz : x2 = A P2 + A Q2 — 2 A P . A Q. cos o. Sphärische Trigonometrie. 96 ft ft \ c c \Q o< W Л Figur 47. А ci О Ct b \ IC & P \ Figur 47 a. Кар. VIL Das schiefwinklige sphärische Dreieck* 97 Im Dreieck OPQ ist der Winkel bei О gleich also ebenso: x2 = O P2 + O Q2 — 2 O P . O Q . cos a. Durch Vergleichung beider Ausdrücke für x2 er hält man: OP2 — AP2 + O Q2 — AQ2 + 2 AP . AQ . cos« — 2 O P . O Q. cos a. Die Dreiecke OAP und OAQ sind bei A recht winklig, folglich ist, wenn der Padius der Kugel wieder mit P bezeichnet wird: OP2—AP2 = OA2=P2 ; OQ2—A Q2 = OA2 = P2; OA ОА -д-р = cos AOP=cos c ; 0q cosAOQ = cosb; also OP P cosc’ OQ= P eosb* Endlich ist A P = P tg AOP = P tg c ; AQ = PtgAOQ=Ptgb. Durch Einsetzen in die letzte Gleichung und Di vision durch 2 P2 folgt : cos а 1 + tg b tg c cos а = cos b cos c* sinb Da tg b = ist, erhält man nach Multiplikation cos b mit cos b cos c die erste der Formeln (I). 2. Ist b grösser, c kleiner als 90°, so ist in dem Nebendreieck, welches c mit ABC gemeinsam hat: a' = 180° — a, b' = 180° — b, c' = c, a' — 180° — а , ß* = 180° — ß, y'=y. b' und c' sind kleiner als 90°, folglich ist nach dem zuletzt Bewiesenen: cos a' = cos b' cos c' + sin b' sin c' cos cs', Hessenberg, Ebene und sphärische Trigonometrie. 7 Sphärische Trigonometrie. 9в; nnd nach den Formeln (I, II) des § 6 : cosa' =—cosa, cosb' = — cosb , cos c'= + cos c cosa'=— cos«, sinb' = + sinb, sinc'= + sine. Setzt man diese Werte ein, so erhält man wieder (I). 3. Sind b und c beide grösser als 90°, so verfahre man ebenso mit dem Nebendreieck, welches a mit ABC gemein hat. Die Formeln (II) entstehen aus (I) durch Auflösen nach cosa, cosß, cos/. § 34. Funktionen der halben Winkel; Sinussatz. Aus (II) folgt: cos a — (cos b cos c — sin b sin c) ~ Z sin b sin c cos a — cos (b + c) sin b sin c = 2 sin * (a + b + cj sin — \| — a + b + cj 1 + cos a — 2 cos2 sin b sine sin s sin sa sin b sin c — cos a -f- (cos b cos c + sin b sin c) sin b sin c _ cos (b — c) — cos a sin b sin c __ 2 sin — ^a — b cj sin -^a + b — cj 1 — cos a = 2 sin2 to a sm b sin с sin Sb sin sc = 2 sin b sin c * Mithin ist cos — .= 1/ sin s sin sa ; sin =]/sin s() sin sc . (HI) 2 V sin bjsin c sin b sin c Кар. VII. Das schiefwinklige sphärische Dreieck. 99 НИ ем Hieraus folgt durch Division: __ n/sin s. sin sa sin sb sin sc cot (IV) sin s. sin sa ? ] sin Sb si n sc’ und durch Multiplikation: a . - У sin s sin sa sin 8ь sin sc = sin о = 2----------—i ;------------------2 sin cos sin b sin c 2 oder sin а __ 2 V sin s sin sa sin sb sin sc (V) sin a sin b sin c. sina Diese letzte Formel enthält den sphärischen er weiterten Sinussatz. Die rechte Seite ist aus den drei Seiten völlig symmetrisch gebildet, es ist also: sin а _ sin ß_sin у sin a sinh sin c? (VI) sin а : sin ß : sin у — sin а : sin b : sin c Die Sinus der Seiten verhalten sich wie die Sinus der gegenüberliegenden Winkel. (Sinussatz.) Anmerkung. Man hat für die hier auftretenden Grössen noch einige nützliche Bezeichnungen eingeführt. Man nennt den Ausdruck S = \|/sin s sin sa sin s^ sin sc den „Eckensinus“ der zu dem Dreieck gehörigen Ecke mit den Winkeln a, b, c. sin a sin b sin c Den Quotienten bezeichnet man als „Mo2b dul“ M des Dreiecks. Es ist sin а = M sin a, sin b = M sin ß, sin c =r M . sin y. Endlich sei noch •j/sin Sa sin s^ sin Sc __^ Г sin s gesetzt, so dass man hat: к . а к ß : tg — tg¥ —-----sin sa 2 sin Sb ’ sin sc" ■ Sphärische Trigonometrie. 100 г—I (N § 35« Reciproke Formeln. Indem man den ersten Cosinussatz auf das Polar dreieck anwendet, erhält man den sogenannten zweiten Cosinussatz : (VII) cos a = — cos ß cos / + sin ß sin y cos a COS a + COS ß COS/ (VIII) cos a = sin ß sin / Setzt man ferner in Analogie zu s, sa etc.: ^cs+/?+/j = 0', —a + ß + yj = 0 — а = ол, тН so wird das s' und das s'a des Polardreiecks zu: s' = - -(l80°— « + 180° — ß+1800 — yj = 270° — a, S'i = —180° + « + 180°—ß+lS0° —/)= 90° иа, womit die Formeln (III) und (IV) übergehen in a i/cos а-ß cosaT sił=F' — cos a cos <7a ; cos¥ “ I sin ß sin / ’1 sin ß sin / tsY = ]/ — COS (7 COS Ua COS Oß cos oy } (X) (V) wird zu: sin a 2 ]/— cos a cos <7a cos oß cos о„ sin а sin a sin ß sin / § 36. Rechnerische Herleitung des zweiten Cosinussatzes. Cotangentensatz. Setzt man den durch den ersten Cosinussatz gegebenen Wert für cos a in die Formel für cos b ein, so ergiebt sich nach leichter Reduktion: cos b sin c = cos c sin b cos a + sin a cos ß. (XI) Solcher Formeln giebt es 6. Sie heissen die sphäri schen С о s i n u s f o r m e 1 n. Da sin a = M sin а u. s. f., wird nach Division durch M : Кар. VII. Das schiefwinklige sphärische Dreieck. 101 cos b sin 7 == cos c sin ß cos a -f- sin a cos ß, oder, wenn man die Ecken A und C vertauscht und etwas anders ordnet: cos ß sin 7 = — cos 7 sin ß cos a + sin a cos b. (XII) Dies ist die reciproke Formel zu (XI). Nimmt man sie zusammen mit der durch V ertauschung von В und A daraus hervorgehenden und eliminiert cos a oder cos b, so erhält man den zweiten Cosinussatz. — Aus (XI) folgt, nach Division durch sin b unter Be achtung von sin a : sin b = sin а : sin ß : cot b sine = cos c cos a -f- sina cot ß oder (XIII) sin c . cot b — sin a cot ß = cos c . cos a. Dies ist der sogenannte Cotangentensatz. Es giebt 6 solcher Formeln. Durch Vertauschen der Ecken A und C erhält man die zu (XIII) reciproke Formel: sin а. cot b — sin у cot ß — cos а . cos 7. § 37. Uebergang in die ebene Trigonometrie : Ein sphärisches Dreieck, welches im Vergleich zur ganzen Kugel Oberfläche sehr klein ist, kann näherungsweise als ebenes Dreieck betrachtet werden. So ist z. B. bei kleinen geodätischen Messungen von der Krümmung der Erdober fläche nichts zu merken; die Abweichungen von der Ebene sind kleiner als die Fehler, die bei der Messung auftreten. Es müssen also die Formeln der sphärischen Trigonometrie für sehr kleine Dreiecke nähe rungsweise mit denen der ebenen Trigonometrie in Uebereinstimmung geb r ach t werden können1. In erster Annäherung kann man die zu den Seiten gehörigen Winkel gleich Null setzen, so dass die Sinus der 1 Wenn ein sphärisches Dreieck sehr klein ist, so bedeutet dies, dass seine Seiten sehr klein sind ; die Winkel können dabei beliebige Werte zwischen 0 und 180° annehmen ; ihre Summe wird um so näher an zwei Rechte herangehen, je kleiner das Dreieck ist. 102 Sphärische Trigonometrie. Seiten alle Null, die Cosinus der Seiten gleich 1 werden. Die Formeln (I) bis (VI) werden dadurch entweder identische Gleichungen, wie (I), (1 = 1) oder unbestimmt, wie (III), В (oosf=?} Aus (VII) und (XII) dagegen folgt — cos а = cos ß cos у — sin ß sin у sin а = sin ß cos у + cos ß sin y. Dies sind die Additionstheoreme, wenn man а = 180 — ß — у setzt. ln zweiter Annäherung kann sin а = ^-gesetzt werden. Denn solange а als geradlinig betrachtet werden kann, fällt es mit der Sinuslinie zusammen: Das Drei eck О В C ist bei C rechtwinklig, also ВC sin а = QB. Der Cosinus ist gleich 1 zu setzen. Hiermit erhält man aus den Formeln (III) bis (VI) die entsprechenden Gleichungen der ebenen Trigonometrie; der Cosinussatz aber bleibt identisch erfüllt, da man das Glied sin b sine cos a Null setzen muss, weil bc ausserhalb der eingehaltenen Genauig keit liegt. Setzt man jedoch in dritter An näherung cos а = 1 — 2 sin2-^Ci 0 1 a2 Figur 48. 2 R2 ’ so folgt aus dem Cosinussatz nach leichter Umformung und Multiplikation mit 2R2: •fr)- Кар. VII. Das schiefwinklige sphärische Dreieck. a2 = b2 + e — 2 b c cos a -f 103 b2c2 [2 ft“ Das letzte Glied ist im Vergleich zu den anderen un endlich klein und kann vernachlässigt werden, so dass man den ebenen Cosinussatz erhält. Durch diese Uebergänge wird zugleich die Bezeichnung der sphärischen Sätze in ein besseres Licht gerückt. Der sphärische Cosinus- und Sinussatz, sowie die sphärischen Cosinusformeln (XI) gehen nämlich in die ebenso benannten ebenen Formeln über. § 38. Auflösung der sphärischen Dreiecke. ic a Erster Fall: Gegeben a, b, c. Man berechne s, sa, Sb und sc und aus (IV) die Tangenten der halben Winkel: log tg - - = -^-\|logsinsb+bgsins0 — logsin s —log sinsaj Da a, ß, y kleiner als 180° sein sollen, sind > 2 spitze Winkel; die aus der Tabelle ent2’ nommenen Werte sind die einzig möglichen und rich tigen. Es giebt also nur eine Lösung. I logtg to P Zweiter Fall: Gegeben «, ß, y. Man berechne erst u, aaßf und aus (X) a b c da a grösser als 90° wird, setze man 2 ’ 2 5 ï 180°— = u'; es wird: = I {log cos o'+log cos - log cos <Tß — log COSOyJ. Es kommt auf dasselbe hinaus, wenn man aus a, ß, y unäcbst die Seiten a' = 180° — a, b' und c' des Polarzdreiecks ausrechnet und aus diesen die Winkel des selben. et', ß\ y\ Dann ist a = 180° — ce' also Sphärische Trigonometrie. 104 a л a' T = 9°-T’ a' tg-2"=cot- und nach (IV): CM to ; c3 log tg — — log cot- = - - jlog sin s/ + log sin s'a — log sin s'b — log sin s'cJBei diesem Verfahren hat man die bequemere Interpolation mit log sin und log tg, die positive Tafel differenzen besitzen. Da übrigens s'a das Komplement von oa ist, ist ein thatsächlicher Unterschied zwischen beiden Wegen nicht vorhanden. Zur Probe auf richtige Rechnung dient in den beiden ersten Fällen am besten der Sinussatz: log sin a — log sin a = log sin b — log sin ß — log sin c — log sin y. Dritter Fall: Gegeben ab/. Man erhält c aus dem ersten Cosinussatz, sodann a und ß nach dem Sinussatz oder besser nach den For meln (IV), da der Sinussatz zwei Winkel liefert. Vierter Fall: Gegeben c, a, ß. Man erhält у nach dem reciproken Cosinussatz, sodann a und b nach Formel (X). Für durchgehende logarithmische Rechnung im dritten und vierten Fall sind geeignete Formeln in diesem Abschnitt noch nicht entwickelt. Vergl. hierzu Kapitel IX und XIII. Fünfter Fall: Gegeben a, b, a. Im Interesse einer bequemen Determination be trachte man, wenn a und b nicht beide spitz sind, das jenige Nebendreieck, in dem die entsprechenden Stücke Кар. VII. Das schiefwinklige sphärische Dreieck. 105 spitz sind. Ist z. B. a stumpf, b spitz, so wähle man das Nebendreieck mit den Stücken a' — 180° — a, b' = b, c' = 180° — c, a‘ = 180° — a, ß' = ß, / = 180° — /, welches mit dem ursprünglichen die Ecken A und C gemein hat. Ist b stumpf, a spitz, so nehme man das jenige, welches В und C mit ABC gemein hat. Sind endlich a und b beide stumpf, so nehme man das an AB anliegende Nebendreieck mit den Seiten a' = 180° — а, b' = 180° — b, с' = c und den Winkeln «' = 180° — a, ß' = 180° — ß, y* — y. Aus den Stücken des Nebendreiecks kann man jedft’zeit wieder die des ursprünglichen berechnen. Wir können uns mithin auf den Fall be schränken, dass a und b spitz sind. Man er hält dann aus dem Sinussatz log sin ß = log sin b — log sin a -f- log sin «. Da zu jedem Sinus zwei Winkel gehören, ergeben sich zwei Lösungen für ß, die aus der Tafel gefundene und deren Nebenwinkel. Die Determination ist unter der Voraussetzung, dass a und b spitz sind, genau dieselbe, wie in der ebenen Geometrie: 1. Ist a > b, so ist nur der in der Tafel aufge schlagene Winkel brauchbar, dieser aber sicher. 2. Ist a = b, so darf a nicht stumpf sein, und es ist ß —a. 3. Ist a b, so muss a spitz sein. Für sin/? kann ein Wert grösser als 1, für logsin/? also eine positive 106 Sphärische Trigonometrie. Grösse herauskommen. Dann existiert kein (reales) Dreieck. Ist dagegen log sin ß negativ, so sind beide Werte für ß brauchbar. Ist log sin ß = 0, so ist ß = 90° und es giebt nur ein Dreieck. Aus a, b, a, ß kann man c und y mittelst der Co sinusformeln berechnen. Eliminiert man z. B. sine aus den beiden Formeln cos a sin b = sin a cos b cos y 4- sin c cos a, cos b sin a = sin b cos a cos y + sin c cos /?, so folgt: cos ß tg b — cos a tg a cos y cos ß tg a — cos a tg b ’ und aus dem Polardreieck: cos htgß — cos a tg a cosc cos b tg a — cos а tg ß * Zur logarithmischen Rechnung geeignetere Formeln werden in Kapitel IX angegeben werden. Sechster Fall: Gegeben а, а, ß. Man kennt im Polardreieck a', a'. b' und kann da her nach dem fünften Fall ß\ y* und c' berechnen. Dann ist b = 180° — ß' \ c = 180° —/ ; у — 180° — c'. Man kann auch in Analogie zum fünften Fall zunächst für eine bequeme Determination a und ß als spitz annehmen. Sollten sie es nicht sein, so betrachte man die Nebendreiecke. In einem von ihnen sind die a und ß entsprechenden Winkel beide spitz. b ergiebt sich aus dem Sinussatz, und zwar ist log sin b = log sin ß — log sin a -f- log sin a. Von den beiden Werten für b ist nur der spitze zu ge brauchen, wenn a>\|î. Ist a ^ ß, so muss a spitz sein. Ergiebt sich dann für log sin b ein negativer Wert, so sind Кар. VIII. Das rechtwinklige sphärische Dreieck. 107 beide Werte von b brauchbar. Wird sin b = 1, so ist b = 90° und nur eine Lösung vorhanden. Ist endlich log sin b > 0, so giebt es kein (reelles) Dreieck, у und c erhält man, nach dem b gefunden, wie im fünften Fall. Achtes Kapitel. Das rechtwinklige sphärische Dreieck. §39. Auflösung der rechtwinkligen sphärischen Dreiecke. Ist in einem sphärischen Dreieck у — 90°, so nennt man c die Hypotenuse, a, b die Katheten. Ist etwa noch а = 90°? so sind auch c und a Hechte nach Satz 3 des § 32, und ß ist gleich b. Yon diesem einfaclien Fall kann im folgenden abgesehen werden. Zur Berechnung der sphärischen rechtwinkligen Dreiecke dient folgendes Formelsystem: Erster Fall: Gegeben c, a. tga cosc ; cosb = I J sin c\| 5 008'“tgc cos a Zweiter Fall: Gegeben c, a. {sin а = sin]c sin csj ; cot ß = cos c tg cs ; tg b = tg c cos cs. D r^i t ,t e r F а 11 : Gegeben a, b. tga tg b tg os ; cos c = cos a cos b. sinb ; tg/? = sina Vierter Fall: Gegeben a, cs. sina tga coses sine == sinb ; sin/? = sines’ tg CS cos a Fünfter Fall: Gegeben a, ß. tga tg c = , tg b = tg ß sin a ; cos a = cos a sin ß. 108 Sphärische Trigonometrie. Sechster Fall: Gegeben «, ß. cos a ; cos ib = G0Sß ; cos c = cot a cot /?. cos а sin/? Die eingeklammerten Formeln des ersten und zweiten Falles bestimmen a bezw. a nicht eindeutig. Es gilt aber der Satz: Kathete und Gegenwinkel sind gleichzeitig spitz oder stumpf. Es ist nämlich (fünfter Fall) cos a = cos а . sin ß und da sin ß stets positiv ist, haben cos « und cos а stets das gleiche Vorzeichen. Die vorstehenden Formeln erhält man sämtlich aus den Fundamentalgleichungen des allgemeinen Dreiecks durch die Specialisierung siny = l, cos у = 0. Aus der dritten Formel des Cosinussatzes wird cos c = cos a cosb, aus dem Sinussatz: sina sinb sin/? sin« sin c’ sine und aus den Cosinusformeln: tgb cos а = cos ß = tga tgc tgc Aus diesen fünf Gleichungen kann man alle an deren herleiten. Es wird z. B. sina 1 sina 1 tg a tg а =--- — •----- --- ------- ------------- -- ------- ; tgf = tgb tg b cos c tg b cos a cos b sin b sina Hiermit sind schon alle Beziehungen zwischen zwei Seiten und einem Winkel, sowie zwischen drei Seiten erschöpft. Man erhält noch sinb cosc cos а Snc * cosb ~ Sm ^ C0S a ’ c°s /? = sin а cos b, Кар. VIII. Das rechtwinklige sphärische Dreieck. 109 und durch Multiplikation: cot a cot ß = cos a cos b = cos c. In allen Fällen, mit Ausnahme des vierten, ist das rechtwinklige Dreieck eindeutig bestimmt. Denn ausser in den eingeklammerten Formeln des ersten und zweiten Falles treten nur die Cosinus, Tangenten und Cotangenten der gesuchten Stücke auf, und diese Funk tionen bestimmen ihre Winkel zwischen 0° und 180° eindeutig ; sie sind für spitze Winkel positiv, für stumpfe negativ. Dass von den zu den eingeklammerten Sinus gehörigen Werten immer nur einer brauchbar ist, war schon gezeigt. Im vierten Falle giebt es zwei Dreiecke, da zu jedem der Sinus zwei Werte gehören. In der That hat eines der Nebendreiecke — dasjenige, welches an В C anstösst — die Seite a und a und einen rechten Winkel, ge nügt also auch den gestellten Anforderungen. Die beiden Lösungen sind aber nur brauchbar, wenn a und a gleich zeitig spitz oder stumpf sind, sonst sind beide unbrauch bar. — Die Zusammengehörigkeit der Werte für c, b und ß ergiebt sich folgendermassen : Zu dem spitzen Wert von b gehört der spitze von ß, zu dem stumpfen Wert von b der stumpfe von ß. Infolge der Gleichung cosc = cos a cosb ist cosc negativ, also c stumpf, wenn von den Stücken a und b eins spitz und eins stumpf ist; dagegen ist cosc positiv, also c spitz, wenn a und b gleichzeitig spitz oder stumpf sind. Hiermit sind die Bedingungen für die Lösbarkeit der Aufgaben noch nicht erschöpft. Im ersten, vierten und sechsten Fall müssen noch die Werte, die man für 110 Sphärische Trigonometrie. die Funktionen der gesuchten Stücke erhält, echte Brüche sein. Im ersten Fall erhält man die Bedingungen sin a < sin c, positiver Wert von tg a <C positiver Wert von tg c, я COSC. я cos а > Es genügt, wenn eine dieser Bedingungen erfüllt ist. Die anderen stimmen dann auch. Man braucht auch nicht die Funktionen der Winkel selbst nachzuschlagen, da man von der Grösse der Winkel auf die der Sinus schliessen kann: Yon zwei Winkeln hat der den grösseren Sinus, der näher an 90° liegt (Fig. 5). Es giebt also folgende 4 Möglichkeiten: c spitz c stumpf Es muss c > a sein Es muss a+c<C180° sein a stumpf Es muss a+c > 180° sein Es muss c < a sein a spitz Im vierten Fall sind die Bedingungen dieselben, wie in dem eben besprochenen, wenn man nur a statt c schreibt: Da aber a und a stets gleichzeitig spitz oder stumpf sind, folgt einfacher: a und a spitz : а > a, a und a stumpf : а < а. Im sechsten Fall ist zur Lösbarkeit notwendig : positiver Wert von cos a <C sin/? cos ß <C sin а r » « cota.cot^< 1. Auch hier zieht das Erfülltsein einer Bedingung das der anderen nach sich. Für die Winkel gilt : 1. ß spitz : 90° — ß < a < 90° + ß; 2. ß stumpf : ß — 90° < a < 270° — ß. vKap. VIII. Das rechtwinklige sphärische Dreieck. 111 Im zweiten, dritten und fünften Fall dürfen die gegebenen Stücke zwischen 0° und 180° beliebige Werte haben. § 40. Die Napier’sche Regel. Die Gleichungen des rechtwinkligen Dreiecks lassen sich in eine einfache von Napier gegebene Regel zu sammenfassen. Es seien a* und b* die Komplemente der Katheten a und b. Dann folgen am Dreieck die Stücke in dieser Reihenfolge aufeinander : ъ, a, C, ß, a, b* . . . Ton diesen fünf ist der Cosinus eines jeden gleich dem Produkt aus den Cotangenten der beiden ihm be nachbarten und auch gleich dem der Sinus der beiden von ihm getrennten Stücke. Diese Regel enthält alle Beziehungen, die zwischen irgend drei Stücken bestehen. Denn drei Stücke liegen entweder aneinander (wie a, c, ß), oder es liegen zwei aneinander, das dritte von ihnen getrennt (wie а, с, а). Man überzeuge sich an den Formeln des vorigen Para graphen, dass die Regel in jedem Einzelfalle zutrifft. Die Napier’sche Regel handelt nur von der Aufeinander folge der Stücke, nicht aber davon, welches der Stücke Hypotenuse, welches Kathete, welches Winkel ist; anderer seits enthält sie alle notwendigen Bedingungen dafür, dass die fünf Stücke einem rechtwinkligen Dreiecke angehören. Daraus folgt, dass man die Bedeutung der fünf Stücke unter Beibehaltung der Reihenfolge abändern darf, und dass die so vertauschten Stücke wieder ein rechtwinkliges Dreieck bilden. Z. B. giebt es ein rechtwinkliges Dreieck mit den Stücken а = 73° 53' 38", b = 39° 57' 4", c = 77° 43' 18" а = 79° 29' 45", ß = 4Vbi6". Sphärische Trigonometrie. 112 Es ist also b = c= a= a* = ß= 50° 2'56" 79° 29'45" 77° 43'18" 41° 5' 6" 16° 6'22" und es giebt noch vier Dreiecke mit den Stücken 79°29'45" 77°43'18" 41° 5' 6" 16" 6'22"j50° 2'56" 77°43'18" 41° 5' 6" 16° 6'22" 50° 2'56" 79°29'45" 41° 5' 6" 16° 6'22" 50° 2'56" 79° 29'45" 77° 43' 18" 16° 6' 22" 50° 2'56"79° 29' 45" 77°43'18" 41° 5' 6" Dies ist auch geometrisch leicht einzusehen. Trägt mjtn von C undB auf CA und CB und (event.) ihren "Verlängerungen C Л 'A C, A, Figur 49. über A und В hinaus CAi =: 90°, BCj = 901 ab, so ist in dem bei C rechtwinkligen Dreieck AjCB die Kathete At C ein Quadrant (90°), also der Winkel bei В ein rechter und Aj В ein Quadrant. In dem Dreieck Cj At В sind also Cj В und Аг В Quadranten, mithin die Winkel bei At und Сг rechte. Nennt man den Punkt A als zu dem Dreieck Ct Aj А gehörig noch nebenbei Bn so ist in dem bei Сг rechtwink ligen Dreieck Aj Bt Cj : Кар. VIII. Das rechtwinklige sphärische Dreieck. 113 = 90° — c, d. h. ax* = c, aL = Ci Bi Cj — b, = 90° — b, с, = В, A, b, = 0,^ = <^0 = 90°-/?, * bj* = ß9’ a,= 90° —<CA1B = 90° — а, „ ^ = a, А = «> » ßt = «• Setzt man an das Dreieck At BL Cx in der gleichen Weise durch Verlängerung von Ct A1 und Bj Ax bis auf 90° ein Dreieck Aa B2 C2 an, so ist in diesem analog a2 = Cj — b, «2 = at = c c2 = bx* = ß, ß2 = a1 — a* Ь2=Л =ct. * * * ■ ^ i: e «? ■? cP 00 c3 M «О . rO cf «P cT 5* * * * ю да я ö а О üi a* = ß= О 2* c= н а— С в Ъ* = ÿ >^ wЙ P Oи Man erhält im ganzen folgende Uebersicht: с в. ß С; с, в; >4 Q В3 'Г, Figur 50. Aus den beiden Formeln cos c — cotg a cotg ß — sin a* sin b* Hessenberg, Ebene und sphärische Trigonometrie. 8 114 Sphärische Trigonometrie. ergeben sich durch Anwendung auf die fünf Dreiecke alle anderen Relationen zwischen den Stücken с, a, ß, a, b. Das fünfte Dreieck stimmt mit dem ersten in allen Stücken wieder überein. Bei der Konstruktion erhält man auch der Lage nach das alte Dreieck zurück, wie leicht zu beweisen ist. Die Figur der fünf Napier’scben Dreiecke besitzt interessante Eigenschaften. In dem Fünfeck C, Ct, C2, die Seiten, in dem Fünfeck A, A1} A2, A3, A4 die Diagonalen alle gleich 90°. Der Leser versuche zur Uebung diese Sätze zu beweisen. g 41. Geometrischer Beweis der Fundamentalformeln des rechtwinkligen Dreiecks. Zieht man wie in § 33 an b und c die Tangenten und nimmt zunächst an, dass b und c spitz sind, so schneiden die Tangenten die verlängerten Badien von C und В in Q und P. Die Ebene des Dreiecks APQ steht aber senkrecht auf OA, also auch auf der Ebene PS b \ 0 c Ä c\o C a \ а Q о к p Figur 51. Figur 51 a. OAQ. Da das Dreieck ABC bei C rechtwinklig ist, steht auch die Ebene OPQ auf OAQ senkrecht, dem- Кар. VIII. Das rechtwinklige sphärische Dreieck. 115 nach auch die Schnittlinie PQ von APQ und OPQ. Daher sind die Winkel OQP und AQP rechte. In den beiden bei A rechtwinkligen Dreiecken О AP und OAQ ist aber (wie in § 33) OQ = В : cos b OP = В : cosc, AQ = В . tgb. AP= В. tgc, 34 In dem bei Q rechtwinkligen Dreieck OPQ hat man daher: O Q __ cos c cosPOQ = cos а OP cosb В tg а ferner PQ = OQ tga = cosb ’ В sina = OP sina cos c In dem bei Q rechtwinkligen Dreieck APQ ist P AQ = a, also A Q_tg b cos a tg с sina sina sin a AP cos c tg c sin c ’ PQ _ tga _ tg a tga AQ“~cosbtgb sinb Anmerkung. Da irgend ein zu APQ paralleler Schnitt A'P'Q' durch die Ecke an den Seitenverhältnissen und Winkeln des Tetraeders O A P Q nichts ändert, hätte man auch, wie es in vielen Lehrbüchern geschieht, P' irgend wo auf О В annehmen, auf O A und О C die Lote P' A' und P'Q' fällen und an dieser Figur dieselben Schlüsse ziehen können. Vielfach wird P' nach В gelegt. — Die Verallge meinerung der Formeln auf Dreiecke, in denen b und c nicht spitz sind, ergiebt sich leicht durch Betrachtung der Nebendreiecke. У С 116 Sphärische Trigonometrie. § 42. Das Quadrantendreieck. Wenn in einem Dreieck eine Seite ein Rechter ist, so nennt man es ein Quadrantendreieck oder recht seitiges Dreieck. DasPolardreieck eines Qua drantendreiecks ist rechtwinklig. Man kann also die Auflösung des Quadrantendreiecks auf die des recht winkligen zurückführen. Ist c diejenige Seite, welche gleich 90e ist, so lautet die Napier’sehe Regel des Quadrantendreiecks : Sind a* und/?* die um 180° vergrösserten Komplemente von a und ß. so ist der Cosinus eines jeden der Stücke Ф ß, a, y, b, a, ß* . . . entgegengesetzt gleich dem Produkt aus den Cotangenten der anliegenden und auch dem aus den Sinus der von ihm getrennt liegenden Stücke. Durch diese Regel gehören wieder je 5 Dreiecke zu sammen, z. B. A Aj A2 ; A1 A2 A3 ; A2 A3 Aé ; A3 A4 А ; Aé A Aj in Fig. 50. § 43. Das schiefwinklige Dreieck im Zusammenhang mit dem rechtwinkligen. 1. Fällt man das sphärische Lot hc = CHc von C auf a in dem schiefwinkligen Dreieck ABC, so ist in den rechtwinkligen Dreiecken ACHc und BCHc: sin hc = sin a sin ß — sin b sin a, oder sin a : sin ß = sin а : sin b. Dies ist der Sinussatz. 2. Ferner ist für AHc = bc, BHc = ac cos hc = cosb cosbe cos а cosac Кар. VIII. Das rechtwinklige sphärische Dreieck. 117 Da ac + bc = c, folgt weiter cos b cos (c — bc) cos а = cos bc = cos b cos c + cos b sin c tg bc. C ь * А-£ 7. /з Figur 52. » tgbc Da cos« -—7-, wird tg bc = tg b cos a, also tg b cos а = cos b cos c -f“ sin b sin c cos «. Dies ist der erste Cosinussatz. 3. Setzt man <£ACHc = 71? <BCHc = 72, so ist cos ß — cos ho sin cos а = coshc sin/j, also cos а_sin yr __sin (y — y2) = sin 7 cot 72 — cos 7. cosß sin/2 sin72 Da cot /2 cot ß = cos а, wird mithin cos а — sin / sin ß cos а — cos ß cos 7. Dies ist der zweite Cosinussatz. Wenn etwa a oder ß stumpf ist und hc ausserhalb des Dreiecks C b at hc A-&- 7Г Hc Figur 53. liegt, ist der Beweis leicht entsprechend zu modifizieren. Sphärische Trigonometrie. 118 Neuntes Kapitel. Weiteres Formelmaterial für das sphärische Dreieck. § 44. Die Gauss’schen Gleichungen und die Napier’schen Analogien. Nach Formel (III) des Kapitel VII ist a sins sin sa sins sin Sb cos — cos —ß = i I// sin b sin c sin a sin c 2 -1sin sa sin Sb sin s sin a sin b * sin c ' Das Vorzeichen der Wurzel ist positiv. Denn die halben Winkel sind spitz, also ihre Cosinus und damit die linke Seite positiv ; s, sa, Sb, sc, a, b, c sind kleiner als 180°, also ihre Sinus ebenfalls positiv. Nach Formel (III) ist aber die Wurzelgrösse gleich sin —, mithin & ß . y sms a cos — cos ^ = sm ~ • ——. 2 2 2 sin c Ebenso findet man: sin s sin sc sin sa cos 2sinT = ]/ sin a sin b sin c y sin sa = COS • . 2 sine sin sa sin Sb sinsc sin ~ sin y = y sin a sin b sin c y sin sc = sin 2 sine ‘ Hiernach wird: cos a+ß 2 a ß Ct . ß = c°s — cos -g- — sln “g- Sln Y —f sin s — sin sc sine (i) (II) (HI) Кар. IX. Weiteres Formelmaterial für das sphär. Dreieck. 119 - : sin toi ft cos • : sin to ft oder: > a— b = cos —2~ • cos toi ft toi ft Beachtet man, dass s — sc = c, s + sc = a + b ist, so folgt weiter: a+b 2 sin cos 2 a+ .7 cos 2' = SmT' c 2 sin cos — = cos a ^ ^ : cos cos a 2 ^ : sin I = sin . a+ß sm-g- : cos sin : cos ^ = sin a-g-— • sin ’ Genau analog erhält man: (IV) 9 • toN> to+ Dies sind die sogenannten Gauss’schen Glei chungen. Durch Division der ersten und dritten, zweiten und vierten erhält man , cc -j- ß a-b a+ b tg —y~ s cotg = cos —g— : cos 2 ’ (V) . а—ß . a —b . a + b te-2 : cotg = sin —g— s Sln —2~> und durch Division der ersten und zweiten, dritten und vierten : a—ß , a+b . c a+ß tg g 2 2 “ cos —2~ 2 cos 2~> (VI) tg + - : tg- 2 = sin “-g-- : sin ct+^ Die Formeln (V) und (YI) sind die Napiersehen Analogien. Bemerkenswert ist, dass man aus diesen Formeln durch TJebergang auf die Nebendreiecke oder das Polardreieck keine neuen Relationen erhält. Sphärische Trigonometrie. 120 § 45. Formeln für s, sa, a, oa, p, pa, r, ra. Setzt man in (I) sin c — M sin y, so wird daraus 1 a 7 8•ins. . У ß T. cosCOS-cos = 8m~2 cos 2 2 2 M sin cos-~r A oder sin s = 2I cos -Ц- cos cos У_ 2 • Ebenso erhält mau aus (II) und (III) : A (VII) sin sa = 2 M cos y sin sin . (VII) Diese Formel entspricht den Gleichungen (VI) in Kapitel 1У. Durch Uebergang auf das Polardreieck erhält man für M' = 1 : M : a b c — cos о = 2 M'. sin — sin — sin —, COS (7a = 2 M'. sin cos А c b cos 2 (УШ) c E 0 W А F Figur 54. И Aehnliche Beziehungen entstehen durch Betrachtung des Inkreises. Berührt er die Seiten a, b, c in D? E, F, so ist AE = AF == У Х + У = с> B F — B I) = i у + z = а, CD = С E = У z + X = b, ^ N also X — Sa > y = Sb, z = sc. Кар. IX. Weiteres FormelmatenaJ für das sphär. Dreieck. 121 In dem rechtwinkligen Dreieck AO F ist tg ^ = tg* e : sin sa, tge = sin Sa.tg tgCa ЬЭ 8 also nach (IV), Кар. VII: %e = ]/sin Sa sin Sb sin sc к (§ 34, Anm.). (IX) sin s Ganz analog wird für die Radien (>a, (>ь? der Ankreise, die die Inkreise der Nebendreiecke sind: /sin s sin sb sin sc % e» = ]/ (X) sin sa Mit (VII) wird noch: sin ~y sin = 2M sin ct ß у = 2 M sin <j- cos™ cos—. (XI) А i 4 M r 1 öfc N Figur 55. Istr der Radius des Umkreises, M sein Centrum, und liegt dies im Innern des Dreiecks, so ist <MAB = MBA — f, 1+4=У <MBC = <MCB =\|, n+ £ — « <MCA = <MAC = n, S+i=ß also £ °a, V = % Ç «Г- Sphärische Trigonometrie. 122 I n 2 P Liegt der Mittelpunkt ausserhalb des Dreiecks, so erhält man für einen der Winkel J, rj, £ den entgegen gesetzten Wert, —c'a, —0ß oder —oy. Fällt man von M das Lot MN auf BC, so ist im Dreieck BMN: cos <£ NBM = tg - - : tgr, d. h. cotg r = COtg Y • C0S a0L. und nach (X), Кар. VII: cotg. r = 1/cos °ol cos gß cos qT _ (XII) — cos о Nach (VIII) dieses Kapitels erhält mal noch а а c b cotg r = cotg—- . cos oa = 2 M' cos - — cos — cos —, V 2 2 2 2 l(XIII) a b . c = 2 M' cos — sin —sin cotgra ra, fb) *c sind die Radien der Umkreise der Neben dreiecke. Man erhält sie, indem man je zwei Seiten mit ihren Supplementen vertauscht. Die Beziehung zwischen In- und Umkreisen wird erläutert durch folgenden Satz, dessen Beweis keine Schwierigkeiten bietet : Die Polare jedes Punktes des In- oder Umkreises berührt den Um- oder Inkreis des Polardreiecks und umgekehrt. § 46. Die L’Huilier’schen Formeln. Schreibt man die dritte Gaussische Formel folgendermassen : -4(«+л cos 4"(a~ ь) 1 sin y (180° — y) so wird 1 cos Tc Кар. IX. Weiteres Formelmaterial für das sphär. Dreieck. sin (a + ß) — sin -gf (180° — y) sin ~2~ (ce -f- ß) -J- sin 123 cos-^j- (a — b) — cos-g- c 1 cos-g- (a — b) -j- cos Tc (180° — y) und nach § 20, (XVI) und (XVI,t): 1 tg ^(a ~\r ß 7 180°) = tg g- sa tg y Sb. tgx(“ + ^-y + 180°) Nun ist a + ß + У —180° = s der sphärische Exzess und « + ß — 7 + 180° = 360° - (2 7 — s\ und die letzte Formel geht über in tg \ e tg -j- (2 y — e) = tg ~2 sa tg -y Sb. Ebenso wird aus der Gaussischen Gleichung cos "2"(a-ß) cos^-(a + b) sin y y cos Tc 1 abgeleitet : tg -j- e • cotg -j- (2 y — e) = tg y s tg -g- sc. Durch Multiplication und Di vision erhält man aus diesen beiden Formeln : tgT = ]/tg Ts tgTSa tgTSb tsTSc (XIV) tg-\|>(2y — e) = \|/co%i-scotg~sc tgi-s» tg-i-Sfc Dies sind die L’Huilierschen Gleichungen. Geht man zum Polardreieck über, so ist für e 360 — (a + b + c) = d zu setzen ; 2 y — s geht über in a + b — c = 2 sc ; sin (270* — o), sa in (90° — c'a). Dadurch erhält man: Sphärische Trigonometrie. 124 d to 4— сто " ста Hs? +- <Й I fo ю ю b j<M + Sc If (XV) tg7= (XV) teT = j/_ cot^46° + -\|) cot(46» - -^)tg(45»-^)tg(45»—\|-) Man kann in diese Formeln sehr viel mehr Symmetrie bringen, e ist der sogenannte sphärische Excess, d der sphärische Defekt des Dreiecks. Das an В C anliegende Nebendreieck hat den sphärischen Excess cc 180° -ß+ 180° — 7 + a -180° = 2 a — г, = te f = t, tgr= ta> tg-^ = tbj tg (lc — 4 so erhält man statt der Formeln (XIV), (XV) : r = 1/ !i \ t ■•“У-t tbtc «. 5 ; Г te т = T>te ~r = “> te ~ = p, tg 4 und den sphärischen Defekt da = - 180° + b — 180° + с — а + 360° = 2 sa. Ferner ist s — 2 а — 180° % = 180° -2 d = 360 — 2 s. Setzt man noch t = jA« rß rr tu =F r rß Гу .. (XVI) Кар. IX. Weiteres Formelmaterial für das sphär. Dreieck. 125 Diese Formeln können auch zur Berechnung des Drei ecks aus den Seiten oder Winkeln dienen. Denn es ist d + da e + fa а— ; а = 180° 2 2 § 47. Logarithmische Rechnung zum Auflösen der Dreiecke. In § 38 konnten nur zur Berechnung des Dreiecks aus 3 Seiten oder 3 Winkeln durchgehende logarithmische Rechnungen angegeben werden. Die Gauss’schen Formeln und Napier’schen Analogien gestatten, auch die Fälle 3 bis 6 logarithmisch zu erledigen. Dritter Fall: Gegeben a, b, y. а—ß Nach (V) dieses Kapitels erhält man und 2 daraus a, ß. c findet man nach dem Sinussatz oder nach irgend einer der Gauss’schen Formeln oder nach (VI) dieses Kapitels. Vierter Fall: Gegeben «, ßy c. —b a+b Man erhält — —— nach (VI) dieses Kapitels 2 ’ danach y aus dem Sinussatz, den Gauss’schen Formeln oder (V) dieses Kapitels. Fünfter Fall: Gegeben a, b, «. Man findet ß nach der ausführlichen Anleitung in § 38. c ergiebt sich aus einer der Analogien (VI), y aus einer der beiden anderen oder unter Verwendung von c aus dem Sinussatz oder einer Gauss’schen Formel. Sechster Fall. Gegeben cc, ßy a. Nachdem b bestimmt ist, verfährt man genau wie im fünften Fall. Dritter Teil. Berechnung und algebraische Anwendung der trigonometrischen Punktionen. Zehntes Kapitel. Elementare Berechnung der trigonometrischen Funktionen. § 48. Die regulären Polygone. Im regulären n-Eck ist der einer Seite gegenüber360° liegende Winkel —-. Fällt man auf eine Seite AB n des n-Ecks das Lot OP vom Mittelpunkt aus, so ist 180° AP 1 Sn 1 360° 180° und sin < AOP = n n ОA 2 r 2 n wenn sn die Seite des dem Kreise vom Radius r ein beschriebenen regulären n-Ecks ist. ü $rt Figur 56. 1. Im Sechseck ist s6 = r, also sin 30°= ~ = 0,50000. Кар. X. Elementare Berechnung der trig. Funktionen. 127 Nach der Formel cos2 a -f- sin2 a = 1 ergiebt sich cos 30° =^ = 0,86603. 0 r, r/z Г/2 Figur 57. Hieraus erhält man nach § 20 die Funktionen von 15° und aus den Additionstheoremen die aller Viel fachen von 15°. 2. Im regulären Zehneck ist AOB = 36°, folglich ОБА = OAB = 72°. Halbiert man OB А und verlängert die Winkelhalbierende bis zum Schnitt mit OA in Q, so sind die Dreiecke OBQ und BQ А gleichschenklig. Es wird nämlich 0 3t / (36° 36° \Q J<r\' 72° В Figur 58. <QAB = <AQB = 72° und also <OBQ = <BOQ = 36°, si0 — AB = BQ =; QP. 128 Berechnung der trigonometrischen Funktionen. Da Л ABQ^BOA, wird AB BO , d. h. AQ BA r —S10 oder r sio’ sio2 + rs.o + r’ = 0’ r 1/5? S„ = -2±\|/T Da nur das obere Wurzelzeichen brauchbar ist, hat man У5-1 S10 2 r und sin 18° łA—= 0,30902. Hieraus erhält man cos 18°; iflicli dem Additions theorem ferner die Funktionen von 18°— 15° = 3° und aller Vielfachen von 3°. Sie sind am Schlüsse des Buches in einer Tabelle zusammengestellt. Die Funktionen von 1° und seiner Vielfachen, die nicht durch 3 teilbar sind, lassen sich nicht durch rationale Zahlen und Quadratwurzeln aus solchen darstellen. Dagegen kann man die Seite des Siebzehnecks durch quadratische Glei360° chungen finden. Für cos ergiebt sich folgender Aus druck : (1/17-1) +^2.17-2 \|Ö7 — -i- f 17 + 3 fW— V2.17 — 2 )/Ï7 - 2 У 2.17 + 2 УТ7 (Gauss, Disq. ar., 365.) § 49. Funktionen sehr kleiner Winkel. 1. Wenn man die Funktionen von 1° auch nicht durch quadratische Gleichungen finden kann, so kann man sie doch näherungsweise mit jederbeliebigen Genauigkeit berechnen. Aus dem Sinus und Co- Кар. X. Elementare Berechnung der trig. Funktionen. 4 ’ Betrachten wir jetzt die Reihe: + CO 3 2 09 u. s. w. 3° « 'S 3° sinus von 3° erhält man nach § 20 die von —, 129 L +l 00 16 ^ 32 04 Sie ist eine geometrische Reihe mit dem Anfangs3 1 glied-^-und dem Quotienten —— . Die Summe ihrer m ersten Glieder ist nach der aus der Algebra be kannten Formel r-l KN th CO KN i-(—)' n -КГt ^------ j positiv, H <N Ist m eine gerade Zahl, so ist also die Summe kleiner als 1 ; ist m ungerade, so ist sie grösser als 1. Mit wachsendem m nähert sie sich der 1 beliebig. Da wir die Funktionen von —, — u. s. w. berechnen können, können wir auch die von 1 — КГ fur beliebiges m berechnen. Mit wachsendem m werden sich die ersten Decimalstellen nicht mehr ändern ; die Aenderung wird sich immer später und später bemerk bar machen, so dass wir die Funktionen von 1° auf beliebig viele Decimalen ermitteln können. Wir er halten z. B. и MKl" 0,01744445 ... sin 1— <N sin гч ii = 0,01745638 ... Hessenberg, Ebene und sphärische Trigonometrie. 9 130 Berechnung der trigonometrischen Funktionen. to so dass sin 1° sicher mit 0,0174 beginnt. Durch Inter polation zwischen beiden Grenzen erhält man : 11 Zuwachs auf 3. тЧ , 'N also auf 11 1193 Einheiten der letzten Stelle, 398 0,01745638 398 sin 1° = 0,01745240. Dies Resultat ist auf 7 Stellen genau. 2. Ein einfacheres Verfahren kann man für sêhr kleine Winkel anwenden, bei denen der Sinus näherungs weise mit dem Arcus übereinstimmt. Es ist für solche sin a < arc a. a Demnach auch sin — arc7T 1 cos a — 1 — 2 sin — > 1 arc a (arc a)2 2 Sodann ist tg a > area, also sin a > cos a . arc a (arc a)3 > area 2 ' 71 Es ist z. В. arcl° = 0,0174533 . .. l«0 (arc l0)8 = 0,0000026583 .. • ? 2 also liegt sin 1° zwischen 0,017453 . .. und 0,017450 . .. Кар. X. Elementare Berechnung der trig. Funktionen. 131 Ferner ist arc 1' = 0,00029089. (arc 10: beginnt mit 11 Nullen, also ist vorstehen 2 der Wert in allen Stellen mit sinl' übereinstimmend. 3. Von den Funktionen sehr kleiner Winkel aus kann man diejenigen grösserer Winkel mittelst der Additionstheoreme berechnen, z. B. aus den von 3 zu 3° berechneten mit Hilfe des Sinus und Cosinus von 1° die Funktionen aller Vielfachen von 1°. Praktische Bedeutung hat diese Methode nicht mehr. Die höhere Mathematik giebt uns in den Reihenentwicklungen Hilfs mittel zur sofortigen Berechnung der Logarithmen der trigonometrischen Funktionen und der Funktionen selbst, die von hervorragender Einfachheit sind. Elftes Kapitel. Der Moivre’sche Salz § 50. Begriff des Vektors. Es kommt in der Geometrie und Physik häufig vor, dass man ausser der Länge einer Strecke auch ihre Richtung in Betracht ziehen muss. Man hat für den Inbegriff einer Strecke und ihrer Richtung die Be zeichnung „Vektor“ eingeführt. Zur Angabe eines Vektors gehören also mehrere Grössen. Erstens seine Länge. Diese ist immer eine absolute (positive) Zahl. Zweitens die nötigen Bestimmungsstücke für seine Richtung. Die beiden Enden des Vektors müssen als An fangs- und Endpunkt unterschieden werden. Die 132 Berechnung der trigonometrischen Funktionen. Richtung des Vektors ist die vom Anfangs- zum End punkt. Ein und dieselbe Strecke kann zwei verschiedene Vektoren darstellen, die gleich lang und einander ent gegengesetzt gerichtet sind. Man nennt solche Vektoren „entgegengesetzt gleich“. Wenn eine Strecke die Länge Null hat, ist ihre Richtung unbestimmt. Man bezeichnet daher alle Vektoren von der Länge Null schlechthin mit 0. Im folgenden beschränken wir uns auf Vektoren, die in einer Ebene liegen. Zu ihrer . Bestimmung denken wir uns einen ganz beliebigen herausgegriffen und als „Einheitsvektor“^ oder o Figur 59. „Einheit“ schlechthin bezeichnet. Seine Länge wählen wir als Masseinheit, seine Richtung als Nullrichtung. Irgend ein anderer Vektor hat dann die Länge r und bildet mit dem Einheitsvektor den Winkel cp, den wir entgegengesetzt dem Sinne des Uhrzeigers messen. Diesen Vektor bezeichnen wir mit r , den Einheitsvektor also mit 10. Man nennt zwei Vektoren gleich, wenn sie in Lauge und Richtung übereinstimmen. Soll r^ gleich sein, so muss also r = s, cp = v> + к . 360° sein, wobei к eine beliebige ganze Zahl, positiv oder Кар. XI. Der Moivre'sche Satz. 133 negativ, ist. An Stelle beider Gleichungen schreiben wir abkürzungsweise Eine Verwechslung mit dem Gleichheitsbegriff der Zahlen ist dadurch ausgeschlossen, dass r^ und s^ keine Zahlen sind. Man kann einen Vektor auch mit einem einzelnen Buchstaben, u, v, w, z, bezeichnen, und muss sich nur gegenwärtig halten, dass dieser Buchstabe nicht eine Zahl, vielmehr das Aggregat zweier Zahlen, r und <p, bedeutet. § 51. Addition von Vektoren. Es seien jetzt AB und BC zwei beliebige Vektoren, u und v. Der zweite ist mit seinem Anfangspunkt an den Endpunkt des ersten angesetzt. Dann nennt man C B' 0 Jo é fB A n Figur 60. den Vektor mit dem Anfangspunkt A und dem End punkt C die Summe der beiden anderen und bezeichnet ihn mit u + v« Eine Verwechslung mit dem gewöhn lichen Summenbegriff der Zahlen ist ausgeschlossen, da ja u und v keine Zahlen sind. Ist AB' gleich und parallel ВC, so istB'C gleich und parallel AB; daher ist AB' wieder v und B'C u. Bei dieser Lage der Vektoren ist AC gleich v-\|-u; man darf also die Summanden vertauschen. 134 Berechnung der trigonometrischen Funktionen. Sind AB, BC, CD, DE beliebige Vektoren u, v, w, z, so bezeichnet man AE als die Summe derselben mit u-{■ v + w z- Da AE auch die Summe von AB, BD, DE, oder von AC, CD, DE, oder AC, CE ist, ist u + v + w + z = u + (v + w)+z = (u + v) + w -f- z = (u + V) + (w + z), • demnach auch gleich u + (w + v) + Z = (v + u) + w + z = (w + z) + (u + v), das heisst =u+w+v+z =V+U+W+Z =W+Z+U+V E z D n % $ C ? 3 A и Figur 61. u. s. w. In einer Summe von Vektoren dürfen die einzelnen Vektoren beliebig unterein ander vertauscht werden. Die Summe entgegengesetzt gleicher Vektoren ist Null, wie man sich durch Konstruktion sofort überzeugt. Kap. XL Der Moivre’sche Satz. 135 Ferner ist u -f- 0 = u, was auch ohne weiteres anschau lich ist. Den u entgegengesetzten gleichen Vektor wollen wir mit —u bezeichnen. ^Soll nun ein Vektor x die Bedingung u+x=w erfüllen, so addiere man zu w noch —u. Es wird w + (— u) = U + (-- u) + X = 0 + X = X. Für w -f- (— u) schreibt man einfacher w — u und bezeichnet auch w — u als die Differenz von w und u. § 52, Multiplikation von Vektoren. Dreht man einen beliebigen Vektor rÿ um einen Winkel гр und vergrössert bezw. verkleinert r im Mass- V5/ r <£_ Figur 62. stabe s:l, so entsteht ein neuer Vektor von der Länge rs und dem Richtungswinkel + den man das Produkt von r_ mit s„,. ip nennt und mit bezeichnet. Da rqp • ^ip — (r S) (p _j_ у wird, ist (I) r(f * 8гр sip • rcp, man darf also in diesem Produkt die Faktoren vertauschen. Als Produkt von beliebig vielenVektoren r^, Sy, t^ . . . bezeichnet man den Vektor (rst...) +^+ Die drei Vektoren u = AB, v = BC und u-f-v = AC bilden ein Dreieck ABC. Dreht man dies um 136 Berechnung der trigonometrischen Funktionen. einen Winkel ip und vergrössert es im Massstab s, so dreht man jede Seite um diesen Winkel und vergrössert sie im selben Massstab. Daher sind die Seiten des neuen Dreiecks A'B'C' u . s f, v-s ib. (u+v)>. Die dritte ist aber nach wie vor die Summe der beiden anderen, d. b. es ist (u +v) = + T8^( (И) Die Multiplikation und Addition von Vektoren befolgen also dieselben Gesetze, wie die der gemeinen Zahlen. Sie gehen auch in die Operationen des gewöhnlichen Rechnens über, wenn man die Vektoren der Richtungen 0 und 180° be trachtet. Es ist ro + so = (r + s)o ro • so = (rs)o. und da r180 = — r0 ist, treten die Vektoren der Rich tung 180° an Stelle der negativen Zahlen. Wir wollen daher den Index 0 im allgemeinen fortlassen und entsprechend für r180 —r schreiben. Die Vektoren in einer Ebene erweisen sich hiermit als Verallgemeinerung der gewöhnlichen Zahlen. Man nennt sie daher auch imaginäre oder besser komplexe Zahlen, wiewohl sie, streng ge nommen, Aggregate von zwei Zahlen sind und zu ihrer Bestimmung die Angabe von zwei Zahlen erforderlich ist. Unter imaginären Zahlen sollten eigentlich nur die Vektoren der Richtung -)- 90° verstanden werden. Kap. XI. Der Moivre’sche Satz. 137 § 53. Zerlegung* der komplexen Zahl in reellen und imaginären Teil. Zieht man durch den Anfangspunkt A eines Vek tors u eine Parallele zur Nullrichtung und fällt vom Endpunkt В das Lot BC darauf, so entstehen die Vek toren AC = ! und CB = 7, so dass U = 1+ nВ & ?>a к H Figur 63. I hat die Richtung 0 oder 180°, r\ die Richtung + 90°. Es ist also £ eine positive oder negative Zahl x, 4 ein positives oder negatives Vielfaches von 190, r\ = y . 190, x und y nennt man die Komponenten von u. Den Vektor 190 bezeichnet man durch weg mit i und nennt ihn die „imaginäre Ein heit“, im Gegensatz zu der „reellen Einheit“, 10. Es lassen sich also alle komplex en Zahlen in der Form u = x-f iy darstellen, und dieser Thatsache verdanken sie den Namen „komplex“. Aus der Länge r und dem Richtungswinkel cp eines Vektors findet man seine Komponenten durch die Glei chungen y = r cos ер, x = r sin cp. (III) Wenn zwei Vektoren gleich sind, stimmen sie in r vollständig, in cp bis auf Vielfache von 360° überein; 138 Berechnung der trigonometrischen Funktionen. cos ф und sin (p haben daher dieselben Werte, mithin auch X und y. Die Vektorengleichung x + yi = a + bi ist also gleichbedeutend mit den beiden gewöhnlichen Zahlengleichungen X = a, y = b. Aus (XIII) ergiebt sich: Y(p = X -f- i y = r (cos (p + i sin ç>). (IV) cos (p + i sin cp nennt man den Richtungsfaktor der komplexen Grösse, r ihren absoluten Betrag. Es ist nach dem Pythagoras: г = + Ух- + у-. (V) te § 54. Summation der Sinus und Cosinus einer arith metischen Reihe топ Winkeln. Ein Kreisbogen vom Radius R und Mittelpunkt О sei in n-gleiche Teile A0 A1? At A2, A2 A3, ... An—iAn geteilt. Der Winkel A0O A, = AŁ O A2 etc. sei gleich «. Die Sehnen A0At, A1A2 .. . u. s. f. haben die Länge r = 2Rsin-^-; bildet die erste mit einer beliebigen Nullrichtung den Winkel cp, so bildet die nächste mit derselben Richtung den Winkel cp a, die dritte den Winkel cp -f- 2 a, die letzte cp -\|- (n — 1) «. Die einzelnen Sehnen, als Vektoren aufgefasst, sind also mit rcp, rq> + a, r<p-f-2a ••• Ycp -f (n — i) a zu bezeichnen. Ihre Vektorensumme ist der Vektor A0An. Seine Länge ist, da er dem Centriwinkel n a gegenüberliegt, na = 2 R sin 2 • Mit A0At bildet er den Winkel Aj A0 An, der als Peripheriewinkel über dem Bogen Кар. XI. 139 Der Moivre’sche Satz. A An gleich П — a ist, mit der Nullrichtung also den * 2 AVinkel—— * а + <p. Mithin ist : 71 R О А ci л ci г А n л. ci л/ ГЗА г. Figur 64. тср H™ rqp -f а “Ь гф -f 2 а “Ь • • • г<р -f (п — 1) а ~ s п — i 2 а + <р oder 2 Esin — jlç> + lqp_pa + • • • lç> + (n -1) « J nа = 2Esin — J Setzt man für 1^ cos ^ + i s^n hebt 2 E fort und vergleicht Eeelles mit Eeellem, Imaginäres mit Imagi närem, so folgt: cos y+COS (<p-f a) + COS (qp+2 а) + • • • COS (q> + (n —1) а) . На ~2~ . COS [ą> + " 2 1 «) . а sinT (VI) 140 Berechnung der trigonometrischen Funktionen. sin cp + sin (qp+a) + sin (g>--\-2 a) + . •. sin {cp -j- (n —1) a} . na sin-g- (VII) . sin (cp + ” 2 1 “) . CI 8111 “2 § 55. Der Moivre’sche Satz. Funktionen der Viel fachen eines Winkels. Wendet man Formel (IV) auf (I) an, so folgt, in dem sich rs weghebt: (cos cp + i sin tp) (cos гр -f- i sin гр) — COS {cp + гр) (VIII) + isin (g?+ </>)• Man kann diese hochwichtige Formą}, als die „Moivre’sche Form des Additionstheorems“ bezeichnen. (Der eigentliche „Moivre’sche Satz“ wird" sogleich abzuleiten sein.) Entwickelt man nämlich die linke Seite von (VIII) und beachtet, dass 1 до * ^90 I, go I ist, so erhält man das Additionstheorem, indem man die reellen Teile beider Seiten der Gleichung und ebenso die imaginären für sich gleich setzt. Die Moivre’sche Formel ist eines der besten Hilfsmittel, um die Additions theoreme auswendig zu lernen! Indem man гр mit —гр vertauscht, erhält man noch (cos gp —(— i sin cp) (cos гр — i sin гр) = cos {cp — гр) (IX) -f- i sin {cp — гр) und wenn man auch —cp für cp setzt: (cos (p — i sin <p) (cos гр — i sin гр) — cos {ср-\-гр) — i sin {cp -j- гр)» Für cp = гр wird aus (IX) : (cos g?-{-i sing?) (cos g? — isin<p) = 1. (X) (XI) Кар. XI. Der Moivre’sche Satz. 141 Offenbar ist für beliebig viele Winkel a, . (cos a -f- i sin «) (cos ß -f- i sin ß) (cos у -f- i sin y) . .. = cos («-)-/? + y-f .. .) + ism(o + /? + y+ ...) Setzt man a = ß — у = ..., so wird daraus (cosа -f- isin/?)» rr cosma + isinma. (XII) Dies ist der eigentliche „Moivre’sche Satz“. Er zerfällt in zwei Sätze über reelle Grössen, wenn man die linke Seite nach dem binomischen Lehrsatz ent wickelt. Dabei treten die Potenzen von i auf : i = i, i=—1) i* = i.i = —i, i = (-l).(-l)=l, i5 = i4.i = i, i»=—1, i7 = —i, i8=l u. s. w. Vergleicht man, nachdem diese Werte eingesetzt sind, Peelles mit Reellem, Imaginäres mit Imaginärem, so erhält man: m /m\ m -2 a sin а COS Ill a = COS а — (2)cos /m\ -M^lcos 111 —4 . 4 asm а -+... (XIII) -3 n‘ —1 /m\ „ sin m а = (/in\ а sin а j 1 COS a sin а — (з)cos i /ш\ + ( 5 ) cos ш —5 . 5 a sin а---- \- ... Beide Formeln lassen sich noch umformen, indem man cosmce als Eaktor vor die rechte Seite setzt: cos m a = cosm a (i-0v.+Gb,-®.+-j {XIV) sin ma = cosma Durch (XIII) und (XIV) hat man die Funktionen der Vielfachen eines Winkels durch die Potenzen der Funktionen des einfachen Winkels ausgedrückt. 142 Berechnung der trigonometrischen Funktionen. Man kann auch umgekehrt jede Potenz eines Cosinus oder Sinus durch die Funktionen der Vielfachen seines Winkels ausdrücken. Setzen wir zur Abkürzung vorüber gehend cos a -f- i sin а = also nach (XI) 1 cos а — i sin а = , T so wird 1 2 cos а = I -f- -jr, /, l\m ■+(?)»■ — 2 Es ist aber — (m — 2) Kl —m W n n 2“ cos m also = cos Д а -\|- i sin Д а ^ = cos Да — i sin Л a. Setzt man diese Werte wieder ein, so hebt sich naturgemäss alles Imaginäre weg und man hat cosm a als Funktion der Vielfachen von cs bis ma einschliesslich. Ebenso wird 2 i sin a = g — -j 3 (2i)msinma = r +...±rm Wird g wieder nach dem Moivre’schen Satz aus gedrückt, so hebt sich, je nachdem m gerade oder un gerade ist, alles Imaginäre oder alles Reelle fort und man erhält sinma durch die Cosinus oder Sinus der Vielfachen von а bis та ausgedrückt. Кар. XII. Unendliche Reihen und Produkte. 143 Zwölftes Kapitel. Unendliche Reihen und Produkte zur Darstellung der trigonometrischen Funktionen. § 56. Im folgenden sind die Winkel nicht mehr nach Graden, sondern im „natürlichen Win keim as su7 d. h. durch den Arcus gemessen gedacht. Ein rechter 7C Winkel wird mit —, ein gestreckter mit n bezeichnet. Der Winkel a hat das Bogenmass 2n «° = x. Unter dieser Voraussetzung gelten die zwei bemerkenswerten Reihenentwicklungen, die für jedes x konvergieren: COS X — 1 X2 , X* 2! ‘ 4! И тн Xs X5 I sin x = — a7+ 5! Xe X8 X7 X9 б!-- 8! - + ... 7! "^"”9? + •♦• (1) (П) iH ÎC Darin bezeichnet n ! das Produkt der n ersten ganzen Zahlen 1.2.3... n = n! (spr. n-Fakultät). Auf eine Herleitung dieser Formeln muss verzichtet werden. Umgekehrt kann man auch den Bogen durch den Sinus oder die Tangente ausdrücken. Es ist: 1 sin8x 1.3sin6x 1.3.5sin7x x = sin x H--------f... (Ш) 1 2 3 ' 2.4 5 2.4.6 7 tg3x + 4-tg5x — — tg’xH----(IV) = tgx — Die erste Reihe konvergiert für jeden Wert des Sinus, d.h. für — 1 < sin x < -f 1, 144 Berechnung der trigonometrischen Funktionen. und liefert den zwischen + 90° und — 90° gelegenen Bogen, d. h. 71 ^ = ^~ П2 2 =x а ► M а Die zweite konvergiert für -1 <tgx< + l und liefert <<+- -• 1 Indem man für sinx die Werte 1, w, i . erhält man Beihen für —-, —r- und ebenso indem 2 4 6’ man tgx = l setzt, die berühmte Leibnitz’sche Beihe: а тН h+ (V) i 1 — —+——1 3^5 7 11 * die aber für praktische Zwecke zu langsam konvergiert. 05 = Andere interessante Entwicklungen sind noch: COS X = \i — Ui- 1 \l_ «-h X X2 X2 (3)T » Ir X2 • • (VI) I (VII) (•I °) — o gesetzt ist. Ferner: (VIII) il cot X 1 •l!1 (2 n) worin zur Abkürzung - + X2 -4-ЭД to а Sin X I - ^Ье! + ( i 2л M +{l3 л 2л - x) ^—лл-f-x}{—2 л ' 2тг + х 4-xl 1 1 = 2x f 1 (2x2 ^ха-/г2^х2 — 4л Зл— x, I—Зл 3 Tr —x 1 — 9л2 •••} (IX) Die Entwicklung von tg und cot nach Potenzen von x und die damit im engsten Zusammenhang stehende Кар. XIII. Die Methode der Hilfswinkel. 145 von log sin X kann hier nicht angegeben werden. Das Vorstehende soll nur dazu dienen, den Leser zur Be schäftigung mit den Methoden der höheren Mathematik, speciell der Integral- und Differentialrechnung, anzuregen. Dreizehntes Kapitel. Die Methode der Hilfswinkel. § 57. Beispiele. Es ist vielfach bei Rechnungen, die nicht durch gehend logarithmisch geführt werden können, von Vor teil, durch Einführung von Hilfswinkeln die trigono metrische statt der logarithmischen Tafel zu benutzen. Dies würd an einer Reihe von Beispielen klar werden. 1. Gegeben loga, logb, gesucht log(a-J-b). Auflösung : a und b sind positive Zahlen. Man hat a-f- b = a^l + 1 Setzt man — = tg2 <jp, so wird 1 + cos2<p a 1 a -{- b mithin log tg q> = — (log b — log a), cos2 <P log (a + b) = log a — 2 log cos <p. Würde man zu loga, logb die Numeri aufschlagen, diese addieren und wieder den Logarithmus nehmen, so hätte man die Tafel dreimal zu benutzen. Bei der trigonometrischen Methode geschieht dies nur zweimal. Wenn man es versteht, zu dem Logarithmus einer Funktion den einer anderen Funktion desselben Winkels sofort aufzuschlagen, ohne erst den Winkel nachzusehen, io Hessenberg, Ebene und sphärische Trigonometrie. 146 Anwendung der trigonometrischen Funktionen. so benutzt man die Tafel sogar nur einmal. Manche Tafeln enthalten daher besondere Täfelchen zum Inter polieren zwischen log sin, log cos und log tg. 2. Gegeben log a, logb, a^>b. Gesucht log (a — b) Auflösung: Setze — = sin cp, d. h. log sin cp — log b — log a, a Z so wird a — b = acos2(p, log (a — b) = 2 log cos cp + log a, 3, Andere Auflösung von 1 und 2 : b sei auch in 1 die kleinere Zahl. Man setze rû C 3 = cos 2 cp, d. h. log cos 2 cp = log b — log a. ■Q Es wird: a-\|-b = 2acos2<p, log(a-\|-b) = log 2 -\|- log a + 2 log?os<p a — b = 2asin2<p, log (a — b) = log 2-f-log a 2 log sin (p. 4. Zu berechnen x = a sin« + k cos «. Man setze a = rcos<p, b = rsing>, so wird asin(<jp + a) x = r sin (<P -j- a) ; tgqp = COS cp a -}- b 5, Zu berechnen x = a—b ö S Man setze — — =tg9p, logtg<p — logb — loga. dl Es wird, da tg 45° = 1 1 + tgqp _a-f-b tg (45° + çï) = X. 1 — t gcp a — b 6. x = j/аЛ+Ь2. Setze a = xcos<p, b = xsingp. tg<p = Es wird l°g tg <jp = log b — log a, a x = cW ^gx = log^ —iogcosç). Кар. XIII. 147 b, a > b. » о 7. X = Die Methode der Hilfswinkel. Setze — = cos cp, log cos qp = log Ъ—log a. Es wird pQ X = a sin qp, log x = log a-flog sin qp. a -f b а > b. Setze, wie in 3, - =cos2qp. 8. X a — b’ Es wird x = + tg<p, log(+x) = logtgqp. 9. c = ya2+b2— 2abcos/ (§10, zweiter Fall) c2 = (a -f b)2 — 2 ab (1 -f cos y) o 3 -V = (a-f b)2 — 4 ab cos2 ^ . 4 a b cos2 -~ Setze = sin2 qp, ^ 1 ! Ill log sin <p = log 2 -f Y loga+ ^ logb tO -f log cos ---- log (a -f b). Es wird c = (a -f b) cos cp, log c = log (a -f b) -f log cos cp. 10. c = bcosa+l/a2 — b2sin2« (§ 10, dritter Fall). b sina sin qp, so wird Setze c = b cos a + а cos qp. Da nach der Definition von cp b sin cp sin а ist, setze man diese Grösse gleich d. Es wird: c = d sin (cp + a). 1—t 11. Die Logarithmierung von 7 im § 26 (erste 1 und zweite Hauptlage) geschieht nach 5, die von cos/? + 2t (dritte Hauptlage) nach 1 bis 3. Ist in 148 Anwendung der trigonometrischen Funktionen. cosfi = cos Л-j- 2 t Л stumpf, so kann man auch ——- — cos2 œ setzen. — cos Л T Dadurch wird cos fi = — cos Л (2 cos2 cp — 1) = — cos Л cos 2 cp. Ist in cos Л = cos fi— 2 t t sin2y; es wird ebenso fi spitz, so setze man cos^ cos Л — cos fi cos 2 cp. 12. cosc=cosacosb-j-sinasinbcos7 (§38, dritterFall). Verfährt man nach 1, so hat man, falls y spitz: tg a tg b cos y = tg2 cp cos a cos b cos c cos2 cp zu setzen. Man kann aber auch so verfahren : = sin a cos y ^ cotga cosb j- sin bj cosc COS y cotga COS y = cotg cp cos a cos {cp — b) cos cp 13. cos y = — cos ß cos ce -f sin « sin ß cos c (§ 38,vierter Fall). Nach 1 ist die Berechnung verschieden je nach den Vorzeichen der Cosinus. Setzt man wieder cosc sin a cos y . cos {cp — b) siny cos y = sin a cos c {-^■coaß+airiß) cot« COS c = tg <p _ sin a cos c sin (ß — <p)_ cos a sin (ß - q>) cos/ COS y siny Кар. XIII. Die Methode der Hilfswinkel. 149 cT 14. у aus der Gleichung a sin y -f- b cos у — c zu berechnen. Verfährt man nach 4, so wird tg cp ='- S 3 r sin (cp + а) = c, c cos <p Sin (<jP + a) — ———. di Hieraus ergiebt sich 9? + / un(I daraus y. In 6, 7, 10, 12, 13 besitzt der ffilfswinkel cp eine einfache geometrische Bedeutung. § 58. Trigonometrische Auflösung der Gleichung zweiten Grades. Die beiden Wurzeln xt, x2 der Gleichung X2 — 2 a x + b — 0 genügen den Bedingungen xi + x2 = 2 a, x1 . x2 = b. 1. Ist b positiv, so setze man, um die zweite identisch zu befriedigen : (I) xx = tgqpj x2 = у b cot qp. Es wird dann aus der ersten Gleichung: 2a tg cp -f- cot cp — yr Die linke Seite ist sin cp cos _sin2 cp -f- cos2 cp 1 2 cos cp' sirup sin cp cos cp sin cp cos cp sin2<p’ Man erhält also zur Bestimmung von cp die Gleichung sin 2 cp = (II) ä Infolge dieser Beziehung ist auch Xj=2a sin2 cp, x2 = 2 a cos2 <jp, m woraus unmittelbar ersichtlich ist, dass xx + x2 = 2 a wird. 150 Anwendung der trigonometrischen Funktionen. 2. Ist b negativ, so setze man Xj = У— b . tg qp, x2 = — У— b cot (p. Es wird: xi + x2 = — 2 i— b cot 2 qp, y=b tg2qp (IY) (V) Das Vorzeichen von ]/—b wählt man am besten dem von a entgegengesetzt, damit tg 2 cp positiv, 2 qp spitz wird. Infolge der Gleichung (V) wird auch COS2 qp sin2qp x1 = — 2 a (VI) » x, = + 2a cos 2 qp cos 2 qp woraus man erkennt, dass x1 + x2 = 2a ist. 3. Ist in (II) У b > a, so gehört zu tn 2 <P kein reeller Winkel. In diesem Fall sind die Wurzeln imaginär Man setze: Xj = Г (cos qp + i sin qp), X2 — Г (COS qp — i sin qp). (VII) h S COSqp = ! Г = УЬ, OB Dann ist § 59. Trigonometrische Auflösung der Gleichung dritten Grades. Setzt man in der reduzierten Gleichung dritten Grades x3 = 3px + 2q (VIII) X = у + z, so geht sie über in y3 + 3y2z + 3yz2 + z3 = 3p(y + z) + 2q, oder 3yz(y + z) + y3 + z3 = 3p(y + z) + 2q. Man kann ihr genügen, indem man (IX) У z = p, y3 + z3 = 2 «1 setzt. Die dritten Potenzen von у und z, Uj = У3, genügen den Bedingungen U2 = z3 Кар. XIII. Die Methode der Hilfswinkel. 151 1 U2 =P;=:U1+U2=2(1 und sind daher die Wurzeln der quadratischen Gleichung u2-2qu + p3 = 0, (X) die man die quadratische „Resolvente“ der Gleichung (VIII) nennt. Die Wurzeln u1? u2 der Resolvente findet man nach der Anleitung des vorigen Paragraphen. Sie sind reell, wenn q2>p3. Alsdann giebt es für y und z je einen reellen Wert, der leicht zu berechnen ist, da man die Logarithmen von Uj und u2 kennt. Ferner kann noch Jx = y-(cos l -x.(4+'S) 2.360° y2 = y^cos---- -— j-isin sein. Die zugehörigen Werte von z sind 2‘ = 2-(-т-#)’ z = z-(-T+i!î)’ weil yx Zj = y2 z2 = y z = p reell sein muss. Ist dagegen q2 < p3, so sind ux und u2 imaginär, u4 = r (cos q> + i sin (p), ua = r (cos (p — i sin <p), worin nach (VII): ___ r = + tp3, <1 cos cp = ;py= ist. (XI) Somit wird y = /r . (^oos-g- + isin-~j, z = j/r .(cos y —isin со 3,- x = y + z = 2\|/r .cos — ) 9 cosT. Für -j- kann es aber im ganzen drei wesentlich verschiedene Werte geben, nämlich Anwendung der trigonometrischen Funktionen. 152 cp ___ cp . 360 Vi — T’ у* ~~ з +~1T’ cp 360 so dass man insgesamt 3 Werte für x erhält: xl = 2 Vp cos = + 2 Vp cos ^ x2 = 2 Vp cos ip2 = — 2 Ур cos ^60°— x3 = 2 Vp cos j (XIT) = — 2 Ур cos ^60°+-\|-j Die Winkel können unmittelbar in der Tabelle auf geschlagen werden, wenn q positiv ist. Ist q negativ, so ist cp stumpf, Ф — 180° — cp' •# wo cp' sein spitzer Nebenwinkel. Dann ist cos Cp‘ = — q Vp3 + X, = + 2Ip“cos^ « •-» ) (XIII) x2 = — 2 Vp cos <p' 3 X3 = + 2 Vp cos (б0° + I j Dieser Fall, in dem die Wurzeln der quadratischen Resolvente f imaginär sind, ist bekannt als „Casus irr educibilis“. § 60. 1. Beispiele zu § 58 und 59. x3 = 9x + 28. p = 3, q = 14. Resolvente: u2—28u-\|-27 = 0 а =14, b = 27. Кар. XIII. Die Methode der Hilfswinkel. 153 1 -g- log 27 = 0.7156819 log 14 = 1.1461280 tO log sin2<jp = 9.5695539 —10 2<p = 21° 47' 12",45 q> = 10° 53' 36",22 log tg<p = 9,2843181—10 log 27 = 0.7156819 log Uj = 0.0000000 (Uj = 1) logu, = 1.4313638 К = 27) 1 2 log Uj = 0.0000000, y = 1 -h log u2 = 0.4771213, z=3 X, = 4, x2 u. x3 = — 2 + i 2. X3 = — 7,398636 X + 14. I log ( b) = CO to — p = —2,466212, q = 7. Kesolvente : u2 — 14 u p3 = 0 a =7, Ъ = — (2,466212)3. - bg (— p) = 0.5880456 log a = 0.8450980 log !g2 <jp = 9.7429476 —10 2<p — 28° 57' 18“,1 <p = 14° 28' 39",05 logtg cp = 9.4119544 — 10 log (— u, ) = 0.0000000 (u, = — 1) log(+ua) = 1.1760912 (u2 = + 15) 154 Anwendung der trigonometrischen Funktionen. 4 log (—u,) = 0.0000000,y= -1,000000 Ó Y log U2 = 0.3920304, z =+ 2,466212 X, = 1,466212 x2 u. x3 = 0,733106 + i Уз . 1,733106. 3. X3 = 2Д X + 20. p = 7, q = 10. B-esolvente : u2 — 20 u + 343 = 0 a = 10, b = 343. log a = 1.0000000 1 — l0g b = 1,2676740 A log cos <p = 9,7823530 — 10 * <p = 57° 19' 11",31 8- CO - -=19° 6' 28",77 log cos 19° 6' 23",77 = 9,9753910 —10 log cos 40° 53' 36",23 = 9,8784810 —10 log cos 79° 6'23",77 = 9,2764211 —10 V, log p = 0.4225490 log 2 = 0.3010300 log X, = 0.6989700 log (—xa)== 0.6020600 log (— xs) = 0.0000001 x,=5, x2 = —4, x3 = —1. 4. X3 = 39x —70. p = 13, q = — 35 = — q'. log q' = 1.5440680 Y log p = 1.6709151 log cos (f‘ = 9.8731529 — 10 Кар. XIII. Die Methode der Hilfswinkel. <p‘ = 41° 41' 37",23 Y = 13° 53' 52", 41 log cos 46» 6' 7",59 = 9.8409684 —10 log cos 13» 53' 52",41 = 9.9870963 —10 log cos 73» 53' 52",41 = 9.4430382—10 % log p =0.5569717 log 3 = 0.3010300 log X, =0.6989701 log (-x2) = 0.8450980 log Xg = 0.3010299 xt=5, x2= — 7, x3 = 2. 155 Anhang. 156 Anhang. Uebungsbeispiele. Tafel I. Bechtwinklige ebene Dreiecke. а а ъ 2° 27' 55" 0,58100 13,497 13,509 22° 37' 12" 5,3846 12,923 14,000 30° 30' 36" 6,6000 11,200 13,000 35° 53' 52" 12,751 17,616 21,746 36° 52' 12" 9,000 12,000 15,000 38° 21' 47" 13,497 17,051 21,746 51° 38' 13" 13,825 10,943 17,632 53° 7' 48" 11,200 8,4000 14,000 54° 6' 8" 10,943 7,9210 13,509 59° 29' 24" 12,923 7,6154 15,000 67° 22' 48" 12,000 5,0000 13,000 87° 32' 17,616 0,75840 17,632 с ш- 5" 21,746 17,632 17,632 16,470 17,632 13,509 11,993 13,509 13,509 5,904 21,746 21,746 1,6000 14,000 13,000 15,000 4,0000 15,000 13,000 14,000 2,2308 7' 48" 7' 48" 92° 27' 55" 38° 21' 47" 125° 53' 52" 92° 27' 55" 5" 87° 32' 14° 15' 0" 59° 29' 24" 112° 37' 12" 67° 22' 48" 59° 29' 24" 38° 21' 47" '49° 10' 18" 33" 25' 57" 38° 21' 47" 53° 53° 7° 53' 24" 7' 48" „TS ,П oQI 53° 7 oTQ 15,000 /9 otQ 14,000 f/8 ,9 13,000 ß „8 a „98 Ю8 oOSI c „98 ,TS o9 b //ST /£8 oSTT a Tafel IL Schiefwinklige ebene Dreiecke. Uebungsbeispiele. 157 Tafel III. 158 В echtwinklige sphärische Dreiecke. // w ю 15 38 6 16 6 22 36 10 39 38 57 12 38 57 12 40 9 21 41 5 6 41 5 6 41 32 38 44 44 17 47 37 21 47 37 21 50 56 50 56 52 54 52 5 54 56 15 43 56 15 43 56 52 23 56 52 23 58 40 13 60 22 24 60 22 24 61 33 4 64 9 43 68 12 58 77 43 18 77 43 18 79 29 45 80 14 41 о // 9 45 19 10 30 15 21 47 2 9 45 19 21 47 2 28 26 56 10 30 15 25 50 17 25 50 17 31 19 47 28 26 56 31 19 47 12 16 42 29 37 36 12 16 42 33 44 17 33 7 37 37 54 6 29 37 36 33 44 17 42 22 39 33 7 37 39 57 4 42 22 39 48 27 22 51 2 48 37 54 6 39 57 4 48 54 54 51 2 48 о // 12 16 42 12 16 42 29 37 36 37 54 6 33 7 37 29 37 36 39 57 4 33 7 37 33 44 17 33 44 17 39 57 4 37 54 6 48 54 54 42 22 39 51 2 48 42 22 39 48 27 22 45 15 43 51 2 48 48 27 22 45 15 43 53 49 21 49 50 39 49 50 39 48 54 54 53 49 21 74 21 54 73 53 38 73 53 38 74 21 54 о / // 38 57 12 41 5 6 38 57 12 15 38 6 36 10 39 47 37 21 16 6 22 41 32 38 41 5 6 47 37 2^ 40 9 21 44 44 17 16 6 22 40 9 21 15 38 6 44 44 17 41 5 6 47 37 21 38 57 12 41 32 38 52 5 54 38 57 12 47 37 21 50 2 56 56 15 43 56 52 23 38 57 12 41 5 6 56 50 52 54 о / С И / ß // 54 52 56 50 56 52 23 77 43 18 60 22 24 50 2 56 77 43 18 56 15 43 56 52 23 52 5 54 60 22 24 56 15 43 79 29 45 61 33 4 80 14 41 58 40 13 64 9 43 58 40 13 68 12 58 64 9 43 56 15 43 68 12 58 61 33 4 60 22 24 56 52 23 60 22 24 80 14 41 79 29 45 77 43 18 77 43 18 со о a b a с 159 Tafel IV. i О СО ю й S CD О т СП О СО СО rt< 05 ю CD » ß От U СО b и СО CD i >, а с О 4 И ч 2 7 54 50 2 56 52 5 54 О 44 56 4 41 50 2 56 52 5 54 4 34 28 5 59 56 15 43 60 22 24 4 3 15 5 30 41 32 38 44 44 17 6 32 15 6 54 36 10 39 40 9 21 8 40 9 16 22 40 32 33 52 5 54 15 38 7 16 22 52 5 54 61 33:4 15 38 7 17 7 44 44 17 56 52 23 16 32 17 41 41 5 6 47 37 21 24 2 33 68 12 58 80 14 41 17 20 21 35 50 2 56 56 52 23 25 26 22 52 40 21 56 52 23 20 35 27 4 41 6 60 22 24 26 40 29 11 56 43 77 43 18 21 34 24 38 12 56 15 43 43 2 36 40 21 50 13 58 50 2 38 12 55 1 2 56 15 43 47 37 39 24 41 5 6 60 22 24 45 26 21 56 52 23 79 29 45 36 10 40 41 60 20 54 60 22 24 47 37 41 60 22 24 79 39 38 38 57 41 38 44 44 17 57 10 4 52 5 44 17 56 52 23 80 14 41 41 32 46 59 50 2 56 56 52 23 47 39 50 2 56 52 5 54 63 21 53 58 40 50 2 56 52 5 54 99 57 42 15 38 52 5 54 61 33 4 83 34 56 50 2 56 15 43 77 43 18 119 37 37 38 57 56 52 23 79 29 45 107 37 55 50 56 52 23 80 14 41 103 59 30 52 68 12 58 80 14 41 128 11 15 52 а О 7 о // 163 53 38 118 26 56 138 54 54 123 7 37 123 7 37 129 57 4 121 19 47 127 54 6 102 16 42 123 7 37 100 30 15 129 57 4 123 44 17 132 22 39 102 16 42 86 34 33 77 43 18 102 16 42 115 50 17 77 43 18 109 45 36 88 42 27 111 47 4 79 29 25 84 53 48 159 44 26 105 6 41 138 54 54 119 15 Гб 113 53 57 138 5 42 Tafel У. 160 Die Sinus und Cosinus der Vielfachen yon 3° im ersten Quadranten. о Sinus Cosinus о со со о. 1 2 s, r, —12 r2 V2 (t, У1Г- S2) Sl t2 r, + s, r2 V2 (ti+s2 m 'hi 90 87 84 81 78 75 72 69 66 63 60 67 54 51 48 45 Cosinus Sinus 0 о s2 rxj- t4 r2 V2 (t2 У_з — Si ) V2 (sj V 2 — t2 У 2 ) V2( t, -st}/3) ^1 Г1 V2 (si V Я “f- 12 ) V2 (Sl УУ+12 V If) Va (tx V 3 + s2 ) Г2 ri ti S2 ^2 r\| Sj r2 V* (s, У 3_— t2 ) V2 (t, У 2 - s2У¥) V» s2 r2 + t, r2 + S2 Г2 Si TjJ- tj r2 V(t,r»+ s,_) V (t, V 2 + s2 fF) /2 У 3 t, r, - s2 r2 Zur Abkürzung ist gesetzt: r, = /4 . У2(\|/3 + 1) r2 = 4* -Ÿ2 (у 3 - 1) _/У4.Уб+1) h.Ÿ2]/3 + f5 tx = 1//4 . 5 + fb /4 . (fb - l) s2 li . f2]/ 3 - /5 t2 = ‘/4 . У 2 t 5 - У5 Textaufgaben. 161 Textaufgaben. 1. Der Schatten eines senkrecht aufgestellten Stabes ist b Meter lang, der Stab selbst a Meter. Unter welchem Winkel treffen die Sonnenstrahlen die Erdoberfläche? 2. Um die Höhe eines Turmes ST (Baumes etc.) zu messen, hat man 50 m weit vom Eusse S in dem Punkte R die „scheinbare Höhe“, d. h. den Winkel SRT = 32°7' gemessen. Wie gross ist ST? \T s: Q Figur 65. о 3. Um die Höhe zu messen, wenn S unzugäng. lieh ist, hat man auf der Geraden RS von einem zweiten Punkt P ebenfalls die scheinbare Höhe des Objektes gemessen und gleich 21° 38' gefunden. Wie gross ergiebt sich in diesem Palle ST? 4. Um die Erhebung eines Luftballons T über die Ebene zu messen, hat man von zwei Punkten R und Q aus die Winkel TRS = 30°7', SRQ = 22°36', SQR = 49° 27' und die Entfernung QR = 250 m gemessen. Wie gross ist ST? Anmerkung. Wie hat man sich die Messung der Winkel zu denken, wo doch der Punkt S im Terrain gar nicht markiert ist? Hessenberg, Ebene und sphärische Trigonometrie. 11 Textaufgaben. 162 5. Wie gross sind in der letzten Aufgabe die Winkel TK,Q, TQB, BTQ? (Sowohl mit der ebenen wie mit der sphärischen Trigonometrie zu finden.) 6. Von drei in einer Geraden liegenden Punkten ORQ aus hat man gleichzeitig die Winkel TOS, TRS, TQS gemessen. Bekarmt sind ferner die Entfernungen OR = a, RQ = b Wie findet man ST? 7. In einer Ebene sei C von A und В aus sicht bar, aber unzugänglich. Gemessen ist AB = c, <£; CAB = a, CB А = ß. Wie findet man CA? . A sei von В aus weder sichtbar noch zugänglich, z. B. liege ein Berg dazwischen. Man hat CA = h, CB = a und A CB = y gemessen. Wie gross ist^AB ? 9. Gieht es im selben Falle keinen Punkt C, von dem aus A und В zugänglich sind, so kann man zwei Punkte, C und D wählen, deren Entfernung CD be kannt ist, und die Winkel ACB, BCD; BDA, ADC messen. Wie findet man daraus AB? 10. In einem ähnlichen Falle sei AB messbar (z. B. sei durch den Berg ein geradliniger Tunnel gebaut), dagegen C von D aus unzugänglich. Wie findet man aus AB und denselben Winkeln, wie im vorigen Bei spiel, CD? (§ 26, erste Hauptlage. Diese Aufgabe ist die „Hansensche“.) 11. Das Dreieck der Punkte ABC sei bekannt. Von D aus misst man die Winkel A DB, B DC. Wie gross ist CD? (Pothenotsche Aufgabe, § 26.) 12. Eine Bahn hat die Steigung : a, d. h. auf a Meter horizontales Fortschreiten kommt m Steigung. Wie findet man den Winkel cp, den die Bahn mit der Horizontalen bildet? 8 1 1 Textaufgaben. 163 / 'S а Figur 66. 13. In einer einfachen Weiche ist das Geleis G i geradlinig, G2 in einem Kreis geführt. An der Krenzungs stelle К der inneren Schienen bilden diese einen Winkel «, dessen Tangente gleich t ist. Wie findet man aus der „Spurweite“ s hiermit die Länge 1 = AK der Weiche? In Praxi ist t = 1: 9 oder 1 : 10 (Neuntel- und Zehntel-Weiche), s = 1,435 m. c ^ > К g2 R d Figur 67. 14, Ein Geleise geht bis A geradlinig, macht darauf eine kreisförmige Biegung vom Radius R und Winkel a, AB. Sodann geht es von В bis C geradlinig, p Meter weit, dann Textaufgaben 164 Os Ö L erfolgt dieselbe Biegung nach der anderen Seite, CD, so dass bei D das Geleise wieder in die frühere Richtung über geht. Um wieviel ist es dann „verschwenkt“, d. h. wie gross ist h? S l h •H ♦ Figur 68. 15. Wenn umgekehrt h, R, p gegeben sind, wie findet man a? Konstruktiv und rechnerisch! Welchen Sinn hat die zweite Lösung für a? Beispiel: R = 1300m, p = 60m. a = 2°0'0", h = 546,0 cm. R = 1300 m, p —60m, h = 10m, a = 3°42'21". 16. Zwei Punkte P1? P2 auf der Erdoberfläche haben die gleiche geographische Breite cp und die Längen \ on Ti p, fi % % 'uator Figur 69. und 12. Wie gross ist ihre kürzeste Entfernung, wie Textaufgaben. 165 gross ihre Entfernung auf dem Breitengrad? (Erdradius im Mittel 6365000 Meter.) 17. Zwei Punkte Pj und P2 haben die Breiten und (p2, die Längen \ und 12. Wie findet man ihre kürzeste Entfernung e? 18. Zwei Punkte Pj und P2 haben die Breiten (p1 und cp2. Ihre kürzeste Entfernung ist e. Wie findet man den Unterschied ihrer Längen ? 19. Wie gross ist der Winkel zweier Seitenflächen an der Kante des regulären Tetraeders? O' -ТЭ 7^ ^ V SSS i 3 r 1 t SC с * T* o « г* L-Ч c > Ш KZ3 20. Wie gross ist er an der Kante des regulären Pentagondodekaeders ? uszu в. 3. Göschen scbe UerlagsDandluna in Leipzig. ■^I^r Soeben beginnt in unsrem Verlage eine neue Publikation zu erscheinen, die den Namen Sammlung Schubert führt. Der ungeahnte Aufschwung, den in den letzten Jahr zehnten Technik und Naturwissenschaften genommen haben, hat die naturgemässe Folge gehabt, dass sich von Jahr zu Jahr ein lebhafteres Interesse der # Mathematik zugewendet hat. Wenngleich für jedes der einzelnen Gebiete der Mathematik Lehrbücher genug vorhanden sind, so fehlte es doch bisher an einem auf dem heutigen Standpunkte der Wissenschaft und der Lehrmethode stehen den Lehrgänge der gesamten Mathematik, welcher, ein heitlich angelegt, in systematisch sich ent wickelnden Einzel-Darstellungen alle Gebiete der Mathematik umfasste. Dieser Umstand bewog uns, die „Sammlung Schubert“ ins Leben zu rufen, eine Sammlung mathe matischer Lehrbücher, die erstens wissenschaftlich angelegt sind, zweitens den Bedürfnissen des Praktikers Rech nung tragen und drittens durch eine leichtfassliche Dar stellung des Stoffs auch für den Nichlfachmann verständlich sind. Die Form der Darstellung ist so gewählt, dass die einzelnen Bände in gleicher Weise für den Unterricht, wie für den Selbstunterricht, oder zur Repetition ge eignet sind. Soeben erschienen : Band I : Elementare Arithmetik u. Algebra von Prof. Dr. Herm. Schubert. „ IV: Konstruierende und beschreibende Stereometrie von Prof. Dr. G. Holzmüller. я VT: Algebra, Determinanten und ele mentare Zahlentheorie von Ober lehrer I)r. 0. Pund. Im Laufe dieses und des nächsten Jahres folgen alsdann : Band II: Elementare Planimetrie von Prof. Pr. W. Pflieger. я III: Ebene und sphärische Trigono metrie von Oberlehrer Pr. Г. Bohnert. я V : Niedere Analysis von Prof. Pr. Herm. Schubert. » VH: Elemente der synthetischen Geo metrie von Oberlehrer Pr. Böger. » Vin • Analytische Geometrie der Ebene von Prof. Pr. Max Simon. « ix : Analytische Geometrie desRaumes von Prof. Pr. Max Simon. Band X: Differentialrechnung von Prof. Dr. Franz Meyer. XI : Integralrechnung von Prof. Dr. Franz Meyer. XII: Darstellende Geometrie von Dr. John Schröder. XIII: Differentialgleichungen von Prof. Dr. L. Schlesinger. XIV : Praxis der Gleichungen von Prof. C. Runge. XV: Elemente der Astronomie von Direktor Dr. E. Hertwig. XVI: Mathematische Geographie von Direktor Dr. E. Hertwig. XVII: Berechnende Stereometrie von Prof. Dr. Gr. Holzinüller. „ „ XVIII : Geschichte der Mathematik von Prof. Dr. R. Haussner. XX: Versicherungsmathematik Ferd. Paul. von Weitere Bände sind in Aussicht genommen. Leipzig, im Juni 1899. G. J. Göschen’sche Verlagshandlung. cs - Saminfung (Soften. l'«LÄ"80W. ^ " © X (ВоЁфепЧфе Derlags&anMnng, Cetp3ig. ^озЮефЁеШтЬе eeZHj&a. цо©е1ф1ф1еЬ.Ша1егеШ. Don ptof. Dr. :Шф 9JlutÇer. mit Dielen ^ofiiiularen. ^ФеЦегтф. ©4ф1ф1е non ber Ur3cit biö J526 oon prof. Dr. £гапз V. Ärones. ^обс5огЙШ1\|Геп(фа!1 ООП prof. Dr. Ж Зфшрраф. Ш<БеЁф1ф1еЬ. îïïalereiY. non prof. Dr. Н1ф. mutter. Dr. mMimalebre ООПШ. prof. Moppen. Q)berlebrer цбЗифДОфпшд von Kob. ©tern. 07©еЁф1ф1е b. fflaleretl. t \6 piilftif Doit Dr. 'Dans Stegwan». non prof. Dr. ;Шф. Stttutpev. \08 ®с(фгф1е b. îïialereill. j ООП prof. Dr. К\|ф. îïïtttl?er. \ \ 7 (БшефЬфебгаттаШ I: Formenlehre oon prof. Dr. Iban» ttolfcer. I \09 (Бе(фгф1е Ь.Ша1егсШ1. mSurgenfunbeDrÄ. con prof. Dr. ;Шф. îïlutber. Urteile ber greffe über „Sammlung ©iifc!)en". Фeutfфe Sehrergeitg., Berlin: $Каф ben üor* liegenben УапЬфеп fielen toir nidjt ait, bie gange ©ammlung aufg angelegentlichfte ttidit allein gitm (tiebramft in höheren 6фц1еп, fonbern аиф gur ©elbftbetehrnng gu empfehlen. Sßatur: ©3 ift gerabegu ег^аипПф, tüte e§ ber rü^mlidbft befannte Verlag ermöglicht, für fo enorm billige greife fo üorgüglicjh anê* geftattete 2® erf феи gu liefern. Фа§ üorliegenbe ЯЗапЬфеп bringt in tuapper nnb üerftänbl^er gorm ba§ Stiffenśmertefte ber Mineralogie gum 2luébrucf. ©aubere 2ïbbilbungen erlebtem ЬаЗ Verftânbniê. ШоЬиЗ: @3 ift erftaunlidj, mie oid biefe fleine Śłartenfnnbe bringt, ohne an tïarfjeit gu üerlieren, mobei поф gu berütfficfytigen ift, baft oiele Vbbilbnngen ben 9iaum ftar! beengen. Vortrefflich mirb bie fôartenprojeftionâlehre nnb bie topographie gefd)ilbert. Sftationalgeitg.: ift biê jept in ber ЬеШ?феп Sitteratnr mohl поф niфt bagemefen, ba& ein Seinmanbbanb bon faft 300 ©eiten in ЬогрдПфег ФгисЬ* nnb Vapierauêftattung einetjt ÿreiê щ ^ареп mar< mie ihn bie „©ammlung @5öfrf)en" itèrent tfcpefien Vanbe, Maç д,.&> tocpk J$efdjid)tc ber bcntfdjen Sitteratur für bett betrag bon fage adjtjig Pfennige ber beutfcpen Sefermelt bietet. Vraft. ©djulmann: Ein sJJteifterftiicf fttrjen mtb biinbigen, unb boep flarett unb oielfageuben 51u§brucf£ mie bie „Seutfcpe Sittęraturgefcpidge" bon $rof. sîft. totp ift and) bte oorliegenbe „Seutfdje Êefdjidjte im ättittelalter", 97atur: gn ber Sternie bon Dr. tlein empfangt ber ©d)iil faft niepr, mie er al§ Anfänger bebarf, minbeftenê aber fo biel, baß er ba§ Siffenêmiirbigfte al§ unentbehrliche Ermtblage jum Verftänbniffe ber Epernie empfängt. . . tun ft f. 5t He (Wutdjen): t. timmiep bepanbelt in feinem Häubchen, „geid)eitf djule" benannt, in f napper, ferniger, fad)lidj= jielbemußter gorm ba§ meite Eebiet be3 bilbmäßigeit geiepnenä unb 5ftalen£. . . . Eieicp nupbringenb nnb in reiepftem SJtaße bitbenb für Seprer, ©cpüler unb Siebpaberfiutftler, möchte ich ba§ mirflicp borjitgüdje Serf mit mannen auerfennenbeit Sorten ber Ein führung in ©фиГе, §au3 unb Scrfftatt jugänglid) maepen. Sie 5Ыftattung ift babei eine fo bornehme, bag mir ber $rei§ bon 80 Pfennigen für baê gebunbene Ser! bon 138 ©eiten fl. 8° mirflid) lädjcrlicp billig erf epeint. sticht meniger al§ 17 tafeln in Son-, garben- nnb Eolbbrud, fomie 135 Voll- unb Sejtbitber ittuftrieren ben änßerft gefunben Sehrgang biefer geidjenfcpule in feiufühtenber Seife, ©фтаЬ. Sfterfur: ^rof. É, Zahler in Utm legt un3 eine Sarfteüuitg ber ebenen (Geometrie bor, bie bk jur 51u§meffung be§ treifeê einfcpließlicp geht. Vefoubere ©orgfalt ift ber 51u§mapl unb 5lnorbnung ber giguren 51t teil gemorben, bereit faubere Shkfüprmtg in 2 garbeit angenehm berührt. E10 b u § : фосгпеё, Urgefd)id)tc. Ser bemährte gorfeper auf oorgefdjidjtlidjent CÖebiete giebt pier in fnappfter gorm bie leprreidje gu* fammenftelluug beê Siffenêmerteften ber Urgefdjicpte. Vortrefflich geeignet jur Einführung nnb junt Ueberblicf. gapreêbericpte berEefdjicptêmiffenfdjaft: §ommel, auf bem Eebiet ber altorientalifcpen Eefd)icpte eine anerfanute Autorität, bepanbelt in biefem Väubcpen bie morgen!änbiftpe Ее f cp i cp te mit großer Eenauigfeit nnb miffenfcpaftlicper Erünblicpfeit in fnappfter gorm. Sa§ fteiuc Vücpteiu mug marrn empfohlen merben. S P jgr. gtg. (Я® i f f en f d&. SB e i r.) : „Sie ^flanje"bonI>r.E,Sennert fbnnen mir befteitê empfehlen, gu fürjefter, fnappefter, fehr flarer unb oerftänblicpcr gorm meiß fein Verfaffer atteê Siffenëmertefte über beu inneren unb äußeren Vau unb über bie gebenêberricptungen ber $ganje jur 5titfchanung ju bringen, mojn feine gattj bortrcffl'idjen, felbftge* jeichneten Sejtabbilbnngen augerorbenttith Diet beitragen helfen. Seimarjcpe g eit g.: Saltparilieb, 9Jtit biefer Ueberfeguitg mirb unê eine pod)mill!ommene mtb üon Sitteratnrfrennben längft erfepnte Eabc geboten. . . . Von einer guten Ueberfegnng ift ju öer1äugen, bag fie, finn= nnb jngleicp möglid)ft toortgetren, opne bem Ur text, mie ber beutfdjen ©ргафе Eemalt anjutpun, ben Eeift be§ Origiuak flar mtb ungetrübt miebcrfpicgele. $>icfcr gorberuug gcrcdjt su »erben, tyat 2lltïjof in meifterpafter 2öeife berftanben. SÖIä,tter f. b. bapr. ($ртп.-©фи1т.: ©moboba, ®ricd). ®e= fdj^te. ©djon ber Stfame imb ber Ütuf beê 33erfaffer§ bürgt bafür, ba&mir nict)t etma bloß eine trodene Compilation bor unë haben, überall seigen fitp bie ©puren felbftänbiger Arbeit $raft. ©фul mann: ©opfert, ©djulpragtô. Œê mirb in gc= brängter £)arfteflung ein veidjer, шор1ЬигфЬаф1ег, ben neneften pabagagifĄen S3eftrebnngen gcrcrfjt merbenber -gnpalt geboten nnb für ben, ber tiefer einbringen mili, ift geforgt burd) rcuppaltige Sitteratur- пафшегТе. Seitfфr. f. b. 9tealfd)ulm.: @3 mar ein glüdlicper (Sebanfe ber rührigen SSerfagSpaubluug, bie s2lbfaffung be§ ber (Siitfüprung in bie 3ïritpmetif nnb ШдеЬга bienenben ЗЗаиЬфепЗ iprer „©ammlung" bem рофдeaĄteten gaĄ^ nnb ©djulmamte ^rof. Dr. ©djnbert $u über tragen . . . 4 ®er SSerfaffer mußte bie ©cbmierigfeiten mit großem (îJef^id 31t bemältigen, inbent er burd) einen ftreng fpftemettfdjen 2luf= bau be§ aritpmetifepen £eprgebäube§ ber gaffungêfraft be§ 3lnfänger3 möglid)ft Sftedptung trug nnb babci nur ba§ ®auptfäd)lid)c in'§ 9luge faßte. — gormclfammlnug mtb Oîcpetitoriunt ber üdiatpematif bau 4$rof. £p. SBürtlen .... SDie bnrd) reincu Фгпй nnb gcfrljmarfballe #u$ftattung fiep аиЗзеЦиеиЬе „gormelfantmlung" mirb infolge ipreś reidjen biclfeitigeu Sn^aite^^ iprer smedentfpredjcnben 3lnorbnung mtb arientiereubcu (Mcbcrung aU 9М)}ф1адеЬиф borsüglidje $ienfte leiften. ©ren^boten: Фа§ grembmort im éeutfdjen ban Dr. ЩпЬ. Cleinpanl. (Sin leprreidjcê Шф1сш, ba§ in feinen engen Söänben .... eiue gnidę bon ©pradjbelcprnug bietet, bie jeben feffeln muß, ber nur einigermaßen baê 33ebürfnté fiißlt, fid) über ©prad)binge 2tufflärmtg зи berfdmffen. Фег 3Serfaffer l)at fid) fd)on Ьигф 3apfreid)e botfêtümticpe ЗЗйфег über bie ©ргафе unb il)r £eben befannt gematpt, er pat eine auëgebreitete, fixere Cettntnté ber ©ргаф- unb 3ßortgefd)^te, pat mit Sluêbauer auf biefem (Gebiete gefammelt unb meiß feinen ©toff immer QefdE)icït 31t gruppieren unb bot3utragen. . . . ©taatêan seiger: ®ie dlöiuifdjc $Ш1сга1игде{ф1ф1е ift eine geiftboUe glättsenbe Arbeit (Sinfenber pat biefelbe bon Anfang bis (Snbe mit größtem ($enuß bmïpgelefen unb babei 9lrt unb (Sntmidlmtg beê römifфen ©фгЦйитё unb bamit beS römifфen ®eifteStebenS über haupt beffer unb дгйпЬйфег berftepcu gelernt, als Ьигф тапфеЗ bielftünbige UniberfitätSfodeg ober bidleibige ^апЬЬйфег. Sfteteorologif фе 8 eit {фг ift: £r ab ert pat in ber 9Jteteo* rologie feine fфmierige Aufgabe bortrefff^ gelöft. gn allen gragen bertritt er ben neueften unb lebten ©tanbpuntt. ©фте1зег11фе 2ebrer3citung: S5er bie ^erfpeftibe bon grepberger unb ba§ (^cometrifdje geidnten bon 33eder ЬигфдеЬ1, mirb feine greubc baran haben, ©o biel für fo menig ©elb mirb rnoßl faum anberêmo geboten. ®ie gttuftrattonen finb faitber unb e?:aft. Фег Stejt ift fnapp unb flar unb аиф ba, то er mehr anbeutet alg augführt, anregenb. <B. % (SöTcgettTcßit СР&хХлаЛлпьеи^ Biblioteka Politechniki Krakowskiej if 1-301353 miw %•». Ą Biblioteka Politechniki Krakowskiej 100000298034

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